Memoria CD.indd - ISHAM

ISSN 0120-4157BiomédicaRevista del Instituto Nacional de SaludVolumen 28, Suplemento No. 1, Agosto, 2008Bogotá, D.C., Colombia, S.A.Proceedings of theX International Congress onParacoccidioidomycosisA Centennial CelebrationAugust 7-10, 2008Intercontinental HotelMedellín, Colombia

Cover figuresScanning electron microscope image of a conidium liberated from Paracoccidioides brasiliensismycelium. Magnification: approximately 12.000 X. Unpublished photography by WA Samsonoff,MR Edwards, ME Salazar, LE Cano, A. Restrepo. New York State Department of Health,Wadsworth Center for Laboratories and Research, Albany, NY, USA. and Corporación paraInvestigaciones Biológicas (CIB), Medellin, Colombia.Composite figure obtained by periodic observations of a microculture inoculated with P. brasilienisisconidia. When freshly prepared, conidial structures - still attached to the parent mycelium - can beseen in the lower portion of the illustration. After 72 hours of incubation at 36° C, multiple buddingyeast cells can be observed in the upper portion of the figure. Magnification: 1.000 X. Illustrationprepared by Martha Urán and Diana Molina, Corporación para Investigaciones Biológicas (CIB),Medellin, Colombia.

Biomédica Instituto Nacional de SaludVolumen 28, Suplemento No. 1, Bogotá, D.C., Colombia - Agosto, 2008COMITÉ EDITORIALRUBÉN SANTIAGO NICHOLLSInstituto Nacional de SaludBogotá, D.C., ColombiaCARLOS ARTURO HERNÁNDEZBogotá, D.C., ColombiaLUIS ALBERTO GÓMEZInstituto Nacional de SaludBogotá, D.C., ColombiaEDITORES ASOCIADOSMARÍA CRISTINA FERROInvestigadora EméritaInstituto Nacional de SaludBogotá, D.C., ColombiaMIGUEL A. GUZMÁNBogotá, D.C., ColombiaLEONARD MUNSTERMANNYale University School of MedicineNew Haven, CN, Estados UnidosÁNGELA RESTREPOCorporación para Investigaciones BiológicasMedellín, ColombiaRICARDO SÁNCHEZUniversidad Nacional de ColombiaBogotá, D.C., ColombiaOMAR SEGURAInstituto Nacional de SaludBogotá, D.C., ColombiaSECRETARIA DEL COMITÉ LINDA GRACE MOLANOARNOLDO BARBOSA RAMÍREZHospital Clínic, Universidad deBarcelona, Barcelona, EspañaCentro de Investigação Em SaudeDa Manhiça, Manhiça, MozambiqueANTONIO BERMÚDEZInstituto Nacional de SaludBogotá, D.C., ColombiaJORGE H. BOTEROUniversidad de AntioquiaMedellín, ColombiaVÍCTOR CÁRDENASUniversity of TexasEl Paso, TX, Estados UnidosALBERTO CONCHA-EASTMANOrganización Panamericanade la SaludWashington, D.C., Estados UnidosZOILO CUÉLLARAcademia Nacional de MedicinaBogotá, D.C., ColombiaLUIS GABRIEL CUERVOOrganización Panamericana de laSaludWashington, D.C., Estados UnidosCOMITÉ CIENTÍFICOPATRICIA DEL PORTILLOCorpogénBogotá, D.C., ColombiaANDRÉS DE FRANCISCOForo Global para la Investigación enSaludGinebra, SuizaFERNANDO DE LA HOZUniversidad Nacional de ColombiaBogotá, D.C., ColombiaJOSÉ LUIS DI FABIOOrganización Panamericana de laSaludWashington, D.C., Estados UnidosJORGE HERNANDO DONADOUniversidad Pontificia BolivarianaMedellín, ColombiaJOSÉ FIGUEROAWorld Health OrganizationGinebra, SuizaLUIS FERNANDO GARCÍAUniversidad de AntioquiaMedelín, ColombiaALBERTO GÓMEZPontificia Universidad JaverianaBogotá, D.C., ColombiaENRIQUE GONZÁLEZUniversity of Texas Health ScienceCenter at San AntonioSan Antonio, TX, Estados UnidosJOHN MARIO GONZÁLEZUniversidad de los AndesBogotá, D.C., ColombiaFELIPE GUHLUniversidad de los AndesBogotá, D.C., ColombiaANTONIO IGLESIASUniversidad Nacional de ColombiaBogotá, D.C., ColombiaJORGE JARABID/Secretaría de SaludTegucigalpa, HondurasERNESTO JARAMILLOOrganización Mundial de la SaludGinebra, SuizaMARCELO LABRUNAUniversidade de São PauloSão Paulo, BrasilJAIRO LIZARAZOHospital Universitario Erasmo MeozCúcuta, Colombia

JUAN GUILLERMO MCEWENCorporación para InvestigacionesBiológicasMedellín, ColombiaROBERTO MENDOZAThe Hospital for Sick ChildrenToronto, Ontario, CanadaALVARO MONCAYOUniversidad de los AndesBogotá, D.C., ColombiaRICARDO NEGRONIHospital de InfecciosasFrancisco Javier MuñizBuenos Aires, ArgentinaMARÍA TERESA OCHOAUniversity of California Los ÁngelesLos Ángeles, CA, Estados UnidosJUAN P. OLANOUniversity of Texas Medical BranchGalveston, TX, Estados UnidosBLANCA RESTREPOUniversity of TexasBrownsville, TX, Estados UnidosVÍCTOR E. REYESUniversity of Texas Medical BranchGalveston, TX, Estados UnidosGERZAÍN RODRÍGUEZInvestigador EméritoInstituto Nacional de SaludUniversidad de la SabanaBogotá, D.C., ColombiaGUSTAVO ROMÁNUniversity of TexasHouston, TX, Estados UnidosPEDRO ROMEROLudwig Institute for Cancer Research,Lausanne branchLausana, SuizaÁLVARO RUIZPontificia Universidad JaverianaBogotá, D.C., ColombiaGIOCONDA SAN BLASInstituto Venezolano deInvestigaciones CientíficasCaracas, VenezuelaÁLVARO SANABRIAUniversidad de la SabanaFundación Abood ShaioBogotá, D.C., ColombiaNANCY GORE SARAVIACIDEIMCali, ColombiaROBERT TESHUniversity of TexasGalveston, TX, Estados UnidosORLANDO TORRES-FERNÁNDEZInstituto Nacional de SaludBogotá, D.C., ColombiaBRUNO TRAVIUniversity of TexasSan Antonio, TX, Estados UnidosGUSTAVO VALBUENAUniversity of TexasGalveston, TX, Estados UnidosJUAN MIGUEL VILLALOBOSUniversidade Federal de RondôniaPorto Velho, BrasilJOHN WALKERCideimCali, ColombiaMOISÉS WASSERMANInvestigador EméritoInstituto Nacional de SaludUniversidad Nacional de ColombiaBogotá, D.C., Colombia® Instituto Nacional de SaludLa revista Biomédica del Instituto Nacional de Salud es una publicación trimestral, eminentemente científica. Estáamparada por la resolución número 003768 de 1981, emanada del Ministerio de Gobierno, y con tarifa postal reducidasegún resolución número 1128 del 5 de mayo de 1982.Ninguna publicación, nacional o extranjera, podrá reproducir ni traducir sus artículos ni sus resúmenes sin previa autorizaciónescrita del editor. Ni la revista, ni el Instituto asumen responsabilidad alguna por los puntos de vista expresados por losautores. La revista no publicará ningún tipo de propaganda comercial. Los nombres de equipos, materiales y productosmanufacturados que eventualmente puedan mencionarse, no implican recomendación ni propaganda para su uso y sólose mencionan como identificación genérica.La revista Biomédica aparece reseñada en Index Medicus/Medline de la National Library of Medicine, en el Science CitationIndex Expanded (SCIE) de Thomson Scientific, en SciELO Colombia (Scientific Electronic Library Online), en el índice dela Literatura Latinoamericana en Ciencias de la Salud (LILACS), en la Red de Revistas Científicas de América Latina, elCaribe, España y Portugal (RedAlyC), en Scopus de Elsevier B.V., en el Sistema de Información Bibliográfica RegionalAndina (SIBRA), en CAB Abstracts, Review of Medical and Veterinary Entomology, y forma parte del Índice Nacional dePublicaciones Seriadas Científicas y Tecnológicas Colombianas de Colciencias y del Índice Latinoamericano de RevistasCientíficas y Tecnológicas (LATINDEX).INSTITUTO NACIONAL DE SALUDAvenida Calle 26 No. 51-20Apartado aéreo 80334 y 80080Bogotá, D.C., Colombia, S.A.URL: http://www.ins.gov.cobiomedica@ins.gov.co

Proceedings of theX International Congress onParacoccidioidomycosisA Centennial CelebrationAugust 7-10, 2008Intercontinental HotelMedellín, ColombiaCORPORACIÓN PARAINVESTIGACIONESBIOLÓGICASLa Ciencia al Servicio de la VidaCarrera 72A No 78B - 141 / Tel: (4) 441 08 55 / Fax: (4) 441 55 14http://www. cib.org.co / e-mail: cib@cib.org.co / A.A 7378 / Medellín-Colombia

CommitteesOrganizing CommitteeJuan Guillermo McEwen O. PresidentLuz Elena Cano R. General SecretaryAngela Restrepo M. General CoordinatorDiego Miguel Sierra B. 1st TreasurerMaría del Pilar Jiménez A. 2nd TreasurerJuliana Correa T. SecretaryScientific CommitteesInternationalRicardo Negroni - ArgentinaVera L. Calich - BrazilEva Burger - BrazilMarcelo Franco - BrazilRosana Puccia - BrazilGioconda San-Blas - VenezuelaBeatriz L. Gómez - United StatesAgostinho de Almeida - PortugalColombiaAna María García C.Angel A. González M.Angela M. Tobón O.Angela Restrepo MBeatriz H. Aristizabal B.Catalina de Bedout G.Juan G. McEwen O.Luz Elena Cano RMartha E. Urán J.Myrta Arango A.Editorial CommiteeMyrta Arango A.Catalina de Bedout G.Juan Eugenio Ochoa M.Martha E. Urán J.

Financial CommiteeLavive Rebaje de ÁlvarezDiego Miguel Sierra B.María del Pilar Jiménez A.Gisela María Acevedo T.Marcela Gallego B.Logistic AgencyEntorno Comunicaciones:María Eugenia Puerta V.Olga Lucía Jaramillo E.Graphic Design and CoverJuan Eugenio Ochoa M.Nelcy López M.Diana Molina M.Web pageMartha E. Urán J.www.pcm2008.org.coPowered by Interconsulting S.A.www.interconsulting.com.co

Invited SpeakersArgentina• Ricardo Negroni. Unidad de Micología, Hospital de Infecciosas Francisco Javier Muñiz, BuenosAires, Argentina.Brazil• André Amaral. Departamento de Biologia Celular, Laboratorio de Biologia Molecular, Universidadede Brasilia, Brasilia, DF, Brazil.• Angela M.V.C. Soãres. Departamento de Microbiologia e Imunologia, Instituto de Biociências,Universidade Estatal de São Paulo, Botucatú, SP, Brazil.• Bodo Wanke. Laboratorio/Serviço de Micologia Médica, Centro de Pesquisa Hospital EvandroChagas (IPEC), Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.• Carla Pagliari. Departamento de Patologia, Facultade de Medicina, Universidade de São Paulo,São Paulo, SP, Brazil.• Carlos Taborda. Departamento de Microbiologia, Instituto de Ciências Biomédicas II (ICB-II),Universidade de São Paulo (USP), São Paulo, SP, Brazil.• Celia M. Soãres. Laboratorio de Biologia Molecular, Instituto de Ciências Biologicas, UniversidadeFederal de Goiãnia, Goiãs, Brazil.• Darcy Roberto Andrade Lima. Professor do Instituto de Neurologia da Universidade Federal doRio de Janeiro, Brazil e Director Associado de Pesquisas, ICS, Vanderbilt University, TN, USA.• Eduardo Bagagli. Departamento de Microbiologia e Imunologia, Instituto de Biociências, UniversidadeEstadual Paulista, Botucatú, SP, Brazil.• Eva Burger. Departamento da Imunologia, Instituto de Ciências Biomédicas (ICB), Universidadede São Paulo (USP), São Paulo, SP, Brazil.• Flavio Queiroz-Telles. Departamento de Saúde Comunitária, Universidade Federal do Paraná,Curitiba, PR, Brazil.• Gil Benard. Laboratorio de Alergia Clinica e Experimental e Imunologia. Departamento de Dermatologia,Faculdade de Medicina da Universidade de São Paulo (FMUSP), SP, Brazil.9

• Henrique L. Lenzi. Departamento de Patologia, Fundação Oswaldo Cruz, FioCruz, Rio de Janeiro,RJ, Brazil.• Ildinete Silva Pereira. Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidadede Brasília, Brasilia, DF, Brazil.• João Santana Silva. Departamento de Bioquimica e da Imunologia, Escola de Medicina, Universidadede São Paulo, Ribeirão Preto, SP, Brazil.• José Daniel Lopes. Disciplina de Imunologia, Departamento de Microbiologia, Imunologia eParasitologia, Centro de Microscopia electrônica, Universidade Federal de São Paulo (UNIFESP),São Paulo, SP, Brazil.• Larissa Fernandes. Departamento de Biologia Celular, Laboratorio de Biologia Molecular, Universidadede Brasilia, Brasilia, DF, Brazil.• Ligia B. Vizeu. Departamento de Geografia, Faculdade de Filosofia, Letras e Ciências Humanas.Universidade de São Paulo, São Paulo, SP, Brazil.• María Adelaide Millington. GT de Pneumonias e Micoses Sistemicas, Ministerio de Saúde,Brasilia, DF, Brazil.• Maria Aparecida Shikanai-Yasuda. Laboratorio de Imunologia, Departamento de Doênças Infecciosase Parasitarias, Faculdade de Medicina, Universidade de São Paulo (FMUSP), São Paulo,SP, Brazil.• Maria Heloisa Blotta. Departamento de Patologia Clínica, Faculdade de Ciências Médicas,Universidade Estatal de Campinas (UNICAMP), Campinas, SP, Brazil.• Maria José Mendes-Giannini. Departamento de Análises Clínicas, Faculdade de CiênciasFarmacêuticas, UNESP, Araraquara, SP Brazil.• Maria Sueli Felipe. Departamento de Biologia Celular, Instituto de Ciências Biologicas, Universidadede Brasilia, Brasilia, DF, Brazil.• Rinaldo Põncio-Mendes. Departamento de Medicina, Faculdade de Medicina, UniversidadeEstatal de São Paulo, Botucatú, SP, Brazil.• Roberto Martinez. Departamento de Molestias Infecciosas da Faculdade de Medicina de RibeirãoPreto, Universidade de São Paulo. Ribeirão Preto, SP, Brazil.• Rosana Puccia. Departamento de Microbiologia, Imunologia e Parasitologia, Divisão de BiologiaCelular, Universidade Federal do São Paulo, São Paulo, SP, Brazil.• Rosely Zancopé Oliveira. Fundação Oswaldo Cruz, Instituto de Pesquisa Clínica Evandro Chagas.Rio de Janeiro, RJ, Brazil.• Vera L. Calich. Departamento da Imunologia, Instituto de Ciências Biomédicas (ICB), Universidadede São Paulo (USP), SP, Brazil.10

Central America• Eduardo Arathoon. Director Médico Clínica Familiar “Luis Angel García”, Asociación de SaludIntegral. Hospital General San Juan de Dios, Guatemala City, Guatemala.• Marion C. de Martin. Departamento de Microbiología, Sección de Micología, Universidad dePanamá, Panamá, Republic of Panamá.Colombia• Ana María García. Corporación para Investigaciones Biológicas (CIB) and Universidad PontificiaBolivariana (UPB), Medellín, Colombia.• Angel González M. Corporación para Investigaciones Biológicas (CIB), and Escuela de Microbiología,Universidad de Antioquia (U de A), Medellín, Colombia.• Angela M. Tobón. Corporación para Investigaciones Biológicas (CIB) and Hospital La María,Medellín, Colombia.• Angela Restrepo M. Corporación para Investigaciones Biológicas (CIB), Medelllín, Colombia.• Manuel Elkin Patarroyo. Instituto de Inmunología, Bogotá, Colombia.• Luz E. Cano. Corporación para Investigaciones Biológicas (CIB) and Escuela de Microbiología,Universidad de Antioquia (UdeA), Medellín, Colombia.Europe• Agostinho João Almeida. Life and Health Sciences Research Institute (ICVS) School of HealthSciences, University of Minho, Campus de Gualtar, Braga, Portugal.• Christopher Kibbler. Department of Medical Microbiology, Royal Free Hospital, London, UnitedKingdom.• Neil AR Gow. School of Medical Sciences, Institute of Medical Sciences. University of Aberdeen,Aberdeen, UK.11

United States of America• Beatriz L. Gómez. Mycotic Diseases Branch, Centers for Disease Control and Prevention (CDC),Atlanta, GA, USA.• Daniel Matute. Department of Ecology and Evolution, University of Chicago, Chicago, IL, USA.• Garry T. Cole. Department of Biology, University of Texas at San Antonio, San Antonio, TX,USA.• John E. Bennett. Clinical Mycology Section, Laboratory of Clinical Investigation, National Instituteof Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.• John R. Graybill. Department of Medicine, University of Texas Health Science Center at SanAntonio, San Antonio, TX, USA.• John W. Taylor. Department of Plant and Microbial Biology, University of California, Berkeley, CA,USA.• Joshua Fierer. Research Service, VA San Diego Healthcare, San Diego, CA. Department of Medicine,Division of Infectious Diseases School of Medicine, University of California San Diego, USA.• Karl V. Clemons. Division of Infectious Diseases, Santa Clara Valley Medical Center, CaliforniaInstitute for Medical Research, and Division of Infectious Diseases and Geographic Medicine,Department of Medicine Stanford University, San Jose, CA, USA.• Mary Brandt. Mycology Disease Branch, Centers for Disease Control and Prevention (CDC),Atlanta, GA, USA.• Mia Champion. The Broad Institute of Massachusetts, Institute of Technology, Cambridge, MA,USA.• Steven K. Ault. Communicable Diseases Unit Area of Health Surveillance and Disease Management,Pan American Health Organization, Regional Office of the World Health Organization,Washington DC, USA.• Tom Chiller. Epidemiology Unit, Mycotic Disease Branch, National Center for zoonotic, vector borneand enteric diseases, Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA.• William B. Goldman. Department of Molecular Microbiology, Washington University, St. Louis,MO, USA.Venezuela• Gioconda San-Blas. Centro de Microbiología y Biología Celular, Instituto Venezolano de InvestigacionesCientíficas (IVIC), Caracas, Venezuela.• Gustavo Niño. Centro de Microbiología y Biología Celular, Instituto Venezolano de InvestigacionesCientíficas (IVIC), Caracas, Venezuela.12

ChairpersonsThursday, August 07Symposium 1.Symposium 2.Symposium 3.Symposium 4.Symposium 5.Symposium 6.Symposium 7.Symposium 8.Symposium 9.Symposium 10.Symposium 11.Symposium 12.P. brasiliensis genomics and bioinformaticsChairs: Celia M. Soãres and Daniel MatuteUnderstanding the immune responses to dimorphic fungi in experimentalanimal hostsChairs: John R. Graybill and Luz Elena CanoFriday, August 08Virulence factors and host-parasite interactionsChairs: Gioconda San-Blas and Angel GonzálezImmune responses in paracoccidioidomycosis: Cellular and molecularmechanismsChairs: Maria José Mendes-Giannini and María del Pilar JiménezGranuloma and fibrosis: Tissue markers of paracoccidioidomycosisChairs: Marcello Fabiano Franco and María Mercedes PatiñoFungal cellular biology aspectsChairs: Joshua Fierer and Agostinho J. Almeida.Saturday, August 09The endemic mycoses under consideration by public health agenciesChairs: Andrea Vicari and Gilberto ÁlvarezClinical and epidemiological considerations on the endemic mycosesChairs: Tom Chiller and Ángela RestrepoNew approaches to the clinical and laboratory diagnosis of systemicmycosesChairs: Maria José Mendes-Giannini and Elizabeth CastañedaAdvances on antifungal antibioticsChairs: Ricardo Negroni and Carlos Andrés AgudeloSunday, August 10Agriculture and paracoccidioidomycosisChairs: María Adelaide Millington and Donald L. GreerEvolutive biology of P. brasiliensisChairs: John W. Taylor and Gustavo Niño13

Scholarship WinnersTraveling Award contributed by Drs. Karl V. and Lynda Clemons to one of the ten best abstractsto be presented in the Congress by a Latin American graduate student• Bonfim, C.V.Laboratório de Imunologia Molecular e Celular. Departamento de PatologiaClínica, Faculdade de Ciências Médicas -UNICAMP- Campinas, SP, Brazile-mail: camilinha@gmail.comTLR-2, TLR-4 and DECTIN-1 expression in human macrophages and neutrophils stimulated byParacoccidioides brasiliensis.Scholarships auspiced by theX International Congress on Paracoccidioidomycosis• Ferreira, M.C.Department of Clinical PathologyFaculty of Medical Science – UNICAMP – Campinas, SP, Brazile-mail: mcf@fcm.unicamp.brIncreased T cell regulatory activity in patients with paracoccidioidomycosis• Estefanía, M.N. Martins.Laboratório de Radiobiologia, CDTN / CNEN, Departamento de Bioquímica e Imunologia, UFMG,Belo Horizonte, MG, Brazile-mail: estefaniabio@yahoo.com.brEvaluation of the protection induced by the immunization with radioattenuated yeast cells of Paracoccidioidesbrasiliensis in animal model• Borges, C.L.Instituto de Ciências Biológicas,UFG, Goiânia, Brazile-mail: clayton@icb.ufg.brFormamidase of Paracoccidioides brasiliensis: cytolocalization, purification and analysis of thenative protein.• Felonato, M.Depto. de Imunologia, Instituto de Ciências Biomédicas,Universidade de São Paulo, SP, Brazil.e-mail: felonato@gmail.comCD28 costimulatory molecule deficiency results in more severe Paracoccidioides brasiliensis infectionbut protects mice from life-threatening immunopathology.15

• Bernardino, S.Instituto de Ciências BiomédicasUniversidade de São Paulo, São Paulo, Brazile-mail: sibern@icb.usp.brNitric oxide but not TREG cells plays a major immunoregulatory role in a pulmonary model of fungalinfection• Costa, T.A.Instituto de Ciências BiomédicasUniversidade de São Paulo, Brazil.e-mail: tacosta@usp.brIL-10 deficiency determines a better fungicidal ability associated with overproduction of IFN-gamaand nitric oxide by Paracoccidioides brasiliensis infected macrophages• Silva, J.F.Faculdade de Ciências Farmacêuticas de Araraquara, UNESP, SP, Brazile-mail: silvajf@fcfar.unesp.Gp43 isoforms of Paracoccidioides brasiliensis as ligands of laminin, fibronectin and collagen type I.• Simoneide, S.S.Instituto de Ciências BiológicasUniversidade de Brasília, Brasília, DF, Brazile-mail: simoneide.silva@gmail.comComparative analysis of gene expression during macrophages infection by pathogenic fungi (Paracoccidioidesbrasiliensis and Histoplasma capsulatum).• Theodoro, R. C.Depto. de Microbiologia e ImunologiaIBB- Unesp, Botucatu, SP, Brazile-mail: raquel@ibb.unesp.brPhylogenetic analysis of PRP8 intein and mycological features in Paracoccidioides brasiliensisspecies complex.16

EditorialIn 1971 the Pan American Health Organization (PAHO) convened the First Pan American Symposiumon Paracoccidioidomycosis in Medellin, Colombia. It was in this city and country thatmany concepts -now classic- were revived and where many important advances in the areawere made. In 2008, 37 years later, we meet again in the same city and country to work on thesame subject, this time provided with new knowledge and renewed hopes for progress.In 1908 Adolfo Lutz, a physician trained in Switzerland and who held his medical practicein São Paulo, Brazil, described for the first time a peculiar disorder afflicting two patients andthat resembled a fungal disorder, coccidioidomycosis, described sixteen years earlier (1892)by Posadas in Argentina. Lutz examined tissue sections from Posadas’ original case and noticedthat the endogenous sporulation, characteristic of Coccidioides immitis, was absent fromhis own patients concluding that the Argentinian and the Brazilian mycoses were two differententities. Additionally, Lutz isolated the etiologic agent in its mycelial form and without naming itwrote in his report that “… the tissue forms are different from the ones observed in cultures…”.Thus, Lutz’ keen mind allowed him to discover a new disease, a new agent and its dimorphicproperties, all in a matter of a few months. From the very beginning, Lutz had set an examplefor all the researchers who were to follow, including most of us.One hundred years have elapsed by since Lutz’ discovery, and this century has providedthose of us, interested in paracoccidioidomycosis and Paracoccidioides brasiliensis, a wealthof findings but most importantly, a multitude of doors open to future research, and a richness ofquestions still unsolved. After all, it is the question that matters.Nowadays the fungus has become an important research tool due to its versatility and enduranceand, why not, its capricious moods. Yet patients continue to experiment the “slings andarrows” inflicted by the fungus and suffer its ill effects when their vital organs are attacked.Judging by the abstracts that will be presented during this X International Congress on Paracoccidioidomycosis,a great deal of progress has been accomplished in all fields of knowledgeon this and related mycoses. It appears that several of the previously existing “icebergs” arenow conquerable with thorough research. It remains to be determined whether or not such freshknowledge will solve some of the most significant problems still standing. Be as it may, we willadvance further only if we join efforts in searching common goals.17

Along these lines of thought and accepting that, presently, scientific progress can be achieved onlythrough cooperative efforts and merging of the individual expertise, the X International Congress onParacoccidioidomycosis represents the forum where such progress can be achieved. We invite youto profit from this opportunity in order to establish cooperative studies with your colleagues and findcommon grounds for exploration of those significant points that mean so much to patients, to scienceand to human kind’s progress.All the members of the Corporación para Investigaciones Biológicas (CIB) heartily welcomeyou to our city, to our country. We feel honored with your presence and trust that the programwe have prepared for you will prove satisfactory so that when you leave you may take with youkind remembrances and new knowledge.Ángela Restrepo M, Ph.D.MedellÍn, Colombia, August 7, 2008.18

Scientific Program15:00-18:00 Arrival and Registrations7:00-7:45 RegistrationsDisplaying PostersWednesday, August 06Thursday, August 077:00-7:307:30-7:45Note: Posters can be visited all throughout the congress; presenters will make themselvesavailable for discussion from 7:00 to 8:00 and from 18:00 to 19:00 hours Friday08 to Saturday 09 August.Welcome greetings: Luz E. Cano.General Secretary of the Congress. Corporación para Investigaciones Biológicas (CIB) yEscuela de Microbiología, Universidad de Antioquia (UdeA), Medellín, Colombia.Symposium 1. P. brasiliensis genomics and bioinformaticsChairs: Celia M. Soãres and Daniel Matute7:45-8:058:05-8:258:25-8:458:45-9:05Comparative analysis of Paracoccidioides brasiliensis and other dimorphic fungi:Mia Champion.The Broad Institute of Massachusetts, Institute of Technology, Cambridge, MAS, USA.Comparative genomics in Coccidioides species:John W. Taylor.Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA.Post-genomic contribution to the knowledge on the host-pathogen interaction:Maria Sueli Felipe. Departamento de Biologia Celular, Instituto de Ciências Biologicas,Universidade de Brasilia, Brasilia, DF, Brazil.Genes involved in morphogenesis and cell wall synthesis and regulation in P. brasiliensis:Gustavo Niño.Centro de Microbiología y Biología Celular, Instituto Venezolano de InvestigacionesCientíficas (IVIC), Caracas, Venezuela.9:05-9:25 Questions9:25-9:45 Coffee breakSymposium 2. Understanding the immune responses to dimorphic fungiin experimental animal hostsChairs: John R. Graybill and Luz Elena Cano9:45-10:05Experimental animal models and their contribution to the study of fungal pathogenesis:Karl V. Clemons.Division of Infectious Diseases, Santa Clara Valley Medical Center, California Institutefor Medical Research, and Division of Infectious Diseases and Geographic Medicine,Department of Medicine Stanford University, San Jose, CA, USA.19

10:05-10:2510:25-10:4510:45-11:0511:05-11:2511:25-11:45Toll like receptors and the adaptor molecule MYD88 play an important role in pulmonaryparacoccidioidomycosis: Vera L. Calich.Departamento da Imunologia, Instituto de Ciências Biomédicas (ICB), Universidade deSão Paulo (USP), SP, Brazil.In vivo protection against P. brasiliensis infection with monoclonal antibodies:Carlos Taborda.Departamento de Microbiologia, Instituto de Ciências Biomédicas II (ICB-II), Universidadede São Paulo (USP), São Paulo, SP, Brazil.P10 immunization using Salmonella enterica’s flagellin as adjuvant:Carlos Taborda and Luis R. Travassos.Departamento de Microbiologia, Imunologia e Parasitologia, Instituto de Ciências BiomédicasII (ICB-II), Universidade de São Paulo (USP), São Paulo, SP, Brazil.Pattern recognition receptors in the host immune response to Coccidioides spp.:Joshua Fierer.Research Service, VA San Diego Healthcare, San Diego, CA. Department of Medicine, Divisionof Infectious Diseases School of Medicine, University of California San Diego, USA.A strategy for the generation of a recombinant vaccine against coccidioidomycosis:Garry T. Cole.Department of Biology, University of Texas at San Antonio San Antonio, TX, USA.11:45-12:00 Questions12:00-17:00Silleteros’ parade18:00-19:00 RegistrationsOpening Ceremony, “A Centennial Celebration”Opening lecture: Henrique L. Lenzi. Departamento de Patologia, Fundação OswaldoCruz, FioCruz, Rio de Janeiro, RJ, Brazil.19:00-21:00Official greetings: Juan Guillermo McEwen. President of the Congress. Corporaciónpara Investigaciones Biológicas (CIB) and Facultad de Medicina, Universidad de AntioquiaMedellín, Colombia;Diego Miguel Sierra B. Director General Corporación para Investigaciones Biológicas(CIB), Medellín, Colombia.Birthday Celebration (Cocktail)Friday, August 08Posters7:00-8:00Wake-up call: Tropical posters, do not miss this opportunity to enjoy fresh fruits whilestudying the posters. A good mix.Note: Posters can be visited all throughout the Congress; presenters will make themselvesavailable for discussion from 7:00 to 8:00 and from 18:00 to 19:00 hours Friday08 to Saturday 09 August.20

Symposium 3. Virulence factors and host-parasite interactionsChairs: Gioconda San-Blas and Angel González8:00-8:208:20-8:408:40-9:009:00-9:209:20-9:40Histoplasma virulence mechanisms with parallels in paracoccidioidomycosis:William B. Goldman. Department of Molecular Microbiology, Washington University,St. Louis, MO, USA.Integrated genomics and proteomics strategies bring new insight into P. brasiliensisresponse upon host interaction: Celia M. Soãres.Laboratorio de Biologia Molecular, Instituto de Ciências Biologicas, Universidade Federalde Goiãnia, Goiãs, Brazil.Adhesion and extracellular matrix proteins: Maria José Mendes-Giannini.Departamento de Análises Clinicas, Faculdade de Ciências Farmacêuticas, UNESP,Araraquara, SP, Brazil.Virulence insights from the P. brasiliensis transcriptome: Ildinete Silva Pereira.Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade deBrasília, Brasilia, DF, Brazil.Role of melanin in certain dimorphic fungi: Beatriz L. Gómez.Mycotic Diseases Branch, Centers for Disease Control and Prevention (CDC), Atlanta,GA, USA.9:40-10:00 Questions10:00-10:20 Coffee breakSymposium 4. Immune responses in paracoccidioidomycosis:Cellular and molecular mechanismsChairs: Maria José Mendes-Giannini and María del Pilar Jiménez10:20-10:4010:40-11:0011:00-11:2011:20-11:40Microbicidal mechanisms exerted by macrophages against P. brasiliensis conidia:Angel González M.Corporación para Investigaciones Biológicas (CIB), y Escuela de Microbiología, Universidadde Antioquia (U de A), Medellín, Colombia.Human cells and molecules involved in the microbicidal mechanisms against P.brasiliensis:Angela M.V.C. Soãres.Departamento de Microbiologia e Imunologia, Instituto de Biociências, UniversidadeEstatal de São Paulo, Botucatú, SP, Brazil.The role of CD8+ cells in human paracoccidioidomycosis:Maria Heloisa BlottaDepartamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estatalde Campinas (UNICAMP), Campinas, SP, Brazil.Antigen-specific immunosuppression in paracoccidioidomycosis: adverse effect or desiredoutcome?: Gil Benard.Laboratorio de Alergia Clinica e Experimental e Imunologia. Departamento de Dermatologia,Faculdade de Medicina da Universidade de São Paulo (FMUSP), SP, Brazil.21

11:40-12:00Several reasons for the immunosuppression observed in paracoccidioidomycosis:João Santana Silva.Departamento de Bioquimica e da Imunologia, Escola de Medicina, Universidade de SãoPaulo, Ribeirão Preto, SP, Brazil.12:00-12:20 QuestionsLunch with professors. Historical accounts: CoccidioidomycosisCoordinator: María del Pilar Jiménez A.Coccidioidomycosis Discovery in Argentina: Ricardo Negroni.Unidad de Micología, Hospital de Infecciosas Francisco Javier Muñiz, Buenos Aires,Argentina.12:20-13:20A history of research on coccidioidomycosis in the U.S.: Garry T. Cole.Department of Biology, University of Texas at San Antonio, San Antonio, TX, USA.13:20-14:00 BreakCoccidioidomycosis: Recent records in Latin America: Bodo Wanke.Laboratorio/Serviço de Micologia Médica, Centro de Pesquisa Hospital Evandro Chagas(IPEC), Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.Symposium 5. Granuloma and fibrosis:Tissue markers of paracoccidioidomycosisChairs: Marcello Fabiano Franco and María Mercedes Patiño14:00-14:2014:20-14:4014:40-15:0015:00-15:20B-1 cells modulate the kinetics of granuloma formation induced by P. brasiliensis:José Daniel Lopes. Disciplina de Imunologia, Departamento de Microbiologia, Imunologiae Parasitologia, Centro de Microscopia electrônica, Universidade Federal de São Paulo(UNIFESP), São Paulo, SP, Brazil.Extracellular matrix modulation in paracoccidioidal granuloma: Eva Burger.Departamento da Imunologia, Instituto de Ciências Biomédicas (ICB), Universidade deSão Paulo (USP), São Paulo, SP, Brazil.Pulmonary fibrosis in an experimental model of paracoccidioidomycosis: Modulationthrough therapeutic intervention: Luz E. Cano.Corporación para Investigaciones Biológicas (CIB) y Escuela de Microbiología, Universidadde Antioquia (UdeA), Medellín, Colombia.Immune reactive cells in skin lesions of patients with paracoccidioidomycosis:Carla Pagliari. Departamento de Patologia, Facultade de Medicina, Universidade deSão Paulo, São Paulo, SP, Brazil.15:20-15:40 Questions15:40-16:00 Coffee breakSymposium 6. Fungal cellular biology aspectsChairs: Joshua Fierer and Agostinho J. Almeida.16:00-16:20Immune recognition of the Candida cell wall: The taste of fungus:Neil AR Gow.School of Medical Sciences, Institute of Medical Sciences. University of Aberdeen,Aberdeen, UK.22

16:20-16:4016:40-17:0017:00-17:2017:20-17:40Cell signaling in morphogenesis and virulence of P. brasiliensis: Larissa Fernandes.Departamento de Biologia Celular, Laboratorio de Biologia Molecular, Universidade deBrasilia, Brasilia, DF, Brazil.Paracoccidioides brasiliensis conidia to yeast transition analysis of certain genes involvedin the process: Ana María García.Corporación para Investigaciones Biológicas (CIB) and Universidad Pontificia Bolivariana,Medellín, Colombia.CDC42p controls yeast-cell shape and virulence in P. brasiliensis:Agostinho João Almeida.Life and Health Sciences Research Institute (ICVS) School of Health Sciences, Universityof Minho, Campus de Gualtar, Braga, Portugal.Differential analysis of extracellular vesicles and of possible cell wall-associated proteinsin isolates of P. brasiliensis: Rosana Puccia.Departamento de Microbiologia, Imunologia e Parasitologia, Divisão de Biologia Celular,Universidade Federal do São Paulo, São Paulo, SP, Brazil.17:40-18:00 Questions18:00-19:00 PostersSaturday, August 097:00-8:00 Registration for the new attendants to the Clinical SymposiumPosters7:00-8:00Wake-up call: Tropical posters, do not miss this opportunity to enjoy fresh fruits whilestudying the posters. A good mix.Note: Posters can be visited all throughout the Congress; presenters will make themselvesavailable for discussion from 7:00 to 8:00 and from 18:00 to 19:00 hours Friday08 to Saturday 09 August.Symposium 7. The endemic mycoses under consideration by public healthagenciesChairs: Andrea Vicari and Gilberto Álvarez8:00-8:208:20-8:408:40-9:00Paracoccidioidomycosis in the panorama of the neglected tropical diseases:Steven K. Ault. Communicable Diseases Unit Area of Health Surveillance and DiseaseManagement, Pan American Health Organization, Regional Office of the World HealthOrganization, Washington DC, USA.The evolving public health importance of mycotic diseases: Tom Chiller. EpidemiologyUnit Mycotic Disease Branch, National Center for zoonotic, vector borne and entericdiseases, Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA.Epidemiology issues in invasive fungal infections: A United States prospective:Mary Brandt. Mycology Disease Branch, Centers for Disease Control and Prevention(CDC), Atlanta, GA, USA.23

9:00-9:20Paracoccidioidomycosis in Brazil – surveillance and control program:María Adelaide Millington.GT de Pneumonias e Micoses Sistemicas, Ministerio de Saúde, Brasilia, DF, Brazil.9:20-9:40 Questions9:40-10:20 Coffee breakSymposium 8. Clinical and epidemiological considerations on the endemicmycosesChairs: Tom Chiller and Ángela Restrepo10:20-10:4010:40-11:0011:00-11:2011:20-11:40The impact of histoplasmosis in HIV-infected patients:John R. Graybill. Department of Medicine, University of Texas Health Science Centerat San Antonio, San Antonio, TX, USA andEduardo Arathoon. Clinica Familiar “Luis Angel García”, Asociación de Salud Integral.Hospital General San Juan de Dios, Guatemala City, Guatemala.The coexistence of HIV infection and paracoccidioidomycosis: Roberto Martinez.Departamento de Molestias Infecciosas da Faculdade de Medicina de Ribeirão PretoUniversidade de São Paulo. Ribeirão Preto,SP, Brazil.Treatment compliance in paracoccidioidomycosis: Rinaldo Põncio-Mendes.Departamento de Medicina, Faculdade de Medicina, Universidade Estatal de São Paulo,Botucatú, SP, Brazil.Guidelines for the treatment of paracoccidioidomycosis: Flavio Queiroz-Telles.Departamento de Saúde Comunitária, Universidade Federal do Paraná, Curitiba, PR,Brazil.11:40-12:00 QuestionsLunch with professors. Historical accounts: HistoplasmosisCoordinator: Beatriz L. Gómez G.12:00-13:00Histoplasmosis: Discovery in Panama: Marion C. de Martin.Departamento de Microbiología, Sección de Micología, Universidad de Panamá, Panamá,Republic of Panamá.Histoplasmosis in the United States: John R. Graybill.Department of Medicine, University of Texas Health Science Center at San Antonio, SanAntonio, TX, USA.13:00-14:00 BreakSymposium 9. New approaches to the clinical and laboratory diagnosisof systemic mycosesChairs: Zoilo Pires de Camargo and Elizabeth Castañeda14:00-14:20The value and the promise of molecular biological tools in the diagnosis of aspergillosisand candidiasis: Beatriz L. Gómez.Mycotic Diseases Branch, Centers for Disease Control and Prevention (CDC), Atlanta,GA, USA.24

14:20-14:4014:40-15:0015:00-15:2015:20-15:40Molecular diagnostic approaches in paracoccidioidomycosis and histoplasmosis:Rosely Zancopé Oliveira. Fundação Oswaldo Cruz, Instituto de Pesquisa Clínica EvandroChagas. Rio de Janeiro, RJ, Brazil.Histopathology of paracoccidioidomycosis in the postgenomic era – reflections and perspectives:Henrique L. Lenzi. Departamento de Patologia, Fundação Oswaldo Cruz,FioCruz, Rio de Janeiro, RJ, Brazil.Clinico-immunological and immunopathological correlations in different clinical forms ofthe paracoccidioidomycosis:Maria Aparecida Shikanai-Yasuda. Laboratorio de Imunologia, Departmento de DoênçasInfecciosas e Parasitarias, Faculdade de Medicina, Universidade de São Paulo (FMUSP),São Paulo, SP, Brazil.Clinical diagnosis in paracoccidioidomycosis: Its defferentiation from similar entities:Angela M. Tobón. Corporación para Investigaciones Biológicas (CIB) y Hospital LaMaría, Medellín, Colombia.15:40-16:00 Questions16:00-16:20 Coffee breakSymposium 10.Advances on antifungal antibioticsChairs: Ricardo Negroni and Carlos Andrés Agudelo16:20-16:4016:40-17:0017:00-17:2017:20-17:40Coccidioidomycosis as seen in a nonendemic area: John E. Bennett.Head, Clinical Mycology Section, Laboratory of Clinical Investigation, National Institute ofAllergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.New and experimental antifungal drugs: Gioconda San-Blas.Centro de Microbiología y Biología Celular, Instituto Venezolano de InvestigacionesCientíficas (IVIC), Caracas, Venezuela.Nanobiotechnology: Advances in antifungal therapy. André Amaral.Departamento de Biologia Celular, Laboratorio de Biologia Molecular, Universidade deBrasilia, Brasilia, DF, Brazil.The epidemiology of Candida and Aspergillus infections: Clinical and molecular aspects:Christopher Kibbler.Department of Medical Microbiology, Royal Free Hospital, London, United Kingdom.17:40-18:00 Questions18:00-19:00 Important notice: Posters should be removed from the exhibit hall after discussion.Symposium 11.7:30-8:00Sunday, August 10Agriculture and paracoccidioidomycosisChairs: María Adelaide Millington and Donald L. GreerP. brasiliensis growth and infective propagule production in clayey and sandy soil:Eduardo Bagagli. Departamento de Microbiologia e Imunologia, Instituto de Biociências,Universidade Estadual Paulista, Botucatú, SP, Brazil.25

8:00-8:208:20-8:408:40-9:009:00-9:20On the relationship between land use and paracoccidioidomycosis: Coffee, sugar caneand other cultures: Ligia V. Barrozo.Departamento de Geografia , Faculdade de Filosofia, Letras e Ciências Humanas. Universidadede São Paulo, São Paulo, SP, Brazil.Influence of alternating coffee and sugar cane agriculture in the incidence of paracoccidioidomycosisin Brazil: Flavio Queiroz-Telles. Departamento de Saúde Comunitária,Universidade Federal do Paraná, Curitiba, PR, Brazil.Chemical components derived from coffee and their impact on health:Manuel Elkin Patarroyo.Instituto de Inmunología, Bogotá, Colombia.Impact of regular coffee consumption on alcohol intake and depressive feelings amongstudents: Darcy Roberto Andrade Lima.Professor do Instituto de Neurologia da Universidade Federal do Rio de Janeiro, Brazile Director Associado de Pesquisas, ICS, Vanderbilt University, TN, USA.9:20-9:50 Questions9:50-10:10 Coffee breakSymposium 12.Evolutive biology of P. brasiliensisChairs: John W. Taylor and Gustavo Niño10:10-10:3010:30-10:5010:50-11:1011:10-11:30What can we know about speciation in P. brasiliensis?: Daniel Matute.Department of Ecology and Evolution, University of Chicago, Chicago, IL, USA.Is P. brasiliensis an unique species? Maria Sueli Felipe.Departamento de Biologia Celular, Laboratorio de Biologia Molecular, Universidade deBrasilia, Brasilia, DF, Brazil.Climate variability and acute/subacute paracoccidioidomycosis in a hyperendemic areain Brazil: Ligia V. Barrozo.Departamento de Geografia, Faculdade de Filosofia, Letras e Ciências Humanas, Universidadede São Paulo, São Paulo, SP, Brazil.Overview of the biogeography of P. brasiliensis: Eduardo Bagagli.Departamento de Microbiologia e Imunologia, Instituto de Biociências, UniversidadeEstadual Paulista, Botucatú, SP, Brazil.11:30-11:40 QuestionsFarewell lunch11:40-14:30Poster awards and Adjourn: Ángela Restrepo M. Corporación para InvestigacionesBiológicas (CIB), Medelllín, Colombia.ConcertLunch26

Symposia IndexPage434344Symposium 1. P. brasiliensis genomics and bioinformaticsComparative analysis of Paracoccidioides brasiliensis and other dimorphic fungi:Mia Champion.Comparative genomics in Coccidioides species:John W. Taylor.Post-genomic contribution to the knowledge on the host-pathogen interaction:Maria Sueli Felipe.45Genes involved in morphogenesis and cell wall synthesis and regulation in P. brasiliensis:Gustavo Niño.47Symposium 2. Understanding the immune responses todimorphic fungi in experimental animal hosts49Experimental animal models and their contribution to the study of fungal pathogenesis:Karl V. Clemons.50515253Toll like receptors and the adaptor molecule MYD88 play an important role in pulmonaryparacoccidioidomycosis: Vera L. Calich.In vivo protection against P. brasiliensis infection with monoclonal antibodies:Carlos Taborda.P10 immunization using Salmonella enterica’s flagellin as adjuvant:Carlos Taborda and Luis R. Travassos.Pattern recognition receptors in the host immune response to Coccidioides spp.:Joshua Fierer.55A strategy for the generation of a recombinant vaccine against coccidioidomycosis:Garry T. Cole.57Symposium 3. Virulence factors and host-parasiteinteractions59Histoplasma virulence mechanisms with parallels in paracoccidioidomycosis:William B. Goldman.59606162Integrated genomics and proteomics strategies bring new insight into P. brasiliensisresponse upon host interaction: Celia M. Soãres.Adhesion and extracellular matrix proteins:Maria José Mendes-Giannini.Virulence insights from the P. brasiliensis transcriptome:Ildinete Silva Pereira.Role of melanin in certain dimorphic fungi:Beatriz L. Gómez.27

65Symposium 4. Immune responses in paracoccidioidomycosis:Cellular and molecular mechanisms6768687071737374Microbicidal mechanisms exerted by macrophages against P. brasiliensis conidia:Angel González M.Human cells and molecules involved in the microbicidal mechanisms against P.brasiliensis:Angela M.V.C. Soãres.The role of CD8+ cells in human paracoccidioidomycosis:Maria Heloisa BlottaAntigen-specific immunosuppression in paracoccidioidomycosis: Adverse effect or desiredoutcome?: Gil Benard.Several reasons for the immunosuppression observed in paracoccidioidomycosis:João Santana Silva.Lunch with professorsHistorical Accounts: CoccidioidomycosisCoccidioidomycosis Discovery in Argentina:Ricardo Negroni.A history of research on coccidioidomycosis in the U.S.:Garry T. Cole.74Coccidioidomycosis: Recent records in Latin America:Bodo Wanke.77Symposium 5. Granuloma and fibrosis:Tissue markers of paracoccidioidomycosis79B-1 cells modulate the kinetics of granuloma formation induced by P. brasiliensis:José Daniel Lopes.8082Extracellular matrix modulation in paracoccidioidal granuloma:Eva Burger.Pulmonary fibrosis in an experimental model of paracoccidioidomycosis: Modulationthrough therapeutic intervention: Luz E. Cano.84Immune reactive cells in skin lesions of patients with paracoccidioidomycosis:Carla Pagliari.85 Symposium 6. Fungal cellular biology aspects878789Immune recognition of the Candida cell wall: The taste of fungus:Neil AR Gow.Cell signaling in morphogenesis and virulence of P. brasiliensis:Larissa Fernandes.Paracoccidioides brasiliensis conidia to yeast transition analysis of certain genes involvedin the process: Ana María García.28

91CDC42p controls yeast-cell shape and virulence in P. brasiliensis:Agostinho João Almeida.92Differential analysis of extracellular vesicles and of possible cell wall-associated proteinsin isolates of P. brasiliensis: Rosana Puccia.93Symposium 7. The endemic mycoses under considerationby Public Health Agencies95959596Paracoccidioidomycosis in the panorama of the neglected tropical diseases:Steven K. Ault.The evolving public health importance of mycotic diseases:Tom Chiller.Epidemiology issues in invasive fungal infections: A United States prospective.Mary Brandt.Paracoccidioidomycosis in Brazil – surveillance and control program:María Adelaide Millington.99Symposium 8. Clinical and epidemiological considerationson the endemic mycoses101102103The impact of histoplasmosis in HIV-infected patients:John R. Graybill and Eduardo Arathoon.The coexistence of HIV infection and paracoccidioidomycosis:Roberto Martinez.Treatment compliance in paracoccidioidomycosis:Rinaldo Põncio-Mendes.105107Guidelines for the treatment of paracoccidioidomycosis:Flavio Queiroz-Telles.Lunch with professorsHistorical accounts: Histoplasmosis107 Histoplasmosis: Discovery in Panama: Marion C. de Martin.109Histoplasmosis in the United States:John R. Graybill.111Symposium 9. New approaches to the clinical and laboratorydiagnosis of systemic mycoses113114114The value and the promise of molecular biological tools in the diagnosis of aspergillosisand candidiasis: Beatriz L. Gómez.Molecular diagnostic approaches in paracoccidioidomycosis and histoplasmosis:Rosely Zancopé Oliveira.Histopathology of paracoccidioidomycosis in the postgenomic era - reflections and perspectives:Henrique L. Lenzi.29

116117Clinico-immunological and immunopathological correlations in different clinical forms ofthe paracoccidioidomycosis: Maria Aparecida Shikanai-Yasuda.Clinical diagnosis in paracoccidioidomycosis: Its defferentiation from similar entities:Angela M. Tobón.119 Symposium 10. Advances on antifungal antibiotics121121123124Coccidioidomycosis as seen in a nonendemic area:John E. Bennett.New and experimental antifungal drugs:Gioconda San-Blas.Nanobiotechnology: Advances in antifungal therapy.André Amaral.The epidemiology of Candida and Aspergillus infections: Clinical and molecular aspects:Christopher Kibbler.125 Symposium 11. Agriculture and paracoccidioidomycosis127127129130P. brasiliensis growth and infective propagule production in clayey and sandy soil:Eduardo Bagagli.On the relationship between land use and paracoccidioidomycosis: Coffee, sugar caneand other cultures: Ligia V. Barrozo.Influence of alternating coffee and sugar cane agriculture in the incidence of Paracoccidioidomycosisin Brazil: Flavio Queiroz-Telles.Chemical components derived from coffee and their impact on health:Manuel Elkin Patarroyo.132Impact of regular coffee consumption on alcohol intake and depressive feelings amongstudents: Darcy Roberto Andrade Lima.133 Symposium 12. Evolutive biology of P. brasiliensis135 What can we know about speciation in P. brasiliensis?: Daniel Matute.136 Is P. brasiliensis an unique species? Maria Sueli Felipe.137Climate variability and acute/subacute paracoccidioidomycosis in a hyperendemic areain Brazil: Ligia V. Barrozo.138 Overview of the biogeography of P. brasiliensis: Eduardo Bagagli.30

Poster Index1 Clinical Aspects / Case Reports1-01p 1411-02p 1411-03p 1421-04p 1421-05p 1431-06p 1431-07p 1441-08p 1441-09p 1451-10p 1451-11p 1461-12p 146Clinical epidemiology of paracoccidioidomycosis patients in the region of Botucatu(São Paulo State, Brazil).Lopes DF; Carvalho LR ; Mendes RP.Paracoccidioidomycosis in patients with and without HIV infection. ComparativestudyPaniago AM; Oliveira Al; Pegorare Al; Aguiar ESA; Aguiar JIA; M.r.; Chang RVC; Motta-Castro ARC; Wanke B.Paracoccidioidomycosis in MexicoLópez-Martínez R; Velasco-Castrejón O; Bonifaz A; Cazarín-Barrientos; Saul A; ArenasR; Padilla-DesgarennesMC.Paracoccidioidomycosis in BoliviaVargas F.Epidemiological and clinical features of patients with Paracoccidioides brasiliensisattending at a University Hospital from Huila - Colombia, Between 1999-2007Molano VM; Gualtero S; Duran LF; Gómez CA; Diaz G.Paracoccidioidomycosis: Frequency, distribution, and hospitalization flows due tothe endemic in Brazil (1998-2007).Coutinho ZF; Travassos CMR; Wanke B; Oliveira EXG; Valle ACF; Coimbra CEA.Epidemiological and clinical profile of 137 patients with paracoccidioidomycosis inUberaba, Minas Gerais, Brazil.Neves FF; Gerolin GP; Tavares MG; Castro-Silva MH; Lopes GP; Michelin MA; Silva A;Vergara MI.Paracoccidioidomycosis associated to HIV/AIDS infection in Uberaba, Minas Gerais,Brazil.Gerolin GP; Tavares MG; Castro-silva MH; Lopes GP; Michelin MA; Silva-Vergara MI;Neves FF.Acute severe paracoccidioidomycosis in an urban-living boy with a single exposureepisode: Evidence for a long subclinical evolution before full-blown diseaseBenard G, Barata LCB.First description of a fatal adult respiratory distress syndrome in a severe acuteparacoccidioidomycosis patient.Ravanini JN; Silva LFF; Goular S; Benard G.Sub-acute paracoccidioidomycosis in a 49 year-old woman.Myiamoto D, Kono ASG; Benard G; Yoshida M; Giarolla I; Shikanai-Yasuda MA.Paracoccidioidomycosis presenting as evanescent pleural effusion associated tofocal consolidation: An uncommon presentation.Costa AN; Junior SAD; Takagaki TY; Kairalla RA; Carvalho CRR.31

1-13p 1471-14p 1471-15p 1481-16p 1481-17p 1491-18p 1491-19p 1501-20p 1501-21p 1511-22p 1511-23p 1521-24p 1522-01p 155Nested-PCR, immunoblotting and double immunodiffusion in meningoencephalitisby P. brasiliensis without radiological alterations of the central nervous systemAlmeida RAMB; Marques SA; Mendes RP; Moris DV; Bosco SMG; Macoris SAG; BagagliE; Vicentini-Moreira AP; Kohara VS; Cavalcante RS, Chaves CR; Calvi ESA.Cutaneous sarcoidic lesions and moriform stomatitis lesions in a young patient:Two sides but not the same coin?Fairbanks N; Kono A; Yoshida M; Valente NYS; Shikanai-Yasuda MA; Benard G.Adrenal insufficiency by paracoccidioidomycosis (PCM) - series of casesBredt CSO; Bredt Jr GI; Duarte PAD; Pedott F; Pereira T; De CamposMyiamoto L; RibeiroAA; Soliva E Jr; Volpolini LC.Adrenal insuficiency associated to paracoccidioidomycosis: Report of three autopsycases.Mantilla JC; Serrano A.Scintigraphic evaluation of enteric protein loss in patients with activeparacoccidioidomycosis.Griva BI; Mendes RP.Biliary obstruction due to acute paracoccidioidomycosis: Analisys of two casesKono ASG; Yoshida M; Giarolla I; Jukemura I; Benard G; Shikanai-Yasuda MA.Serious hepatic damage in paracoccidioidomycosis and its treatmentLazo R; Barrezueta J; Lazo J.Acute respiratory failure in AIDS patients: Role of open lung biopsy to the diagnosisof paracoccidioidomycosis (PCM)Bredt CSO; Bredt GI Jr; Campos M; Pavan D; Duarte PAD; Pedott F; Pereira T; De CamposL; Moraes DM; Richetti MA.Paracoccidioidomycosis in a renal transplant recipient: Case report and review ofthe literatureAdriana SG; Galvão MM; Yoshida M; Giarolla I; França FOS; Benard G; Lanhez LE; Shikanai-YasudaMA.Pericardic paracoccidioidomycosis (PCM) associated with pulmonary neoplasia- Case ReportBredt CSO; Bredt GI Jr; Duarte PAD; Luiz AA; Pedott F; Pereira T; De Campos L Myiamoto;Ribeiro AA; Soliva E Jr; Volpolini LC.Disseminated Herpes simplex infection in a patient with acute/subacute paracoccidioidomycosisSouza BS; Duarte LX; Moraes MPT; Coelho KYR; Griva BI; Mendes RP.Lung paracoccidioidomycosis: Analysis of clinical, tomographic and functionalimpairment after adequate treatmentCosta AN; Fernandes CJCS; Suesada M; Salge JM; Kairalla R; Carvalho CRR.2 DiagnosisDiagnosis of paracoccidioidomycosis in patients attended In routine services of auniversity hospitalMoreto TC; Marques MEA; Oliveira MISC; Moris DV; Carvalho LR; Mendes RP.32

2-02p 1552-03p 1562-04p 1562-05p 1573-01p 1613-02p 161Serological diagnosis of paracoccidioidomycosis: Evaluation of negative serumsamples on immunodiffusion test from patients with confirmed diseaseMoreto TC; Vicentini Moreira AP; Passos AN; Kohara VS; Carvalho LR, Mendes RP.Combined use of Paracoccidioides brasiliensis recombinant 27 and 40-kilodaltonantigens in an enzyme-linked immunosorbent assay for immunodiagnosis of paracoccidioidomycosisFernandes VC; Coitinho JB; Fonseca JH; Veloso JMR; Araújo SA; Pedroso EP; Goes AM.Laboratory measurements for therapeutic monitoring of paracoccidioidomycosispatients with different clinical formsCavalcante R; Moris DV; Carvalho LR; Mendes RP.Espirometric evaluation in patients with paracoccidioidomycosis (PCM)Bredt CSO; Bredt GI Jr; Duarte PAD; Pedott F; Pereira T; De Campos L; Suzin F.3 Epidemiology / EcologyDetermination of Paracoccidioides brasiliensis reservarea mapping the migratorystory of 128 patientsMoreto TC; Barrozo LV; Bueno RA; Moris DV; Mendes RP.Space-time cluster of acute/subacute paracoccidioidomycosis bearing relationshipwith the 1982-83 El Niño episode in a hyperendemic area in BrazilBarrozo LV, Mendes RP; Marques SA; Benard G; Silva MES; Bagagli E.3-03p 1623-04p 162Spatial distribution of chronic paracoccidioidomycosis in a hyperendemic area inBrazilBarrozo LV, Gonzalez CR; Santana MS; Mendes RP; Marques SA; Bagagli E.Liquid urease test in human and animal isolates of Paracoccidioides brasiliensisMacoris SAG, Bosco SMG; Theodoro RC; Richini-Pereira V, Bagagli E.4 Genomic / Proteomic / Cellular Biology / Molecular Biology4-01p 165Toward a molecular genetic system for the pathogenic fungus ParacoccidioidesbrasiliensisAlmeida AJ; Carmona JA; Cunha C; Carvalho A; Rappleye CA; Goldman WE; HooykaasPJ; Leão C; Ludovico P; Rodrigues F.4-02p 1654-03p 1664-04p 1664-05p 167RNA interference: A potential tool to study gene function in Paracoccidioides brasiliensis- strategies to construct silencing cassettesFernandes L; Paes HC; Teixeira MM; Rodrigues ACJ; Viana DF; Torres FA; Felipe MSS.Development of vector-based RNA interference (RNAi) for ade2 silencing on thepathogenic fungus Paracoccidioides brasiliensisPolez VPP; Fernandes L; Torres FAG; Felipe MSS.Protocol for establishment of Paracoccidioides brasiliensis RNA recovery from humanpneumocytes cell cultureOliveira A; Silva SS; Souza CR; Felipe MS.Evidence for positive selection in putative virulence factors within the Paracoccidioidesbrasiliensis species complexMatute DR; Quesada-Ocampo LM; Rauscher JT; McEwen JG.33

4-06p 1674-07p 1684-08p 1684-09p 1694-10p 1694-11p 1704-12p 1704-13p 1714-14p 1714-15p 1724-16p 1724-17p 1734-18p 1734-19p 174Phylogenetic analysis of PRP8 intein and mycological features in Paracoccidioidesbrasiliensis species complexTheodoro RC, Bagagli E; Trinca LA.A high degree of polymorphism and recombination of the GP43 gene among phylogeneticspecies of Paracoccidioides genusTeixeira MM; Felipe MSS.Virtual screening of Paracoccidioides brasiliensis genome – a new drug designapproachAbadio AKR, Carvalho MJA, Martins NF, Felipe MSS.Formamidase of Paracoccidioides brasiliensis: Cytolocalization, purification andanalysis of the native proteinBorges CI; Barbosa MS; Parente JA; Feitosa LS; Santana JM; Báo SN; Sousa MV; RichartCAO; Soares CMA.Two subunits of β-1,3-glucan synthase complex: Gene expression (RHO1 and FKS1)and functionality (RHO1) in Paracoccidioides brasiliensisSorais F; Niño-Vega G; San-Blas G.β-1,3-glucan synthase: Recombinant protein, cytolocalization, activity and studiesunder stress conditionTomazett PK; Félix CR; Barbosa MS; Santana JM; Faria FP; Báo SN; Soares CMA;Pereira M.The α-amylase (AMY1) gene in Paracoccidioides brasiliensis, identification andsemiquantitative expressionCamacho E; Niño-vega G; San-blas G.Mating type genes MAT1-1 and MAT1-2 and sexual reproduction in ParacoccidioidesbrasiliensisTorres I; García AM; Mcewen JG; Restrepo A; Arango M.Molecular evidences of sexual reproduction in P. brasiliensisTeixeira MM; Paes HC; Fernandes L; Silva SS; Felipe MSS.α-1,3-glucanase is important in the remodelling of Paracoccidioides brasiliensiscell wallVillalobos H; Niño-vega G; San-blas G.Characterization of Paracoccidioides brasiliensis Chs3 by overexpression in SaccharomycescerevisiaeBarreto L; Sorais F; Niño-Vega G; San-blas G.Autophagy in the human pathogenic fungus Paracoccidioides brasiliensis duringdimorphismPedroso SM; Campos CIB; Nóbrega FG, Morais FV.Niche specific regulation of Paracoccidioides brasiliensis genes in the infectiousprocessBailão AM; Borges CI; Chagas RF; Salem-Izaac SM; Pereira M; Soares CMA.Differential gene expression in virulent and avirulent isolates of ParacoccidioidesbrasiliensisArruda C; Alves BCA, Calich VIG; Moreira-Filh CA34

4-20p 1744-21p 1754-22p 1754-23p 1764-24p 1764-25p 1774-26p 1774-27p 1784-28p 1784-29p 1794-30p 1794-31p 1804-32p 1804-33p 181Gp43 isoforms of Paracoccidioides brasiliensis as ligands of laminin, fibronectinand collagen type ISilva JF; Benard G; Mendes-Giannini MJS.Transcription regulation of the PbGP43 gene with nitrogen in the human pathogenParacoccidioides brasiliensisRocha AA, Puccia R.Identification of Paracoccidioides brasiliensis adhesins through representationaldifference analysisNogueira SV; Bailão AM; Borges CI; Pereira M; Mendes-Giannini MJ; Winters M; Soares CMA.Adhesion profiles and extracellular matrix ligands of Paracoccidioides brasiliensisisolates obtained from armadillos (Dasypus novemcinctus)Silva RP; Bagagli E; Mendes-Giannini MJS.Use of in vivo-induced antigen technology in the identification of Paracoccidioidesbrasiliensis proteins potentially expressed during infectionRezende TCV; Dantas SFIM; Bailão AM; Castro NS; Tomazett MV; Deepe GS Jr.; PereiraM; Soares CMA.Changes in the transcription levels of Paracoccidioides brasiliensis CHS5 and CHS4genes, mycelial phase, respond to external osmolarityNiño-vega G; Sorais F; San-blas G.A Paracoccidioides brasiliensis aconitase is induced by ethanol and acetate andrepressed by glucose as carbon sourcesBrito WA, Castro NS; Pereira M; Soares CMA.Comparative analysis of gene expression during macrophages infection by pathogenicfungi (Paracoccidioides brasiliensis and Histoplasma capsulatum)Silva SS; Felipe MSS.A serine protease S08 of Paracoccidioides brasiliensis is induced during infectionof machophages and nitrogen starvationParente JA; Bailão AM; Philippsen HK; Santana JM; Pereira M; Soares CMA.The high affinity copper transporter of Paracoccidioides brasiliensis is up-regulatedduring macrophage infectionSantos RS; Bailão AM; Borges CI; Salem-Izacc SM; Dantas SFIM; Deepe GS Jr.; Soares CMA.Alternative oxidase is transcriptionally stimulated at 36°C and is required for myceliumto yeast transition and viability of Paracoccidioides brasiliensisJunqueira NM; Souza GND; Alves HY; Santos MP; Di Benedette JPT; Campos CBL.Transcriptional profile of Paracoccidioides brasiliens is induced by oenothein B,and antifungal agent from Brazilian savannah plant Eugenia unifloraZambuzzi PF; Rezende RV; Borges CI; Ferri PH; Santos SC; Soares CMA; Pereira M.Transcriptional profile of murine macrophages infected with pathogenic fungusHistoplasma capsulatumSilva SS; Passos-Silva DG; Teixeira SMR; Junta C; Medeiros AI; Silva CI; Faccioli FH;Passos GAS; Felipe MSS.Analysis and identification of three P. brasiliensis genes possibly implicated invirulenceHernandez O; Garcia AM; Restrepo A; McEwen JG.35

4-34p 1814-35p 1824-36p 1824-37p 1835-01p 1875-02p 1875-03p 1885-04p 1885-05p 1895-06p 1895-07p 1905-08p 1905-09p 191Internalization of magnetic nanoparticles by Paracoccidioides brasiliensis yeastcellsAmaral AC; Braun S; Nunes ES; Bocca AI; Lima ECD; Báo SN; Azevedo RN; De MoraisPC; Felipe MSS.Molecular typing and evolution of the Paracoccidioides genusTeixeira MM; Paes HC; Fernandes L; San-blas G; Felipe MSS.Adhesion and expression of ligands of extracellular matrix (ECM) by different Paracoccidioidesbrasiliensis isolatesSilva JF, Benard G, Mendes-Giannini MJS.The heath shock factor of Paracoccidioides brasiliensis and its complementationof Saccharomyces cerevisiaePaes HC, Matos LF, Teixeira MM, Torres FAG,Felipe MSS.5 Immunology: Patients / Experimental AnimalsPhenotypic and functional characterization of human dendritic cells induced by lowand high-virulence strains of Paracoccidioides brasiliensisFornazim MC; Mamoni RI; Spago MC; Oliveira RTD; Gabetta CS; Nowill AE; BlottaMHSI.Paracoccidioidomycosis patients dendritic cells: Effect of Paracoccidioides brasiliensisantigens on surface molecules expressionSato PK; Oshiro TM; Diogo CI; Passos EC; Sadahiro A; Shikanai-Yasuda MA.Altered expression of STATs 1,3,4,5 and their modulation by cytokines in mononuclearcells from paracoccidioidomycosis patientsRomano CC; Mendes-Giannini MJS; Duarte AJS; Benard G.Increased T cell regulatory activity in patients with paracoccidioidomycosisFerreira MC; Blotta MHSI; Mamoni RI.Polymorphisms analysis of CTLA-4 gene in paracoccidioidomycosis patientsLozano VF; Paes HC; Teixeira MM; Lins TCI; Vieira RG; Blotta MHSI; Goes AM; PereiraRW; Bocca AI; Felipe MSS.Lymphoproliferation, levels of antibodies and cytokines to AB2-β-anti-idiotipic antibodiesin patients with paracoccidioidomycosis (PCM)Furuchó CR; Oliveira CCU; Bertossi DRT; Fonseca CA; Shikanai-Yasuda MA.Paracoccidioidomycosis: Study of cytotoxic immune response in cutaneous andmucosal lesionsC. Pagliari; N.v. Pereira; M. I. S. Duarte; And M.n. Sotto.Pulmonary abnormalities induced by Paracoccidioides brasiliensis in a mousemodel: Application of high resolution computed tomography to evaluation and follow-upof lesionsLopera DE; Naranjo TW; Duque DJ; Diaz-Granados LR; Jiménez RA; Restrepo A, ZuluagaAF; Cano LE; Patiño J; Hidalgo JM.Alterations in pulmonary mechanisms during experimental infection by ParacoccidioidesbrasiliensisFracon JF, Bocca AL; Siqueira IM; Zin WA; Faffe DS; Jerônimo MS; Soares MAS; RussoRC; Figueiredo F.36

5-10p 1915-11p 1925-12p 1925-13p 1935-14p 1935-15p 1945-16p 1945-17p 1955-18p 1955-19p 1965-20p 1965-21p 1975-22p 197TLR-2, TLR-4 and Dectin-1 expression in human macrophages and neutrophilsstimulated by Paracoccidioides brasiliensisBonfim CV; Mamoni RI; Blotta MHSI.Absence of TLR-2 results in less severe paracoccidioidomycosis but increasedinflammatory response caused by PMN and TH17 cellsLoures FV; Calich VIG.MYD88 is a dispensable factor in resistance to Paracoccidioides brasiliensis in amurine model of blood-borne disseminated infectionGonzález A; Yañez A; Gozalbo D; Gil ML.The influence of β-2 integrin in the fungal burden in Paracoccidioides brasiliensisinfected miceNunes J; Catão E; Fraga CIF; Amaral A; Figueiredo F; Felipe MSS; Faccioli LH; Bocca AL.Surface antigens increase susceptibility to Paracoccidioides brasiliensis infectionvia interleukin-4 productionOliveira LL; Cavassani KA; Rocha FA; Vancim JO; Moreira AP; Campanelli AP; MilaneziCM; Martinez R; Rossi M; Oliveira EB; Silva JS.FBP plays a role in the imunosuppression on the course of Paracoccidioides brasiliensisinfectionMariano VS; Oliveira LI; Coltri KC; Vendruscolo PE; Ferraz DB; Roque-barreira MC;Panunto-Castelo A.Paracoccidioides brasiliensis inhibits human neutrophils apoptosisAcorci MJ, Dias-Melicio LA; Golim MA; Bordon-Graciani AP, Peraçoli MTS; SoaresAMVC.Prostaglandin inhibits Paracoccidioides brasiliensis killing by human monocytes:Reversal by activation with IFN-γ, TNF-α and GM-CSF due to increased H 2O 2productionBordon-Graciani AP; Dias-Melicio LA; Acorci MJ; Peraçoli MTS; Soares AMVC.Production of leukotriene B4 by different strains of Paracoccidioides brasiliensisDias-Melicio LA, Biondo GA; Bordon-Graciani AP; MJ Acorci; Peraçoli MTS, SoaresAMVC.Comparative analysis of the infection evolution between isolates Pb 18 and Pb 01of the Paracoccidioides brasiliensisFraga CIF; Siqueira IM; Ribeiro AM; Nunes J; Figueiredo F; Felipe MSS; Bocca AL.Paracoccidioides brasiliensis infection determines dendritic cells to differentiate tothe plasmocytoid subpopulation which induces a more severe pulmonary infectionwhen transfered to resistant micePina A; Calich VIG.IL-10 deficiency determines a better fungicidal ability associated with overproductionof IFN-γ and nitric oxide by Paracoccidioides brasiliensis infected macrophagesCosta TA; Calich VIG.CD28 costimulatory molecule deficiency results in more severe Paracoccidioidesbrasiliensis infection but protects mice from life-threatening immunopathologyFelonato M; Calich VIG.37

5-23p 1985-24p 1985-25p 1995-26p 1995-27p 2005-28p 2005-29p 2015-30p 2016-01p 2056-02p 2056-03p 2066-04p 2066-05p 207Nitric oxide but not TREG cells plays a major immunoregulatory role in a pulmonarymodel of fungal infectionBernardino S; Calich VIG.Role of CCR4 in the experimental infection by Paracoccidioides brasiliensis: Migrationcontrol of CD4+CD25+ T cells to lesion siteRocha FA; Oliveira LI; Massafera-Tristão FS; Milanezi CM; Silva JS.Granulomatous inflammation and fibrosis in mice with chronic pulmonary paracoccidioidomycosis:Immune-related associationsNaranjo TW, Lopera DE; Duque JJ; Diaz-Granados LR; Restrepo A; Cano LE.Alterations of granuloma architecture, cellularity and severity of the early infectionin murine paracoccidioidomycosis by IFN-γ, celecoxib or lumiracoxib treatmentMolina RFS; Nishikaku AS; Albe BP; Burger E.Therapeutic activity of a DNA vaccine using the gene Hsp65 from Mycobacteriumleprae for experimental paracoccidioidomycosisRibeiro AM, Souza ACO; Amaral AC; Fraga CIF; Nunes J; Salles BC; Coelho-CasteloAAM; Figueiredo F; Silva CI; Felipe MSS; Bocca AI.Evaluation of the protection induced by the immunization with radioattenuated yeastcells of Paracoccidioides brasiliensis in animal modelMartins EMN, Bernardo S; Reis AMG; Antero SRA.Modulation of experimental paracoccidioidomycosis by monoclonal antibodiesagainst the major diagnostic antigen, Gp43Pinto FA; Puccia R; Da Silva CJ; Muñoz JE; Travassos LR; Taborda CP.The influence of Fonsecaea pedrosoi cell wall in peritoneal macrophage activationNóbrega YKM; Lozano VF; Figueiredo F; Bocca AL.6 TreatmentTiming of itraconazole therapy onset and its impact on the progression of fibrosisinduced by Paracoccidioides brasiliensis conidia: Results in an experimental modelof paracoccidioidomycosisLopera DE; Naranjo TW; Duque DJ; Diaz-Granados LR; Jiménez RA; Restrepo A, ZuluagaAF; Cano LE.Posaconazole for the treatment of paracoccidioidomycosis: First clinical resultsTobón AM; Agudelo CA; Restrepo A.Variable effect of caspofungin on the mycelial and yeastlike phases of several strainsof Paracoccidioides brasiliensisRodríguez-Brito S; Niño-vega G; San-Blas G.Assessment of the efficacy of voriconazole, comparativily to other antifungals, inparacoccidioidomycosis experimental of the ratsGranzoto DS; Vitali LH; Martinez R.Amphotericin B-PLGA-DMSA nanoparticle: A new alternative to treat paracoccidioidomycosisAmaral AC; Bocca AI; Ribeiro AM; Nunes J; Falcomer CI; Peixoto DIG; Simioni AR; Pinto FI;Lacava ZGM; Azevedo RB; Titze-De-Almeida R; Tedesco AC; De Morais PC; Felipe MSS.38

6-06p 2076-07p 2086-08p 2086-09p 2096-10p 2096-11p 2106-12p 2106-13p 211Fungicidal activity of amphotericin B-magnetic fluid conjugate in vitro against ParacoccidioidesbrasiliensisMedeiros PB; Amaral AC, Saldanha CA; Bocca AI; Guilherme RB; Silva JR; Nunes ES;Lima ECD; Azevedo RB; De Morais PC; Felipe MSS.PLGA-P10 conjugate improved 20-times the immunological protection of this peptideagainst Paracoccidioides brasiliensisAmaral AC, Marques AF; Muñoz JE; Bocca AI; Simioni AR; Titze-De-Almeida R; TedescoAC; De Morais PC; Taborda CP; Travassos LR; Felipe MSS.Biological activity of the endophytic fungi UFMGCB551 against ParacoccidioidesbrasiliensisCampos FF; Rosa LH; Johann S; Cota BB; Rosa CA; Sá NP, Cisalpino PS; Zani CI.Antagonistic effect of farnesol, a Candida albicans quorum sensing molecule, onParacoccidioides brasiliensis growth and morphogenesisDerengowski LS; Braz SV; De-Souza-Silva C; Mello-De-Sousa TM; Báo SN; Kyaw CM,Silva-Pereira I..Killing of Paracoccidioides brasiliensis yeast cells by magnetic hyperthermiaAmaral AC; Braun S; Nunes ES; Bocca AI; Lima ECD; Azevedo RB; De Morais PC; FelipeMSS.Antifungal activity of plants extracts used in Brazilian traditional medicine againstParacoccidioides brasiliensisJohann S; Watanabe GA; Cota BB; Siqueira EP; Zani CI; Pizzolatti MG; Cisalpino PS;Resende MA.Treatment of murine paracoccidioidomycosis (PCM) with trimethoprim-sulfamethoxazolecombination (TMP-SMX)Moura F; Lima CRG; Moreto TC; Carvalho LR; Bueno RA; Moris DV; Mendes RP.Therapeutic DNA vaccine in BALB/c and B10A mice against experimental paracoccidioidomycosisGomes-Rittner GM; Muñoz JE; Taborda CP; Travassos LR.6-14p 2116-15p 2127-01p 2157-02p 215Adrenal function in paracoccidioidomycosis patients treated with azolic derivatives:Results after prolonged post-therapy follow upTobón AM; Restrepo CA; Villa CA; Agudelo CA; Quiceno W; Restrepo A..The association tuberculosis-paracoccidioidomycosis: Impact on the outcome oftheraphyTobón AM; Agudelo CA; Tabares AM; Restrepo A.7 Other mycosesSusceptibility to fluconazole and voriconazole of Candida species isolated from intensivecare units patients in several hospitals in Medellín, Colombia (2001 - 2007)Zuluaga A; De Bedout C; Agudelo CA; Hurtado H; Arango M; Restrepo A; González A.Therapeutic study of gomesin in mice infected with Candida albicansRossi DCP; Muñoz JE; Taborda CP ; Daffre S.39

7-03p 2167-04p 2167-05p 2177-06p 2177-07p 218Study of genotypic and phenotypic characteristics among Cryptococcus gattii serotypeB - VGII molecular type clinical isolates from Cúcuta, Colombia and isolatesof the outbreak in Vancouver, Canada. G.Torres; and P. Escandon.Microecology of Blastomyces dermatitidis: The ammonia hypothesisDJ BaumgardnerEvaluation of an antigen-capture ELISA to detect Histoplasma capsulatum antigenuriain immunocompromised patientsScheel CM; Samayoa B; Benjamin L; Riley P; Lindsley M; Herrera A; Raxcacoj G; LimaS; Miramontes R; Chiller TM; Brandt M; Arathoon E; Gómez BL.An analysis of histoplasmosis in an endemic, resource poor area with a high HIVprevalence rate — Guatemala, 2007Miramontes R; Samayoa B; Herrera A; Scheel C; Gómez BI; Arathoon E; Chiller T.Detection of DNA in peripheral blood from a patient with the ocular histoplasmosissyndrome: A case reportHernández JM; Muñoz C; Hernández D; Montoya C; González A.40

Symposium1P. brasiliensis:Genomics and Bioinformatics

Comparative analysis of Paracoccidioides brasiliensisand other dimorphic fungiMia ChampionBroad Institute of MIT 2 Harvarde-mail: champion@broad.mit.eduParacoccidioides brasiliensis is the causal agent of paracoccidioidomycosis (PCM), one of the mostimportant human systemic mycosis in Latin America. In collaboration with the Dimorphic FungalGenomes Consortium, we have sequenced three strains of Paracoccidioides brasiliensis that arerepresentatives of distinct clades in the species tree: Pb01, Pb03, and Pb18. The genome assembliesof these strains contain 33Mb, 29Mb, and 30Mb respectively, and Pb03 contains 9,063 predictedgenes. The Pb18 genome assembly has been anchored to an optical map consisting of 5 linkagegroups, providing a chromosomal context for this assembly. Analysis of syntenic regions as well asregions of unique genomic content between the Paracoccidiodes strains, including the abundanceand distribution of repeat content, will be presented. Comparison to other dimorphic fungi as wellas more distant fungal genomes will enable description of gene families specific to dimorphic fungi.These analyses will provide novel insights into the diversity between three Paracoccidioides strainsand contribute to our broader understanding of the larger group of dimorphic fungal pathogens.Comparative genomics of Coccidioides speciesThomas J. Sharpton 1 , Jason Stajich 1 , Garry T. Cole 2 , and John W. Taylor 11 University of California, Plant and Microbial Biology, Berkeley, CA 94720-3102 -USA2 University of Texas, Biology, San Antonio, TX 78249 - USAe-mail: jtaylor@berkeley.eduCoccidioides species are ascomycetous fungi found in hot, dry areas of the New World. They associatewith endemic small mammals and have been the focus of much research because, likeParacoccidioides brasiliensis, they can cause life-threatening disease in otherwise healthy humans.We are comparing genomes of Coccidioides species and their relatives to focus the tools of evolutionarybiology on the problem of fungal adaptation to an animal host. Variation in gene sequencesand microsatellite repeats has been used to recognize two species of Coccidioides, C. immitis andC. posadasii, and at least five populations therein. Through the efforts of the Coccidioides community,the Broad Institute and TIGR, genomes have been released for each Coccidioides speciesand their close relative, Uncinocarpus reesii. This collection of genomes provides a rich resourcefor comparative genomics, conditional upon the quality of annotation. With annotated genomes forCoccidioides and Uncinocarpus, we have searched for genes specific to Coccidioides or the outgroup,Uncinocarpus, for genes showing unusual rates of evolution, and for genes showing the effects ofstrong selection. Recent effort by the Broad institute has produced sequence for 13 more Coccidioidesgenomes, making it possible to challenge hypotheses about selection based on comparisonof pair-wise genome comparison through the use of population based methods for detecting selection.We will discuss how all of the evolutionary results relate to adaptation and how these broaderhypotheses may, in turn, be falsified.43

Post-genomic contribution to the knowledge on thehost-pathogen interactionMaria Sueli Felipe, Aldo Henrique Tavares and Simoneide Souza SilvaInstituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil.e-mail : msueli@unb.br or msueliunb@gmail.comThe genome constitutes the informational core of all biological processes and the study of living organismsdepends heavily on our ability to access its contents. For many decades, the experimentalapproach consisted of isolating single genes or regulatory elements and characterizing each one ata time by means of loss-of- function and/or gain-of function experiments; or by identifying effectors(proteins, cofactors or metabolites) and studying their roles and interactions. In keeping with theinformational flow in the cell, genome has been closely followed by transcriptome and proteome.The study of pathogens and their interaction with hosts are of special interest. Computer-aided datamining has enabled unambiguous identification of open reading frames, and transcriptional profilinghas yielded relevant information concerning differential gene expression. Microarray technology, inconjunction with statistical and experimental validation, has been used to identify patterns of geneexpression—selected according to previous genome or transcriptome information—when the microbeis confronted with conditions of interest, such as ex vivo interaction with specific cells or exposureto therapeutic agents, signaling molecules or stressors. The fungus P. brasiliensis is one organismthat has benefited from these approaches. The yeast form of P.brasiliensis acts as a facultativeintracellular pathogen being able to survive and replicate within the phagosome of nonactivatedmurine and human macrophages. This ability has been proposed to be crucial to the developmentof disease. In this talk, I will outline the current post-genomic contribution to the knowledge on thehost-pathogen interaction, with focus on differential expression analysis of P. brasiliensis-macrophagecells. Recently, by using cDNA microarray technology we evaluated the early transcriptionalresponse of this fungus to the environment of peritoneal murine macrophages in order to shed lighton the mechanisms used by P. brasiliensis to survive within phagocytic cells. Of the 1152 genesanalyzed, we identified 152 genes that were differentially transcribed. Overall, our data providedadvances on the comprehension of the plasticity of response and indicate that P. brasiliensis is ableto undergo fast and dramatic expression profile microenvironment changes, as will be discussedbelow. In order to better understand the complex interaction between host cells and P. brasiliensiswe have analyzed also by cDNA microarray the transcriptional profile of 624 previously selected murineimmuno-regulatory genes following fungal infection of macrophages for 6, 24 and 48 hours. Ourmicroarray data have identified many genes that are modulated in particular those related to inflammatorymechanisms, membrane proteins, transcription regulation and apoptosis. Inflammation is apowerful protective mechanism coordinated and controlled by cytokines and chemokines. Increasedchemokine gene expression has been detected by microarray analysis in macrophages infected with,Candida albicans, Aspergillus fumigatus and Mycobacterium tuberculosis. Accordingly, our resultsshow that genes encoding the chemokines Ccl21, Ccl22 and Cxcl1, all of which are associated withpro-inflammatory response, were significantly increased in peritoneal macrophages in response toP. brasiliensis-Pb01, at the early stage of infection. Macrophage genes encoding membrane proteinsinvolved in adhesion and phagocytosis were also induced by P. brasiliensis. In this context, we haveobserved significant induction of Cd14 and Clec1b. This differential expression pattern is probablymodulated by the pathogen and its ultimate result is an improved effectiveness of invasion and/ormacrophage activation. In addition, the gene for the C-type lectin-like receptor 1b (Clec1b), which isalso membrane-related, was induced at all times of macrophage infection with P. brasiliensis, which44

has been confirmed by real-time RT-PCR. Matrix metalloproteases (Mmps) are known to be involvedin tissue remodelling, cleavage of cell-surface receptors and chemokine activation. Considering theirimportant role in collagen and cellular matrix remodelling, it has been suggested that induction of thesemetalloproteases might be an important mechanism to facilitate fungal invasion. The up-regulation ofMmp17 and Adam8 was observed in macrophages infected by P. brasiliensis and probably increasethe effectiveness of host cell parasitism. These data fit with the above described induction of adhesionand phagocytosis-related genes, since P. brasiliensis is a facultative parasite that can surviveand replicate in non-activated macrophages. Increased phagocytosis (Cd14 and Clec1b) and thestimulation of matrix remodeling (Mmp17 and Adam8) are expected to contribute to a more efficientinvasion by the fungus of cell and tissue alike. Apoptosis plays a significant role in the regulation ofpathogenesis. In our experimental approach, several genes have been found to be responsive andup-regulated by P. brasiliensis, including caspases 2, 8 and 3; this phenomenon has been correlatedwith a phenotypic marker of infection resistance, probably as an efficient manner to eliminatethe fungus without tissue damage. Recently, our group used cDNA microarray to evaluate the earlytranscriptional response of P. brasiliensis to the inner environment of peritoneal murine macrophagesat six hours of infection. Thus, considering the analyses of differential gene expression for host andpathogen, at six hours, we propose a model for the P. brasiliensis-macrophage interaction that discussesthe events at molecular level during early infection. In response to the harsh macrophagemicroenvironment P. brasiliensis up-regulates genes (sod3 and hsp60) related to the detoxificationof oxidative radicals and amino acid biosynthesis (metG). In addition, genes encoding five enzymesof the glycolytic pathway have been found to be down-regulated, suggesting a glucose-poor environment.Macrophages at the same time point up-regulate genes related to inflammation (chemokinesand cytokines-Ccl21, Cxcl1 and Ccl22) and phagocytosis-related (Clec1b) genes, probably as aneffort to counteract infection by the fungus. In summary, our results demonstrate that P. brasiliensisis a potent inducer of molecules involved in the initial host response to this organism. The kineticapproach used in microarray study has elucidated a coordinate and temporal basis of host defensemolecules elicited against P. brasiliensis infections. These expression data described should providea foundation for further studying the pathogenesis of this infectious agent and a better knowledgeon the host-pathogen interaction.Financial support – CNPq, FAP-DF, FUB.Genes involved in morphogenesis and cell wall synthesisand regulation in P. brasiliensisGustavo Niño-VegaInstituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuelae-mail: gnino@ivic.veCurrently, our group is working on the analysis of genes involved either in the synthesis (α-1,3-glucansynthase, chitin synthases), hydrolysis (α-1,3-glucanase) or regulation (Rho genes) of cell wallα-1,3-glucan and β-1,3-glucan in Paracoccidioides brasiliensis, and also on genes participating inmorphological events by other mechanisms (septins, actin).The α-glucan synthase gene of P. brasiliensis (PbrAGS) has been cloned in our laboratory (Gen-Bank accession number AY780573). Analysis of its sequence suggests a resemblance to previouslyreported fungal AGS (Proc Natl Acad Sci USA 95: 9161–9166, 1998; Fungal Genet Biol 42: 165-177,2005) As with other α-glucan synthases, three distinct domains can be identified in the PbrAgs1 se-45

quence. The first domain (aa 1-1073) –located extracellularly, according to computer models- showssignificant sequence similarities to α-amylases belonging to family 13 of the glycosyl hydrolases(Biochem J 280: 309–316, 1991) and is denoted as an amylase homologous domain (AHD). In otherfungi, it has been suggested that this domain is involved in remodelling newly formed α-glucan orcrosslinking to the existing cell wall (Proc Natl Acad Sci USA 95: 9161–9166, 1998). The seconddomain (aa1097–1990) is flanked by two hydrophobic sequences (aa1074–1096 and aa1991–2008).It is predicted to be intracellular and contains a glycogen or starch synthases homologous domain(G/SSHD). Therefore, it is likely that it encodes the glucan synthase domain of PbrAgs1. The thirddomain (aa1097–2431) is predicted to span the membrane 8 times. This multi-pass transmembraneregion (MTM) is thought to be involved in the transport of the α-glucan chain across the plasmamembrane (Proc Natl Acad Sci USA 95: 9161–9166, 1998). In accordance with the exclusive presenceof α-1,3-glucan in the yeast-like phase of the fungus, northern analyses show that the PbrAGS1gene is only expressed in the yeast-like phase of P. brasiliensis .Recently, Marion et al. (Mol Microbiol 62: 970-983, 2006) reported that an α-(1,4)-amylase isessential for α-(1,3)-glucan production and virulence in Histoplasma capsulatum. Interruption ofthe AMY1 gene responsible for the α-(1,4)-amylase by insertional mutagenesis, resulted in loss ofα-(1,3)-glucan and loss of virulence (Mol Microbiol 62: 970-983, 2006), a phenotype similar to disruptionof domain 1 of the H. capsulatum AGS1 gene. These results have prompted us to searchfor a similar gene in P. brasiliensis.Also, a P. brasiliensis α-1,3-glucanase-like gene sequence has recently been cloned in our laboratory.It has high identity to fungal α-1,3-glucanases (also called mutanases due to their hydrolyzingactivity towards “mutan”, the so called α-1,3-glucan from Aspergillus nidulans and other fungi. Preliminaryanalysis of the gene sequence and comparison with other fungal α-1,3-glucanases suggestthat we have so far cloned and sequenced about two thirds of the gene.So far, information on genes necessary for the establishment and morphology of the pathogenicphase in P. brasiliensis is not certain, although valuable and increasing information on genes preferentiallyexpressed on this phase is been given by the groups working on the construction of expressedsequence tags (ESTs) libraries on this fungus (Yeast 20:263-271, 2003; Eukariotic Cell 2:34-48,2003; Mol. Gen. Genomics 271:667-677, 2005; J. Biol. Chem. 280:24706-24714, 2005). Here wedescribe the cloning and complete sequence of CHS5, a gene encoding a class V chitin synthase,from the pathogenic fungus P. brasiliensis, and its arrangement within the genome in a head-to-headconfiguration with CHS4, both genes sharing a common 5’UTR. Also, changes in transcript levelsfor both genes, following alteration of external osmolarity, suggest a common regulatory mechanismof transcription. The exploration by northern analises of other genes related to synthesis of cell wallcomponents (CHS3 and FKS1) reveals higher expression in the yeast phase of the fungus: contraryto other CHS genes, CHS3 is only expressed in the pathogenic yeast phase and at the end of themycelium-to-yeast transition in P. brasiliensis, while we found, to our surprise, that FKS1 (the onlyβ-1,3-glucan synthase gene so far reported for P. brasiliensis (Yeast 16:451-462, 2000) has a alsohigher expression in the yeast phase, which, made us wonder if β-1,3-glucan, even when representsa small percentage of the yeast cell wall components, would be essential for its maintenance.46

Symposium2Understanding the immune Responsesagainst dimorphic Fungi in experimentalanimal Hosts

Experimental animal models and their contribution to the studyof fungal pathogenesisKarl V. Clemons, Ph.D.California Institute for Medical Research, San Jose, CA; Division of Infectious Diseases,Santa Clara Valley Medical Center, San Jose, CA; andDepartment of Medicine, Division of Infectious Diseases and Geographic Medicine,Stanford University, Stanford, CA USA.e-mail: clemons@cimr.orgWhat makes a fungus cause disease in humans, and how that disease progresses are questionsthat have been under study for decades. Although in vitro studies allow us to examine specificquestions and fungus host interactions, they cannot replicate the conditions found in vivo. Thus,animal models, as surrogates of human disease, have been used historically in studies of fungalpathogenesis. These investigations encompass virulence, as well as host response to obtain theoverall picture of the evolution of a fungal disease. Critical to the study of pathogenesis are thechoice of animal, route of infection, parameters for evaluation and how closely a model mimics thatdisease in humans. Although numerous laboratory animal species have been used, mice are themost commonly used species currently, and have numerous advantages with respect to affordability,defined genetics, immunological reagents, and availability of gene-knockouts, deficient in a singlecytokine or gene-product. Our ability to modulate host response, severity of infection and geneticallymanipulate the host or the infecting organism allow for the study of specific questions. However,no single model, or even strain of animal, will be useful to answer all questions. For example, ourlaboratory has demonstrated that mucosal candidiasis in SCID mice mimics mucosal infection inhumans with AIDS. The mucosal surfaces of the gastrointestinal tract are heavily colonized, butdisseminated disease does not occur. However, mucosal candidiasis established in normal micepretreated with 5-fluorouracil results in dissemination, which is similar to that seen clinically in patientsreceiving cancer chemotherapy. Furthermore, in studies on the potential of Saccharomycescerevisiae to be a pathogen, we found that virulence potential changed depending on the strain ofmouse used in the model. These data raised the possibility of different strategies of virulence mightexist among various strains of the same species of fungus. Pathogenesis is greatly influenced bythe host response. The use of gene-knockout mice has aided our study of the importance of varioushost proteins such as collectins and cytokines. For example, we examined the role played by IL-10in systemic aspergillosis, where we found IL-10 deficient mice more resistant to infection, but couldnot demonstrate a role for IL-10 when mice were treated with anti-IL-10 receptor. In other studies,we have shown how gender influences the outcome of the establishment of disease by demonstratingthe estradiol in vivo blocks the initial transition of the conidia of Paracoccidioides brasiliensisinto the tissue yeast-form, and that the infecting inoculum is rapidly cleared from the pulmonary tissuesof female BALB/c mice, whereas the organism multiplies and disease progresses in the malecounterparts. Animal models also allow us to examine host events associated with fungal infection.Meningitis in humans due to Coccidioides is a severe and lethal disease, where patients often havestroke events and associated vasculitis. We developed a rabbit model of coccidioidal meningitisthat also results in the development of vasculitis of the large arterial vessels of the CNS. Thus far,we have found associations of increased MMP-9, IL-6, interferon-γ, and iNOS in vasculitic vesseltissue, all of which may exacerbate the inflammatory process of vasculitis. Overall, animal modelsof fungal infection are valuable tools in addressing questions concerning the infectious process andcontribute to our deeper understanding of occurrence, evolution and how they might be controlledor better, prevented and eliminated.49

Toll like receptors and the adaptor molecule MYD88 play animportant role in pulmonary paracoccidioidomycosisVera Lucia G. Calich and Flávio V. LouresDepartamento de Imunologia, Instituto de Ciências Biomédicas,Universidade de São Paulo, Brazil, CP 66.208e-mail: vlcalich@icb.usp.brRecognition of invading microorganisms by the innate immune system is a first step to control oreliminate an infectious process from the host. The initial interaction between immune cells andmicroorganisms is mediated by several types of receptors which recognize molecular patterns ofpathogens and are collectively called pathogen recognition receptors (PRR). The Toll-like Receptors(TLRs) constitute a molecular family that recognizes a wide range of microbes and their productsknown as pathogen-associated molecular patterns. TLRS are expressed in diverse innate immunecells such PMN, macrophages, dendritic cells and lymphocytes. Their activation triggers a signalingcascade that results in an inflammatory response through production of proinflammatory cytokinesand up-regulation of costimulatory molecules expression leading to initiation of antigenic-specificadaptative immune response. As described for other microorganisms, TLRs were shown to beinvolved in host defense against different fungal pathogens. The contribution of individual TLRs tothe immune response against pathogenic fungi depends on several factors such as the route of infection,the fungal morphotype or fungal species. Alveolar macrophages are the first host cells thatinteract with Paraccocidioides brasiliensis, however, the initial fungal-host cells interaction is poorlyknown. Mannose and complement receptors appear to play a role in innate immunity to P.brasiliensisinfection, but the role of TLR2, TLR4 and MyD88 in paracoccidoidomycosis was never investigated.Recent findings from our laboratory demonstrated that, compared with the normal strain (C3H/HePas), macrophages from TLR4-deficient mice (C3H/HeJ) had a lower phagocytic ability whichappears to influence the decreased number of viable P. brasiliensis yeasts recovered after 72h cocultivation.Deficient macrophages secrete lower levels of nitric oxide (NO), IL-12, and MCP-1 butproduced equivalent amounts of TNF-α. In contrast, IL-10 was synthesized in higher amounts byTLR4-deficient macrophages. Consistent with in vitro results, 96h after in vivo pulmonary infectionTLR4-deficient mice presented decreased fungal loads in the lungs associated with lower levels ofNO and pro-inflammatory cytokines (IL-12, and GM-CSF). Similar results were observed at week11 after infection of TLR4-mutant mice: decreased CFU counts associated with low IL-12 levels buthigh IFN-γ secretion. Paralleling its mild infection, the deficient strain secreted low levels of IgG1,IgG2b and IgM P.brasiliensis specific isotypes. Cytospin preparations of lung infiltrating leukocytesat week 2 of infection showed an increased number of PMN neutrophils associated with decreasednumbers of lymphocytes and monocytes. Although differences in CFU counts and synthesis of someinflammatory mediators occur in TLR4-deficient mice, such differences were not sufficient to altertheir mean survival times.We have also comparatively analyzed P.brasiliensis infection of TLR2-normal (WT) and TLR2-KOmice in a C57BL/6 background. In vitro infection of KO macrophages resulted in lower phagocyticindexes and decreased recovery of viable yeasts after 72 h co-cultivation. Macrophage supernatantspresented low levels of NO and MCP-1. Compared with WT mice, 48h and 2 weeks after infection KOmice presented diminished pulmonary fungal loads and NO, and these findings were associated withsome differences in the levels of pro- and anti-inflammatory cytokines. Lung infiltrating leukocytesat weeks 2 and 4 of infection showed a decreased proportion of macrophages but an increasednumber of PMNs and lymphocytes. Interestingly, TLR2 KO mice secreted higher levels of IL-6,TGF-β, IL-23 and IL-17 indicating an enhanced Th17 immunity. In addition, a decreased number of50

CD4 + CD25 + FoxP3 + T cells were detected in TLR2 KO mice. At week 11 of infection TLR2-deficientand normal mice presented similar humoral immunity but the former strain showed increased fungalburden in the lungs. Despite this difference, equivalent mortality rates were showed by both mousestrains.When MyD88 KO macrophages were in vitro infected with P. brasiliensis yeasts for 72h, increasednumber of viable fungi was recovered in comparison with normal cells. This diminished fungicidalability paralleled a decreased synthesis of NO and IL-12. The early in vivo infection reproduced thein vitro findings: higher fungal loads were found in MyD88 KO mice associated with lower levels ofpulmonary NO and IL-12. This appears to indicate that absence of MyD88 molecule causes profoundeffects in cell activation resulting in more severe infection. Mortality studies confirmed the highersusceptibility of MyD88 KO mice to P. brasiliensis infection, since their mean survival time is significantlylower than that of WT mice.As a whole, TLR deficiency caused less severe infections associated with altered secretion of NO,cytokines and chemokines, resulting in altered cellular influx to the site of infection. In both PRR-deficientstrains, the lung inflammatory infiltrate was composed of a diminished number of macrophagesassociated with an increased presence of PMNs. Possibly the phagocytic and killing abilities of thesecells would contribute to the decreased fungal inoculum at the site of infection. Furthermore, the lowlevel of MCP-1 was parallel to the decreased number of lung infiltrating monocytes. An interestingobservation was the enhanced Th17 immunity associated with severe inflammatory lesions anddecreased number of regulatory T cells (CD4 + CD25 + FoxP3+) in the lungs of TLR2 KO mice.Mortality studies showed that TLR deficiency was not able to change the late course of infectionsince no differences were observed between TLR KO and TLR-normal mice. Compensatorymechanisms appear to abolish the immunological differences caused by PRR-deficiencies. Thesame was not true for MyD88 deficiency which causes higher mortality of infected mice. The moresevere infections of MyD88-deficient macrophages and mice are probably due to the intact fungalrecognition mediated by the expression of normal PRR but impaired ability of cell activation resultingin diminished fungicidal ability.In summary, our studies suggest that MyD88 deficiency is more important than TLR2 or TLR4deficiency and P. brasiliensis yeasts appear to use TLRs as a virulence mechanism facilitatingthe access of fungal cells into murine macrophages. Despite their TLR-mediated activation, macrophagesare not able to control fungal growth, and the final balance between fungal growth andactivation of the immune system appears to control disease outcome. Furthermore, our studies havealso suggested that, besides TLR, other PRR play a role in the host immune response against byP.brasiliensis infection.Financial support provided by FAPESPIn vivo protection against P. brasiliensis infectionwith monoclonal antibodiesCarlos P. Taborda 1 , Rosana Puccia 2 and Luis R. Travassos 21Institute of Biomedical Sciences, Departament of Microbiology,University of São Paulo, São Paulo, Brazil.2Departament of Microbiology, Immunology and Parasitology,Federal University of São Paulo, São Paulo, Brazile-mail: taborda@usp.brThe treatment of paracoccidiodomycosis patients with antifungal drugs is the accepted therapy,even if after prolonged periods of drug administration there is no assurance of complete cure andwith relapses being frequent events. The protective function of antibodies against fungal infections is51

controversial. Recently, several studies have established that some antibodies are protective againstfungi as exemplified by two monoclonal antibodies that have entered clinical trials for cryptococcosisand candidiasis treatment. Protective role for antibodies against Paracoccidioides brasiliensis is stilldoubtful. In the present work the effects of monoclonal antibodies against the major diagnostic antigen(gp43) have been analyzed in vitro and in vivo. Passive administration of some mAbs beforeand after intratracheal or intravenous infections resulted in reduction of fungal burden and decreasedpulmonary inflammation. Protection mediated by mAb 3E, the most efficient mAb in the reductionof fungal burden, was associated with increased phagocytosis of yeast cells by macrophages. Theingestion of opsonized yeast cells led to increased NO production by macrophages and enhancedlevels of IFN-γ in the lung of infected mice. The reactivity of mAb 3E against a panel of gp43 derivedpeptides suggested that the sequence NHVRIPIGWAV contains its binding epitope. The protectionof paracoccidiodomycosis has always been attributed to a robust cellular immune response whereasantibodies have been associated to bad prognosis. Here, we show that specific antibodies to a majorantigen of P. brasiliensis can be protective against a challenge of virulent fungi. A better understandingof the parameters that influence antibody-mediated immunity will increase our knowledge of vaccineefficacy and host susceptibility to infection.P10 immunization using Salmonella enterica’sflagellin as adjuvantLuis R. Travassos 1 , Luis C.S. Ferreira 2 , Catarina J.M. Braga 2 and Carlos P.Taborda 21Departament of Microbiology, Immunology and Parasitology,Federal University of São Paulo, São Paulo, Brazil.2Institute of Biomedical Sciences, Departament of Microbiology,University of São Paulo, São Paulo, Brazile-mail: taborda@usp.brThe major diagnostic antigen of P. brasiliensis (gp43) was cloned and sequenced. A peptidesequence, known as P10 contained a T-cell epitope able to induce an IFN-γ-mediated Th-1response. Association of P10 immunization with chemotherapy was studied in Balb/c mice intratracheallyinfected with P. brasiliensis with very encouraging results. Early studies on the gp43and P10 always used complete Freund’s adjuvant (CFA) which is not acceptable for human use.Alternative ways of delivering P10 and using other adjuvants were investigated. In the presentwork, we studied the adjuvant properties of flagellin FliCd from Salmonella enterica Müenchen.P10 was genetically fusioned in the central region of flagellin FliCd or just mixed together withfree flagellin and nasally administered in Balb/c mice. Our results showed that administrationof P10 mixed with flagellin FliCd promoted a better protection than the p10-fusioned flagellin orthe peptide alone in CFA. The analysis of colony-forming units indicated a reduction of fungalburden in the lung with significant production of IL-12 and IFN-γ in lung homogenates. Theseresults may have an impact in P10 vaccination, with the perspective of using an intranasal vaccineof high immunological potency.52

Pattern recognition receptors in the host immuneresponse to Coccidioides spp.Maria del Pilar Jiménez-A. 1 , 2,3 , Suganya Viriyakosol 2 , Lory Walls 2 , Sandip K. Datta 4 ,Theo Kirkland 4,5 , Sigrid E.M. Heinsbroek 6 , Gordon Brown 7 and Joshua Fierer 2,4 .1 Corporación para Investigaciones Biológicas, CIB, Medellín – Colombia.2 Research Service, VA San Diego Healthcare, San Diego, CA.3 Programa de Doctorado en Ciencias Médicas,Universidad Pontificia Bolivariana (UPB)-CIB-CES. Medellín, Colombia.4 Department of Medicine, Division of Infectious Diseases School of Medicine,University of California San Diego, USA.5 Department of Pathology, UC San Diego School of Medicine, San Diego, CA, USA.6 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.7 Institute of Infectious Disease and Molecular Medicine,University of Cape Town, South Africa.e-mail: pjimenez@cib.org.coCoccidioidomycosis is a systemic mycosis endemic in the Sonoran deserts in the new world. The etiologicalagents are Coccidioides immitis and Coccidioides posadasii (Coccidioides spp). Coccidioidesspp., are dimorphic fungi that grow as a mold in the desert soil. Infections are acquired by inhalingspores (arthroconidia) that then germinate in the host and transform into spherules, the pathognomonictissue form of the fungus. The vast majority of infections are either asymptomatic or self-limited, whichimplies that innate immunity is quite effective under most circumstances. However, when exposureis massive, a very high percentage of the resulting infections are symptomatic. African-Americansand Filipinos are at least 10 times more likely to develop disseminated Coccidioidomycosis thanare Caucasians, even if the primary pneumonia is self-limited. This suggests that there is a geneticbasis for the extra-pulmonary dissemination of this infection in humans. There have been a numberof studies of acquired immunity to Coccidioides, both in humans and in experimental animals. It is Tcell dependent and the patients that recover spontaneously develop a Th1 type immune response.Particularly, in the animal models have been established that there are different susceptibility patternsin congenic strains of mice. C57BL/6J and BALB/c mice are the most susceptible strains and there isa correlation between susceptibility and the production of Th2 cytokines. DBA/2N mice are the mostresistant and there is a correlation between resistance and the production of Th1 cytokines.In contrast to what is known about acquired immunity, very little is known about the innate immuneresponse to this fungus. Pattern Recognition Receptors (PRRs) play an important role in theorchestration of the innate immune response by recognizing microbial molecules that are conservedaccross broad taxa and that are denominated Pathogen Associated Molecular Patterns (PAMPs).Several cell wall components of Coccidioides spp. might act as PAMPs by receptors such as Dectin-1, Toll like receptors (TLRs), or the Mannose Receptor (MR).Dectin-1 is the β1-3 glucan receptor that is expressed on macrophages and dendritic cells. β-glucanaccounts for a large percentage of the weight of a Coccidioides spherule. The mannose receptor(MR) is a C-type ligand expressed on antigen presenting cells that is involved in endocytosisof mannosylated proteins and participates in the homeostasis of glycoproteins. Coccidioides spp.contain significant amounts of mannose and 3-O-methylmannose in its cell wall. TLRs are a family of53

conserved non-phagocytic receptors that mediate cellular responses to PAMPs and other ligands. Inthe case of immune response to fungi, the contribution of individual TLRs might vary dependingon fungal species and fungal morphotypes.Few studies have been done to determine how Coccidioides spp interact with the innate immunesystem. It has been reported that spherules activate T cells to secrete pro-inflammatory cytokinesand chemokines such as TNFα and MIP-2, and that this response is both Dectin-1 and TLR2dependant. One study done with human adherent peripheral blood mononuclear cells (PBMC)shown that the MR mediates the cellular immune response to TK27, a glycosylated antigen. It isknown that mannose is the principal monosaccharide of T27K.The goal of this work was to study the role of several PRRs: Dectin-1, MR and TLRs in the innateimmune response to Coccidioides spp., both in vivo and in vitro experimental models.We used formalin-killed spherules and 1,3-β-glucan purified from spherules to stimulate elicitedperitoneal macrophages and myeloid dendritic cells (mDCs) from susceptible (C57BL/6) andresistant (DBA/2) mice. DBA/2 macrophages produced more TNF-α and IL-6 than macrophagesfrom C57BL/6 mice, and the amount of TNF-α made was dependent on both TLR2 and Dectin-1. mDCs from C57BL/6 mice made more IL-10 and less IL-23p19 and IL-12p70 than did DBA/2mDCs. These responses were inhibited by a monoclonal antibody to Dectin-1. DBA/2 miceexpressed full-length Dectin-1, whereas C57BL/6 mice spliced out exon 3, which encodes mostof the stalk. RAW cells transduced to express the full-length Dectin-1 responded better to FKSsthan cells expressing truncated Dectin-1. We compared the isoform of Dectin-1 expressed by 34C57BL/6 X DBA/2 recombinant inbred (BXD RI) lines with their susceptibility to Coccidioides immitis.In 25 of 34 RI lines susceptibility or resistance corresponded to short or full-length isoforms,respectively. Also, we found that resistant (DBA/2) mice had higher levels of Dectin-1 mRNA andmore Dectin-1+ cells in their infected lungs than did susceptible (B6 and BALB/c) mice. Theseresults suggest that Dectin-1 plays an important role in innate immunity to coccidioidomycosisand that different isoforms and differential expression of this lectin contribute to susceptibility ofmice to coccidioidomycosis. As Dectin-1 is expressed on both macrophages and DC it affects thecytokine responses to spherules.We infected MR KO and did quantitative mycology on the lungs and spleens. We found thatMR KO mice infected either i.p. or intra-nasally, in both cases we recovered significantly highernumbers of C.immitis than in control mice. These results suggest that MR is a pattern recognitionreceptor that plays an important role in resistance to C.immitis.We infected MyD88 -/-, TLR2-/-, TLR4-/-, TLR9 -/- and IL-1R-/- mice with C. immitis. We foundthat MyD88 deficient mice on a C57BL/6 background were more susceptible to C. immitis. However,TLR4, TLR9, and TLR2 deficient mice were not more susceptible than C57BL/6 mice tointra-nasal (i.n.) infection with C. immitis as measured by numbers of CFU cultures from lungs andspleens 14 days after i.n. infection. IL-1R-/- mice were more susceptible to dissemination but notpulmonary infection, and they were not as susceptible as MyD88 -/- mice. Thus, IL-1R played asignificant role in resistance to infection, but does not completely account for the MyD88 result.We found that both TLR2 and Dectin-1 were necessary for TNF-α secretion from macrophagesstimulated by β-glucan. The apparent resistance of TLR2 deficient mice may be explained by adefect in the Dectin-1 response to coccidioidomycosis in B6 mice.All these results suggest that an effective host response to Coccidioides spp requires a collaborativerecognition of distinct Coccidioides spp cell wall components by different innate immunereceptors. Genetic polymorphisms in one or more components may explain the increasedsusceptibility of some individuals to this infection.54

A strategy for the generation of a recombinant vaccine againstcoccidioidomycosisGarry T. ColeDepartment of Biology and South Texas Center for Emerging Infectious Diseases.University of Texas at San Antonio, San Antonio, Texas 78249 U.S.A.Coccidioides is an airborne fungal pathogen that can cause mild to severe respiratory disease (coccidioidomycosis;San Joaquin Valley fever) in immunocompetent individuals. The fungus inhabitsdesert soil in the Southwestern U.S. between West Texas and Southern California, as well as certainarid regions of Mexico, Central and South America. About 8 million people reside in the endemicregions of United States. Although rarely a life-threatening disease, Coccidioides causes significantmorbidity in more than 40% of infected individuals, and is responsible for high health care costs relatedto long term treatment. Although about half of the Coccidioides-infected individuals experience onlymild discomfort and do not seek medical intervention, a large body of clinical evidence suggests thatreactivation of the respiratory disease may occur months to years after the original insult. Recoveryfrom symptomatic infection typically confers lifelong immunity to reinfection, suggesting that vaccinationagainst coccidioidomycosis is feasible. We have shown that disease-susceptible BALB/c micecan be fully protected against coccidioidal infection by vaccination with a genetically engineered,avirulent strain of the pathogen. A detergent-extracted protein fraction of the parasitic cell wall ofthis mutant strain protects mice against pulmonary coccidioidomycosis. We have applied a novelstrategy to develop an epitope-driven vaccine based upon a subset of cell wall-derived antigens of thelive vaccine strain of Coccidioides that interface with the host immune system and elicit a protectiveresponse against coccidioidal infection. We have identified candidate T-cell-reactive proteins in thissubset of antigens that drive a robust immune response against pulmonary challenge of transgenicmice which express human MHC class II molecules. These protein vaccine candidates were selectedon the basis that they contain promiscuous epitopes with high affinity for human MHC II complexes,stimulate an in vitro proliferative response of peripheral blood mononuclear cells isolated from skintest-positive patients, and provide protection (survival and reduction of fungal burden) to mice infectedintranasally with a lethal inoculum of Coccidioides spores. We argue that this epitope-driven vaccinecan simultaneously direct the immune response to multiple dominant and subdominant epitopes and,thereby, induce robust and durable protection against coccidioidomycosis.55

Symposium3Virulence Factors andhost-parasite Interactions57

Histoplasma virulence mechanisms with parallelsin paracoccidioidomycosisWilliam GoldmanUniversity of North Carolina at Chapel Hill, NC, USA.e-mail: goldman@med.unc.eduHistoplasma capsulatum is one of the best-studied dimorphic fungal pathogens, and most biologicalwork has focused on specific characteristics that enable the yeast form to be a successful intracellularparasite of macrophages. New molecular genetic tools have allowed us to evaluate the expressionand prove the importance of two yeast phase-specific factors: CBP, a secreted protein that is essentialfor virulence, and α-(1,3)-glucan, a virulence-associated cell wall polysaccharide. However,the mechanisms that explain their role in fungal pathogenicity have remained a complete mysteryuntil recently.We have now used NMR to solve the structure of CBP, revealing that this protein is a proteaseresistanthomodimer and a member of the saposin family of lipid- and membrane-binding proteins.It is likely that CBP is involved in lipid binding, lipid metabolism, and/or membrane remodeling inthe phagolysosomal compartment in which Histoplasma resides. We have taken two approachesto study α-(1,3)-glucan: the first is a forward genetics strategy, using Agrobacterium-mediated insertionalmutagenesis, to identify genes implicated in the regulation, synthesis, and processing ofα-(1,3)-glucan. The second approach uses reverse genetics, combining fungal gene disruption withmammalian RNA-interference, to study the genes involved in production of and response to α-(1,3)-glucan. This work has revealed that α-(1,3)-glucan on the surface of Histoplasma yeasts masksrecognition of the underlying β-glucan by dectin-1, a macrophage pattern-recognition receptor thatis critical in the innate immune response to fungi.Integrated genomics and proteomics strategies bring new insight intoParacoccidioides brasiliensis response upon host interactionCelia M.A. SoaresLaboratório de Biologia Molecular, ICB,Universidade Federal de Goiás, Goiânia, Goiás, Brasil.e-mail: celia@icb.ufg.brParacoccidioides brasiliensis is a major fungal pathogen of humans. The fungus can thrive in a rangeof niches within its human host. These niches can vary in many aspects, such as availability of nutrientsand ambient pH. Also the fungus may have the ability of modifying the ambient by its metabolicactivity, implying that patterns of fungal gene expression could vary temporal and spatially in hostniches. Genome transcriptional expression profiles and proteomics of microbes during infectionprovides information about the host microenvironment to which the microbe is exposed, as well asthe gene expression changes that enable the microorganism to adapt to its host niche. The aim ofthe current study was the identification of genes and proteins expressed during invasion of organssuch as liver, spleen as well as in routes of fungal dissemination. Also the global influence of ironand copper deprivation and extracellular matrix proteins (ECM) in fungal mRNAs and protein levelswas investigated. We observed that depending on the site of infection, P. brasiliensis adapts to theenvironment. P. brasiliensis can modify its global transcriptional profile in a temporal way, as well as59

can adopt a metabolic style on dependence of the host niche. For instance, the fungus can modifyits global transcriptional profile within 10 min of exposure to human blood and suffer a new globaladaptation after 60 min of incubation. Genes expressed in response to liver include those relatedto the glycolytic style, to the cell-wall remodeling and to lipid synthesis. Interestingly upon exposureto human plasma the fungus shifts to a starvation mode. Other experimental conditions that mimiccharacteristics of certain host environments, such as iron and copper starvation, as well as fungaladhesion to ECM components induce a global modification in the transcriptional and proteomic profiles.By analyzing and comparing transcriptional and proteomics patterns we identified genes andmetabolic routes that could be relevant to the fungal survival in the host milieu. Those genes include,for example, those coding for proteins of iron and copper transport systems, those coding for predictablenovel adhesins such as enolase, as well as those coding for enzymes of specific metabolicroutes over expressed in host niches. The methodological approaches also led to the detection ofspecific pathways related to P. brasiliensis virulence.Financial support: CNPq, FINEP.Adhesion and extracellular matrix proteinsMaria José Mendes-GianniniDepartamento de Análises Clinicas, Faculdade de Ciências Farmacêuticas,UNESP, Araraquara, São Paulo, Brazil.e-mail: giannini@fcfar.unesp.brParacoccidioides brasiliensis possesses multiples factors that can cause damage to the host andthat can contribute to its virulence phenotype. With the objective of understanding the mechanismsthat regulate the interaction of this fungus with host cells, our study emphasis the role of moleculesthat can represent new microbial targets to the infection control. For this reason, interaction betweenP. brasiliensis and epithelial cells were evaluated, with an emphasis on the adherence, inductionof cytoskeletal alterations, and differential signaling activity of the various surface molecules. Thesuccess of colonization is a complex event, generally involving a ligand coded by the pathogen (adhesins)and a cell receptor. An important aspect in the interaction between P.brasiliensis and yourhost is the ability to adhere to matrix extracelular components (ECM). This fungal employs a series ofstrategies to colonize and to disseminate, including ligands. The understanding and identification ofthese molecules it is very important in the discovery of efficient treatments for the systemic mycoses.Differential adhesion to the epithelial cells and extracellular matrix (ECM) were verified dependingon the strain and the fungal growth conditions. In this study, proteomic approaches will allow thecharacterization of adhesins under different circumstances. An increase of adhesin expression wasevident from freshly fungal isolated from human or animal passage as well as armadillos and thatpossessed a higher adhesion capacity. This occurrence could be associated with host adaptationand related to the virulence of P. brasiliensis, showing the potential of this fungal in the host-parasiteinteraction.FAPESP, PADC-FCF and CNPq60

Virulence insights from the P. brasiliensis transcriptomeIldinete Silva-PereiraDepartamento de Biologia Celular, Instituto de Ciências Biológicas,Universidade de Brasília, Brasília/DF - Brazile-mail: ildinetesp@gmail.com or xocolau@unb.brThe ecology of Paracoccidioides brasiliensis, the etiologic agent of Paracoccidioidomycosis, is poorlyunderstood. However, host infection is probably acquired by inhalation of airborne propagules derivedfrom the mycelial saprophytic form of this fungus. Once in the lungs, this microorganism undergoesa dimorphic transition, converting to the yeast form, an essential step for the establishment of thepulmonary infection, which alternatively can be eradicated, contained in a granuloma, or disseminatethroughout the body. These different outcomes of the fungus-host interaction will mainly depend onthe host immunological response and the fungal virulence.The major host defense mechanism against P. brasiliensis infection is the cell-mediated immuneresponse, characterized by the production of TNF-α, IL-12 and IFN-γ that activate macrophages.However, in the absence of such cytokines, or in susceptible hosts, this fungus is able to surviveand replicate within the phagolysosome of nonactivated murine and human macrophages. Thus, thefacultative intracellular life style of P. brasiliensis must be compatible with the inhospitable microenvironmentimposed by phagocytic cells. Only recently, we have started to better understand thefactors required for intracellular persistence of this fungus.In the last decade, genomic approaches have proved to be a landmark in the characterization offungal virulence factors, becoming a starting point for the knowledge of its molecular pathogenesis.A number of potential virulence factors and events are considered important for invasive fungi, includingdimorphism, growth at elevated temperatures, adherence to host cells, cell wall componentsand enzymes production.Genomic and transcriptome sequencing efforts, coupled to sophisticated molecular biological tools,have been used to find direct evidence of whether a given factor is required for fungal virulence. Inorder to consider a gene as a virulence determinant, the infection caused by the null mutant mustbe attenuated, when compared to those caused by the wild-type and reconstituted strains. This approach,which is based on the “Molecular Koch’s postulates”, was originally described for bacteria.In P. brasiliensis, the lack of efficient gene-disruption and transformation systems preclude the studyof putative virulence genes based on these postulates.In this sense, we performed in the P. brasiliensis transcriptome a search for orthologs of virulencegenes found in other pathogenic fungi. BLAST comparative analyses were done among PbAESTs(P. brasiliensis Assembled Expressed Sequence Tag) and the sequences deposited in GenBank.As a result, the putative virulence PbAESTs were grouped into five classes, cell wall-, metabolism-,and detoxification-related, secreted factors, and other determinants.Besides the previously described α- and β-glucan synthases, orthologs to chitin synthase andmannosyl transferases, also important in cell wall synthesis and stabilization, were identified. Amongthe metabolism-related putative virulence PbAESTs, we have identified orthologs of the glyoxylatecycle enzymes. This metabolic pathway allows the utilization of C2 compounds as carbon sourcesto gluconeogenesis, and is involved in the virulence of bacteria and fungi. Further studies, employingsemiquantitative RT-PCR analyses, revealed that these genes were upregulated when P. brasiliensiswas recovered from murine macrophages, without any further in vitro growth. The induction ofthis cycle, in response to macrophages microenvironment, suggests that P. brasiliensis uses theglyoxylate cycle as an important adaptive metabolic pathway. In addition, the levels of glyoxylatecycle transcripts were also increased in response to the cultivation of P. brasiliensis in a mediumwith acetate instead of glucose as the carbon source.61

Among the secreted factors, we were able to find phospholipase and urease orthologs in P. brasiliensistranscriptome. With respect to the enzymes involved in the intracellular survival of P. brasiliensis,orthologs to superoxide dismutase, thiol peroxidase, and an alternative oxidase, were also found.Recently, by using cDNA microarray technology, we evaluated the early transcriptional responseof this fungus to the environment of peritoneal murine macrophages, in order to shed light on themechanisms used by P. brasiliensis to survive within phagocytic cells. The most significantly inducedgene, sod3, encodes for a putative Cu,Zn SOD, which is an enzyme involved in the elimination ofsuperoxide anions. In this context, the upregulation of sod3 expression in internalized P. brasiliensis,as well as after in vitro menadione treatment, provide evidences that sod3 may be needed for theelimination of exogenously and endogenously generated superoxides. Overall, these data reveal atranscriptional plasticity of P. brasiliensis in response to the harsh environment of macrophages, whichmay contribute to the adaptation and consequent survival of this pathogen within these cells.Collectively, our results suggest that P. brasiliensis possesses a vast arsenal of putative virulencegenes allowing its survival in the different host environments.Another important issue related to virulent fungi is the increased incidence of systemic mycosesin healthy and in immunocompromised individuals. Since the treatment of those infections is stillhampered by the high costs, drug side effects and the development of resistant fungal strains, thediscovery of new treatment approaches is of prime relevance.Recent data of the literature reveal that farnesol, one of the quorum sensing molecules of Candidaalbicans, acts as an antagonistic molecule inhibiting growth and virulence of bacteria and fungi. Inthis sense, our group has performed experiments assessing the effects of farnesol on P. brasiliensisgrowth and morphogenesis. This isoprenoid, also present in many essential oils, was able to inhibit P.brasiliensis growth and, when employing concentrations that do not compromise cell growth, it alsoaffected the P. brasiliensis dimorphic transition. Despite of the remaining of intact cell wall, and theunaffected cell permeability, P. brasiliensis cells treated with farnesol exhibited a fully cytoplasmic degeneration,as reveled by electron microscopy. No synergistic effect between farnesol and fluconazolewas observed. Although our data clearly demonstrated the in vitro antimicrobial activity of farnesolagainst P. brasiliensis, additional studies involving animal models of experimental paracoccidioidomycosisneed to be performed to assess its potential use as an adjuvant therapeutic agent.Role of melanin in certain dimorphic fungiBeatriz L. Gómez 1* , Marta E. Uran 2 , R. Morris-Jones 3 , Luz E. Cano 2 , AngelaRestrepo 2 , Arturo Casadevall 4 , Frank Odds 5 , Christopher Kibbler 1 ,and Joshua D. Nosanchuk 41University College London, 2 Corporación para Investigaciones Biológicas, Medellín, Colombia,3King’s College London, 4 Albert Einstein College of Medicine, NY, 5 University of Aberdeen, UK.*Current address: Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta. GA USA,e-mail: bgomez@cdc.govMelanin pigments are enigmatic compounds produced by organisms in all biological kingdoms,including a wide variety of pathogenic fungi, bacteria, and helminths. Melanin synthesis has beenassociated with virulence for a variety of pathogenic microbes and, in the last 10 years, increasingattention has been directed to the study of melanization in human pathogenic fungi. For somemicrobes melanin appears to contribute to virulence by reducing their susceptibility to host defensemechanisms and by altering host immune responses. Microbial melanization can interfere with theactivity of antimicrobial drugs in vitro, and may potentially result in clinical resistance, particularly to62

certain antifungal drugs. The effect of melanin on microbial susceptibility to drugs and host defensesmay be an important consideration in the selection and development of antimicrobial therapy. Workconducted by different groups has characterized melanogenesis in the important dimorphic fungalpathogens Paracoccidioides brasiliensis, Histoplasma capsulatum, Sporothrix schenckii, Blastomycesdermatitidis, Coccidioides posadassi and more recently in Candida albicans. These findings andtheir possible clinical impact will be discussed and reviewed in this presentation with special focuson recent developments in P. brasiliensis, the etiological agent of paracoccidioidomycosis (PCM).Both conidia and yeast cells of this thermally dimorphic fungal pathogen produce melanin or melanin-likecompounds in vitro and in vivo. Our results demonstrate that P. brasiliensis synthesizes andpolymerizes melanin during infection and generates antibodies, principally IgG but not IgM, againstconidia and yeast melanins, as detected in sera and BALs such as reported previously for other fungi.The mouse anti-P.brasiliensis melanin MAbs obtained by our group and the significance of antibodyresponse in vivo will be useful in the study of P. brasiliensis melanization, particularly in regardsto passive immunization for prolonged survival of infected mice and/or modulation of the immuneresponse against this fungal infection. Another important and recent contribution (da Silva MB et al,2006) indicates that P. brasiliensis production of melanin may contribute to virulence by reducing theuptake of yeast cells by peritoneal and alveolar macrophages. This would enhance the resistanceof fungal cells to attack by the immune effector system. Melanized cells were observed to be lesssusceptible to potent antifungal drugs, in particular amphotericin B, ketoconazole, fluconazole, itraconazoleand sulfamethoxazole. We are currently investigating potential role(s) of melanogenesisin the pathogenesis of P. brasiliensis infection.63

Symposium4Immune Responses inparacoccidioidomycosis:cellular and molecular Mechanisms

Microbicidal mechanisms exerted by macrophages againstParacoccidioides brasiliensis conidiaA. Gonzalez 1,2 , Erika Caro 1 , Marta E. Uran 1 , Damaris Lopera 1 and Luz Elena Cano 1,21Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas (CIB);2Microbiology School, Universidad de Antioquia, Medellín, Colombia.e-mail: agonzalezm@cib.org.coIt is known that macrophages play an essential role in the control of Paracoccidioides brasiliensisinfection. In this sense, our laboratory has demonstrated that these phagocytic cells once activatedwith interferon-gamma (IFN-γ) or tumor necrosis factor alpha (TNF-α), exhibit either a fungicidal orfungistatic activity against P. brasiliensis conidia mediated by a nitric oxide (NO) dependent or independentsystem, respectively. In addition, we also showed that the NO-mediated fungicidal mechanismexerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of ironinteraction.Recently, we have demonstrated that the microbicidal activity exerted by IFN-γ activated murineperitoneal macrophages is mediated by a combination of both nitrogen and oxygen reactive species.Thus, IFN-γ-activated macrophages inhibited the conidia-to-yeast transition process, as well as theirviability. When co-cultures of macrophages and P. brasiliensis conidia were treated with superoxidedismutase (SOD) and 5’-aminosalicilic acid (ASA) (O 2-inhibitors), as well as with aminoguanidine (AG)(NO inhibitor), the conidia-to-yeast transition process was reverted indicating that both O 2-and NOwere involved in the fungistatic activity. Nonetheless, the fungicidal effect was observed only whenSOD and AG were added simultaneously to the co-cultures. Therefore, when P. brasiliensis conidiawere exposed to 3-morpholinosydnonimine (SIN-1), a peroxynitrite (ONOO - ) donor, a fungicidal/fungistaticactivity against the fungus was observed; in addition, immunohistochemical studies showedthat nitrotyrosine (an ONOO - nitration marker) was detected only in infected macrophages.The effect of the antimicrobial peptide, lysozyme, against P. brasiliensis was also investigated.When murine alveolar macrophages of the MH-S cell line were activated with TNF-α the transitionprocess was inhibited and the macrophages exerted an important fungicidal effect against P.brasiliensis conidia. However, when a selective inhibitor of lysozyme was added, these microbicidaleffects were reverted. Therefore, when P. brasiliensis propagules were exposed directly to differentconcentrations of lysozyme, a dual effect was observed. Physiologic concentrations of this enzymefacilitated the conidia to yeast transition process while inflammatory concentrations impaired sucha process. Interestingly, when yeast cells were exposed to lysozyme, irrespective of concentration,the multiple-budding ability was strongly impaired. Furthermore, ultra-structural changes such assubcellular degradation, lipid vacuoles fusion, and damage to lamellar structures with interruption ofthe fibrilar layer, were observed in lysozyme-exposed conidia.In vivo preliminary studies have shown that in mice infected with P. brasiliensis conidia and forthe first 4 days post-infection, bronchoalveolar lavage fluids showed an increase in TNF-α productionand/or in its mRNA levels, as observed in mouse lungs; simultaneously, lysozyme expression inphagocytic cells was augmented with a parallel decreasing of the fungal loads. Contrariwise, duringthese first days post-infection the inducible nitric oxide synthase mRNA levels and NO productionbecame detected only at their basal levels.All these findings indicate that the various microbicidal mechanisms exerted by activated macrophagesare operating in an integrated manner to attain control of P. brasiliensis infection especiallyduring the initial process, and that these mechanisms are depending on the specific cytokineinvolved.Financial support: Colciencias, Banco de la República, U. de Antioquia.67

Human cells and molecules involved in the microbicidalmechanisms against Paracoccidioides brasiliensisAngela M.V.C. SoaresDepartment of Microbiology and Immunology, Bioscience InstituteSão Paulo State University- Unesp- Botucatu, São Paulo- Brazile-mail: acsoares@ibb.unesp.brIn the last years our laboratory has studied the modulatory and effector molecules involved in P.brasiliensis killing by human phagocytic cells. First, experiments were carried out with the objectiveto study the effect of the cytokine IFN-γ on activation process of monocytes for high (Pb18) and low(Pb265) virulent P. brasiliensis killing. Nonactivated cells displayed a very low fungicidal activityagainst both strains of the fungus. This activity was increased after priming cells with the cytokine.However, killing against low virulent strain was significantly higher than that detected with low virulent.These results were explained by higher capacity of this strain to induce TNF-α production byhuman monocytes. Further studies were carried out with the objective to study the modulatory effectof prostaglandins upon fungicidal activity of monocytes against the two strains. It was concludedthat prostaglandins release by human monocytes after Pb18 and Pb265 challenge, inhibit TNF-αproduction by human cells. To compensate this inhibitory mechanism the cells must be activatedwith cytokines such as IFN-γ. However this compensatory effect was more efficient to Pb265, since itinduces higher TNF-α production when compared to Pb18. More recently we showed that activatedmonocytes kill P. brasiliensis by a mechanism dependent on H 2O 2release. Then, we asked if theinhibitory effect of prostaglandins on human P. brasiliensis killing could involve H 2O 2inhibition. Forthis, human monocytes were no activated or activated with IFN-γ, TNF-α or GM-CSF in the presenceor absence of indomethacin, challenged with Pb18 or Pb265 and evaluated for fungicidal activity andH 2O 2, TNF-α, IL-6 and IL-10 release. We concluded that TNF-α inhibition by prostaglandins, leadhuman monocytes to release lower levels of H 2O 2.However, this effect was reverted by cytokinesactivation, with monocytes activated with TNF-α or GM-CSF and challenged with Pb265 showinghigher results when compared to the other treatments. In view of these results, we asked if NO,another metabolite involved in P. brasiliensis killing, could be modulated by prostaglandins. Twoapproaches for NO detection were used: the quantification of this metabolite in culture supernatantsand evaluation of inducible nitric oxide synthase (iNOS) mRNA expression by real-time RT-PCR.Differently of the assays with H 2O 2, a clear association between prostaglandins and NO inhibitionwas not detected, and other assays have been made to better characterize this effect.The role of CD8+ cells in human paracoccidioidomycosisRonei Luciano Mamoni, Lanny C. Burlandy-Soares, Maria Heloísa S.L. BlottaDepartamento de Patologia Clínica – FCM – UNICAMP, Campinas, SP, Brazile-mail: heblotta@fcm.unicamp.bCellular immune response mediated by Ag-specific IFN-γ secreting CD4+ T cells has long been establishedas an essential component of protective immune response against P. brasiliensis. However,there is an increasing evidence of the participation of CD8+Tcells in both the murine and human P.brasiliensis infection. A protective role for CD8 + T cells was suggested in experimental PCM since its68

depletion induces a more severe and/or disseminated disease in both, resistant and susceptible mice.Moreover, an elevated number of CD8 + T cells is detected in the lungs of patients with pulmonaryPCM, in addition to high concentration of MIP-1α in BAL, a chemokine known to selectively attractthis cell subset. Ag specific CD8+ T cells can act as cytokine-secreting cells producing IFN-γ andTNF-α, which stimulate macrophage fungicidal activity. Moreover CD8+cells are able to kill infectedcells through the release of cytolitic molecules such as perforin, granzymes and granulysin.In this study we analysed cytotoxic granules expression as well as fungicidal and cytotoxic activityof CD8+ cells from patients with PCM, infected individuals living in the endemic area, but without anyclinical manifestation of the disease (PI), as well as controls (C). The ex-vivo evaluation of intracellulargranules expression by flow cytometry showed that PCM patients presented a lower frequencyof CD8+ cells expressing granzyme A, B and perforin compared to PI individuals. Granulysin mRNAexpression was elevated in PBMC from controls and PI individuals and decreased in PCM patients,mainly in the ones with severe forms of the disease. In accordance, sera from PCM patients showedreduced levels granulysin concentration as compare to PI and controls individuals, which reverseafter antifungal treatment.To determine the fungicidal activity, CD8+ and CD56+ cells were cultured with P. brasiliensis yeastcells (high virulent, Pb18 and low virulent, Pb265). CD56+ and CD8+cells from PI individuals wereable to inhibit the growth of P. brasiliensis, against Pb18 and Pb265, and in higher degree than PCMpatients. In the control group only CD56+ cells were able to inhibit the growth of both strains of P.brasiliensis. In general Pb265 was more susceptible to lysis than Pb18.It has been demonstrated that lymphocyte-mediated antifungal activity was critically dependenton IL-15, a cytokine with growth factor activity for T cells and NK cells. IL-15 is a potent chemoattractantand controls both proliferation and survival of naive and memory-phenotype CD8 + T cells. Todetermine whether IL-15 could enhance the fungicidal activity, purified CD8+and CD56+ cells isolatedfrom patients with PCM, PI and control individuals were stimulated or not with IL-15 in the presenceof yeast cells. Cells from PI and patients with PCM displayed an increased fungicidal activity againstPb265 after IL-15 treatment. In controls IL-15 addition resulted in increased fungicidal activity onlyin CD56+ cells against both, Pb265 and Pb18. When the CD8+ and CD56+cells were pretreatedwith SrCl 2to induce degranulation and deplete their granules, the ability to kill P. brasiliensis (Pb18)was abrogated. However, pretreatment with concanamycin and EGTA, which inhibit the perforincytotoxicity pathway, had only a slight effect over the ability to kill Pb18. These data indicates thatthe fungicidal activity against P. brasiliensis is mainly mediated by granules, positively induced byIL-15 and more elevated in PI individuals than in patients with PCM. .CD8+ and CD56 cells from PI individuals were able to kill target cells (CD14+) infected with Pb265in a dose dependent manner and more intensively than cells from PCM patients. The cytotoxic activitywas specific since cells from controls did not display considerable activity even in high numbers.Only CD8+ cells showed a dose-dependent capacity to kill Pb18-infected cells while CD56+ cellswere able to kill target cells infected with Pb265, but not with Pb18. In general the percentage oflysis was slightly higher in relation to Pb265 compared to Pb18 yeast cells and CD8+ cells exhibiteda greater cytotoxic potential than CD56+ cells. Similar to fungicidal activity the cytotoxic responseagainst infected cells by CD8+ and CD56+ cells was positively influenced by IL-15 and in a dosedependentmanner. Cells from PI individuals presented a higher response as compared to PCMpatients. Moreover, the response of both CD8+ and CD56+ cells to Pb265 was stronger than toPb18. In relation to the control group IL-15 had a modest effect on CD56+ cells. When CD8+ andCD56+ from PI individuals were pretreated with SrCl, CMA or EGTA their ability to lysis target cellswas significantly reduced, showing the dependency of granules and perforin.CD8+ and CD4+ cells from PI individuals were able to specifically proliferate in response to P.brasiliensis antigen or yeast cells (Pb18 and Pb265). Interestingly when CD8+ cells were cultivated inthe presence of CD4+ cells an increased proliferative response of CD8+ cells was detected, showing69

a role for CD4+ cells in the growth of CD8+cells. The addition of IL-15 into the cultures stimulatedwith P. brasiliensis yeast cells replaced the effect of CD4+ cells on CD8+ proliferation. Additionally,treatment of CD8+CD4+ co-cultures with anti-IL-15 partially inhibited the positive effect of CD4+ cellson CD8+ proliferative response. These data indicate that CD4 growth effect on CD8+ T cells mightbe mediated, at least in part, by IL-15.In summary we found that CD8+ cells from patients with PCM express lower levels of cytotoxicmolecules (granzyme A, B, perforin and granulysin) and diminished fungicidal and cytotoxic activityagainst P. brasiliensis yeast cells compared to PI individuals. In general low virulent Pb265 wasmore susceptible to lysis than high virulent Pb18 yeast cells. These factors, in association to othermechanisms that compromise cellular immunity, may account for a deficient cytotoxic activity, and,therefore reduced protective response against the fungus.Antigen-specific immunosuppression inparacoccidioidomycosis: Adverse effect or desired outcome?Camila R. Cacere 1 , A.J.S. Duarte 1 , Maria José S. Mendes-Giannini 2 , Gil Benard 1,31Laboratory of Dermatology and Immunodeficiencies,Medical School of the University of São Paulo.2Laboratory of Clinical Mycology, Pharmaceutical Sciences Faculty, São Paulo State University.3Systemic Mycoses Outpatient Clinic, Division of Infectious Diseases,Hospital das Clínicas da Faculdade de Medicina da USP (HC FMUSP), Brazil,e-mail: mahong@usp.brThe T-cell hypoproliferative reactivity observed in the immune response to P. brasiliensis antigens ofpatients with active paracoccidioidomycosis probably contributes to the failure of the host in controllingthe infection, leading to a disseminated disease. It is, however, largely reversible with treatment in mostpatients. The mechanisms leading to this hyporresponsiveness are not well known. We have previouslydemonstrated that patients’ mononuclear cells in presence of gp43 exhibit enhanced apoptotic levels.I an attempt to explain such findings, we hypothethized that these cells were inadequately activateddue to altered costimulatory molecules expression, such as CD80, CD86, CD28, CD152, ICOS ePD-1. Expression of these molecules were evaluated on T-cells and monocytes of the peripheralblood of patients with active, disseminated PCM (n = 71), and healthy individuals with a past historyof treated and cured PCM (n = 24). These cells were cultured in presence of a Candida albicansmetabolic antigen (CMA), gp43, or kept without exogenous stimuli for 4 days. Our results show thatthe expression of CD28 was comparable between patients an controls’ cells, and that CD152, PD-1e ICOS, all of which known to deliver negative costimulatory signaling and to arrest cell cycle entry,were overexpressed in patients’ T-cells. In parallel, we performed additional experiments where therespective costimulatory signalings were blocked by addition of blocking antibodies specific to each ofthese molecules. Whatever the blocking antibody used, there was no reversal of the hypoproliferativestate of patients’ T-cells. However, while the expression of the CD80 and CD86 molecules on monocyteswas similar between controls and patients, their expression on T-cells was significantly higherin patients. Adding the respective blocking antibodies at day zero of the culture, we could observethat both the gp43 and the CMA responses were inhibited, but differentially according to the antibodyemployed. In both patients and controls the blocking CD86 signaling decreased the response to gp43and CMA of patients and controls, while blocking of CD80 signaling decreased only the response togp43, and only in the control group. These data suggest that different antigens may have differentcostimulatory requirements for antigen presentation. Addition of the antibodies at day 4 of culture70

did not restore the lymphoproliferative response or modified the response of the controls. Our resultssuggest that the hypothesis, raised from other models of prolonged foreign antigen exposure, thatrepetitive and persistent in vivo exposure to fungal antigens, which also occurs in patients with PCM,lead the T-cells to a adaptive tolerant state, which is hardly reverted in vitro. We then investigatedthe fate of such putatively tolerized patients’ cells, by analyzing the role that apoptosis may havein this tolerant state. We observed that expression of the anti-apoptotic molecule Bcl-2 was lowerin patients’ cells, even when the cells were in vitro reestimulated with CMA and gp43, suggestingthat the cells are more susceptible to undergo apoptosis. When then analyzed the expression of theactive form of the caspases 8 and 9 molecules. We first analyzed their expression on cells kept incultures for 4 days with or without stimuli. Unexpectedly, we observed that controls’ cells, and notpatients’ cells, exhibited higher levels of expression of both molecules. To explore further these data,we tested the hypothesis that the patients’ cells were already undergoing apoptosis at an earlierthan 4 days stage. Caspases expression were therefore analyzed ex vivo. In fact, we observed thatTCD3+ cells exhibited markedly enhanced caspase 8 and expression as compared to controls’ cells.These findings may help to explain why we failed to redress the proliferative responses to gp43 inthe experiments where blocking antibodies were added: these cells would be committed to apoptoticdeath, thus refractory to in vitro manipulations, as described for adaptively tolerant T-cells.Several reasons for the immunosuppressionobserved in paracoccidioidomycosisAna Paula Moreira 1 , Karen A. Cavassani 1 , Ana P. Campanelli 1 ; Fabrine S. MassaferaTristão 1 , F.A. Rocha 1 , L.L. Oliveira 1 , L. Panagio 1 , Roberto Martinez 1 and Joao S. Silva 1Medical School of Ribeirão Preto; Ribeirão Preto, Brazile-mail: jsdsilva@fmrp.usp.brThe long-term persistence of pathogens in the host is a hallmark of certain infectious diseases, includingthe Paracoccidioides brasiliensis that causes the paracoccidioidomycosis (PCM). Althoughseveral reasons could be responsible for the persistence of this fungus in the host, one is the profoundimmunosuppression that occurs in the infected patients. Evaluating the mechanisms that lead to theremarkable T cell unresponsiveness to antigens in paracoccidioidomycosis we described that theyare not associated with imbalanced cytokine production or with defective CD28 or Fas expression.However, only T lymphocytes from patients expressed higher levels of CTLA-4 and were AnnexinV + and FasL + . As the addition of anti-FasL to cultures of peripheral blood mononuclear cells (PBMC)resulted in decreased levels of apoptosis, we concluded that an activation-induced cell death, triggeredthrough the Fas-FasL pathway, could be responsible for this modulation. Moreover, the blockageof CTLA-4 and FasL resulted in increased production of interferon-gamma, and the concomitantinhibition of FasL and CTLA-4, but not of transforming growth factor-beta, resulted in significant Tcell proliferation in response to phytohemagglutinin. Together, we concluded that apoptosis mediatedby Fas-FasL and engagement of CTLA-4 are involved in modulation of the immune responsein patients infected with P. brasiliensis. However, as the inhibition of CTLA-4 and blockage of Fasand FasL interaction only partially reversed the T cell response, it revealed that others mechanismscertainly were involved in this phenomenon. Therefore, as natural regulatory T CD4 + CD25 + (Treg)cells are involved in the control of immune responses in the infections with several pathogens, therole of these cells in the control of lesions of patients with PCM was investigated. We found that the71

leukocyte subsets found in the lesions were similar in patients and controls, except for CD11c + CD1a +cells. However, a higher frequency of CD4 + CD25 + T cells expressing CTLA-4, glucorticoid-inducibleTNFR, membrane-bound TGF-β and Foxp3 were observed in PBMC of patients. In accordance, thesecells exhibited stronger suppressive activity when compared with those from controls (94.0 vs 67.5%of inhibition of allogeneic T cell proliferation). It was observed a higher frequency of Treg in PBMCof patients and chemokines receptors (CCR4 and CCR5) accumulate in the P. brasiliensis-inducedlesions. Indeed, the secreted CCL17 and CCL22, both associated with the migration of Treg cells toperipheral tissues, were also detected in the biopsies. Because Tregs are in the lesions of patientswith PCM, the migration of Treg cells is dependent on the axis chemokine-chemokine receptors,and CCR5 ligands are produced in P. brasiliensis-induced lesions, we investigated the role of CCR5in the control of the infection. The results showed that CCR5 -/- mice are more efficient in controllingfungal growth and dissemination and exhibited smaller granulomas than wild-type (WT) mice. In theabsence of CCR5, the percentage of Treg expressing Foxp3, glucocorticoid-induced TNFR (GITR),CD103, CD45 low , and CTLA-4 in the granulomas was significantly decreased. Interestingly, P. brasiliensisinfection resulted in an absence of T cell proliferation in response to Con A in WT but notCCR5 -/- mice that was abrogated by anti-CTLA-4 mAb and anti-GITR mAb. Moreover, the adoptivetransfer of CD4 + CD25 + but not CD4 + CD25 - T cells from infected WT to infected CCR5 -/- mice resultedin a significant increase in fungal load. Altogether, these data provide the first evidence that Tregcells play a role in controlling local and systemic immune response in patients with a fungal-inducedgranulomatous disease advancing our understanding about the immune regulation in human chronicdiseases. Also, CCR5 is a key receptor for the migration of Treg cells to the site of P. brasiliensisinfection in mice, leading to down-modulation of effector immune response and the long-term presenceof the fungus in the granulomas.72

Historical accountsCoccidioidomycosisCoccidioidomycosis: Discovery in ArgentinaRicardo NegroniMycology Unit. Hospital de Infecciosas Francisco Javier Muñize-mail: ricnegroni@hotmail.comThe history of coccidioidomycosis in Argentina began in 1892, when Dr. Bengolea asked a medicalstudent, Alejandro Posadas, to perform a histopathologic study of a skin biopsy taken from a verrucouslesion of patient with tentative diagnosis of fungoid mycosis. Alejandro Posadas was workingunder the direction of Professor Roberto Wernicke, who was to head of histopathology laboratory of“Hospital de Clínicas”. The patient was Domingo Ezcurra, a 36-year-old, cavalry soldier, who wasborn in Argentina. Ezcurra presented a chronic disseminated form of coccidioidomycosis, with a veryshow progression until his death in 1898.Posadas and Wernicke found that the biopsy presented a chronic inflammatory response whichwas identical to the granulomatous reaction observed in tuberculosis, but they visualized a lot of cystic-like parasitic forms very similar, but not equivalent to those usually found in the protozoan of theCoccidia gender. They also described the existence of two types of spherules, those of proliferativeprogression containing protospors inside the spherule and those of cystic appearance with matureendospors, and they related these two types of parasitic forms to the host’s defensive capacity.They were able to reproduce this infection in several animal species, using monkeys, rats, mice,rabbits, cats, dogs and guinea pigs. Monkeys were the most susceptible hosts. After subcutaneousinoculation of maceration of human biopsy, they developed an acute disease, with severe pulmonarylesions which presented a fatal outcome in three weeks. The microscopic examination of thelesions exhibited great amount of proliferative spherules in histopathology. In other animal speciesthe evolution of the experimental infection was chronic, cutaneous and lymph nodes involvementwas often observed at histopathology. They considered that the lung was the portal of entry of coccidioidomycosisand they tried to establish cultures in several natural culture media, but these weresystematically discarded as contaminations due to the growth of mould. Perhaps the only mistakemade in this outstanding research.73

A history of research on coccidioidomycosis in the U.S.Garry T. ColeDepartment of Biology and South Texas Center for Emerging Infectious Diseases,University of Texas at San Antonio, San Antonio, Texas 78249, U.S.A.The history of coccidioidomycosis begins south of the U.S. border. In January 1891, a 32-year-oldsoldier was brought from Northern Argentina to the University Hospital in Buenos Aires with a lesionon his right cheek. The condition was believed to be an unusual case of mycosis fungoides becauseof a coccidia-like parasite associated with the apparent malignant neoplasm. In 1893, a similarcase was observed in the San Joaquin Valley of Southern California which involved an immigrantPortuguese laborer from the Azores. The causative agent in both cases was Coccidioides, originallythought to be a protozoan, but correctly identified as a fungus by Ophuls and Moffitt in 1900 at StanfordMedical School. Fewer than 500 cases of coccidioidomycosis were reported by the CaliforniaDepartment of Health up to 1935 and “Valley fever” was considered a rare infection. This conceptchanged dramatically after a series of epidemiologic studies conducted in the San Joaquin Valley ofSouthern California between 1937 and 1950 led by Gifford, Dickson and Smith. The earlier impressionthat coccidioidomycosis was a rare and often fatal disease, shifted as a result of these studies to anew perception that this mycosis in endemic regions of the U.S. is common and may present as abenign infection or a disseminated, potentially fatal disease. The stage was set to attract researchersinterested in the mycology, ecology, immunology, diagnosis and treatment of coccidioidomycosis.Some of the major pioneering investigators and their contributions to our current understanding ofthe nature of this disease and the biology of its causative agent will be reviewed.Coccidioidomycosis: Recent records in Latin AmericaBodo Wanke1, Márcia S. Lazéra1, A. Deus Filho2, Luciana Trilles1, Maria A. Salmito2,Liline M.s. Martins2, Regina C.l. Macêdo1, Claudia C.f.bezerra1, And Kelsen D. Eulálio, 21Evandro Chagas Clinical Research Institute, Fiocruz,Rio de Janeiro, Brazil. 2Federal University Of Piauí, Brazil.e-mail: bodo.wanke@ipec.fiocruz.brCoccidioidomycosis and paracoccidioidomycosis are systemic mycoses that occur endemicallyonly in the Western Hemisphere. While paracoccidioidomycosis is confined to Latina America,coccidioidomycosis exhibits a broader distribution, reaching its most important endemic area in thesouthwestern United States (US), where it presents an estimated annual incidence of 150,000 newinfections, nearly 60,000 primary acute and 750 disseminated cases.Since coccidioidomycosis is not a reportable disease, its true incidence is unknown. Consequently,statistics on its prevalence and incidence are either fragmentary or simply not available. However,some recent data have been reported in Latin America.Mexico has the largest and most important endemic region following the US, reflecting an estimatedannual incidence of 1,500 new cases of primary and 15 cases of disseminated coccidioidomycosis.In comparison, the estimated incidence in the US is 40 times fold higher than in Mexico. Moreover,similarly to the US, an incresed incidence is expeted in some Mexican regions, mainly those situatednext to the US border.Although clinical cases and epidemiological surveys have demonstrated endemic areas in CentralAmerica (Guatemala, Honduras, Nicaragua and El Salvador) and South America (Colombia, Paraguayand Bolivia), no recent records have been published.74

In South America, from Venezuela to Argentina, coccidioidomycosis is endemic in several discontinuoussemi-desertic regions, but in all these areas the prevalence and incidence rates are quite lowerthan in North America. Venezuela appears to have the highest prevalence and incidence of the mycosisin South America. Unfortunately, the only recent information point to an annual incidence rangingfrom 0 to 13 (mean 4.3) cases/year from 1984 to 2004 (San Blas 2007, personal communication).In argentina, where the first case was reported in 1892, coccidioidomycosis is endemic in largesemidesertic regions extending from Jujuy in the North to Neuquén in the South. Despite many studieson coccidioidomycosis along the last 110 years, there are no precise data on its true incidence.However, a recent multicenter serodiagnostic study points to possibly at least 10 new cases/year.The most recent endemic region of coccidioidomycosis was discovered in the NE semi-arid regionof Brazil. Since 1978 more than 100 human cases were recorded in the states of Piauí, Ceará,Maranhão, and Bahia. An attempt to estimate the incidence in the state of Piauí during a 9 yearsperiod (1999-2007) diagnosed 81 new cases, out of them nearly 90% had the primary acute and10% had disseminated forms of coccidioidomycosis. Although the annual distribution is not uniform,an approximate annual incidence of 9 cases/year can be estimated only for Piauí State. However,adding other states where the mycosis has been demonstrated and the remaining semi-arid regionof northeastern Brazil, the incidence should be estimated as twice higher. The infection was alsodemonstrated in dogs and armadillos (Dasypus novemcinctus), and Coccidioides posadasii wasisolated from the soil of four armadillo burrows. The excavated sites caused outbreaks of acutepulmonary coccidioidomycosis in armadillo hunters. Thus, so far the most important risk activity foracquiring coccidioidomycosis was armadillo hunting with digging them out of their burrows, in nearly90% of the patients.Looking to the estimated incidence in North America (US and Mexico) and comparing their rateswith that estimated outside, as in Venezuela, Argentina and Brazil, primary and disseminated coccidioidomycosesare less important in the latter countries. In comparison, Mexico presents an estimatedincidence 30 times fold higher than the three above mentioned South American countries together.It shoud be considered that the above estimated incidence rates are based on imprecise and evennot comparable data, frequently obtained by different and not standardized methods, pointing to thenecessity of a Pan American surveillance programme on coccidioidomycosis.The same importance should be given to soil studies, attempting the demonstration of contaminatedfoci in wild animal burrows and ancient Indian burial sites, in order to determine the majorenvironmental risk factors. To illustrate this, in Brazil curiously it was found that areas endemic forparacoccidioidomycosis (humid climate) are next to areas endemic for coccidioidomycosis (aridclimate) and, additionally, for both mycoses the nine-banded armadillo Dasypus novemcinctus hasbeen demonstrated as, so far, the most important non-human host.Key words: coccidioidomycosis, epidemiology, incidence, prevalence, ecology.75

Symposium5Granuloma and Fibrosis:Tissue Markers ofparacoccidioidomycosis

B-1 cells modulate the kinetics of granuloma formationinduced by P. brasiliensisJosé Daniel Lopes, Ana Flavia Popi and Mario Mariano.Department of Microbiology, Immunology and Parasitology,Universidade Federal de São Paulo, São Paulo, Brasil.The concept, origin, fate and biological significance of granulomatous inflammation are still controversial.Kindler and others objectively defined the typical granulomatous lesion induced by Mycobacteriumtuberculosis as an isolated newly formed organ originating through specific assembly of differentiatedmacrophages named epithelioid cells. The multicellularity, cell pleomorphism, participation ofthe immune system modulating the lesion and the complexity of factors involved in the mediation ofgranuloma formation limit the comprehensive interpretation of the process. Strong evidence indicatesthat most macrophages in granulomas derive from blood monocytes. It is unclear, however, to whatextent proliferation, migration and activation of other cell types influence the onset of lesions. Thecharacteristic of Paracoccidioidomycosis (PCM), a disease produced by P. brasiliensis, is the typicalgranulomatous formation in immunologically non-compromised patients. However, the nature andfunction of cells which participate in their formation are as yet poorly understood.Results from our and other laboratories have implicated B-1 cells in the pathogeny of inflammation,tumor growth and immune regulation. B-1 cells are a unique type of hematopoietic cells, present inthe peritoneal and pleural cavities of mice, that can be identified by CD11b + IgM hi IgD low phenotypeand can be further subdivided by differential expression of cell-surface antigen CD5 into B-1a (CD5 + )and B-1b cells (CD5 – ). These cells migrate to distant sites of inflammation, produce large amounts ofautoreactive IgM and secrete large amounts of IL-10. After migration, B-1 cells home at distant sitesof inflammation and become macrophage-like cells. However, the influence that these cells mighthave on the kinetics and fate of the inflammatory process is not known.Considering that macrophages are pivotal in the inflammatory reaction, we decided to investigatethe possible influence B-1 cells could have on macrophage activities in vitro. Results showed thatperitoneal macrophages from Xid mice, a strain deprived of B-1 cells, have higher phagocytic indexesfor zymosan particles when compared with macrophages from wild-type mice. Experiments usingco-cultures of B-1 cells and macrophages from Xid mice in transwell plates demonstrated that B-1cells down-regulate macrophage activities. As B-1 cells are one of the main sources of interleukin(IL)-10, we demonstrated that adherent peritoneal cells from Xid mice produce significantly lesseramounts of this cytokine when compared with cells from wild-type mice. When B-1 cells from IL-10 knock-out mice and macrophages from wild-type mice were co-cultured in transwell plates, thephagocytic index of macrophages was not altered, demonstrating that B-1 cells influence the effectorfunctions of macrophages in vitro via IL-10 secretion.Gp43, the main antigen secreted by P. brasiliensis, the causative agent of PCM, is a high mannoseglycoprotein. The role played by gp43 in the pathogenesis of the disease is not completely known.We have described the influence of this molecule on the interaction between peritoneal murinemacrophages and Pb. Phagocytosis of Pb, live or heat-killed, by adherent peritoneal cells from both,B10.A (susceptible) and A/Sn (resistant) mice, was evaluated. Addition of different concentrations ofgp43 to the culture medium inhibited, in a dose-dependent pattern, phagocytosis of live or heat-killedPb by peritoneal macrophages from both B10.A and A/Sn mice. Gp43 also inhibits phagocytosis ofzymosan particles but did not interfere with the uptake of opsonized sheep red blood cells. It wasalso shown that both gp43 and heat-killed Pb have an inhibitory effect on the release of NO by zymosanstimulated macrophages. Finally, it was demonstrated that gp43 inhibits the fungicidal abilityof macrophages from both lineages. Preliminary results showed that peptides derived from gp43,79

chosen for being exposed at the molecule surface, were equally able to produce these effects, aswell as to increase H 2O 2expression by bone-marrow derived macrophages. Based on these data,it was suggested that gp43 can be considered one of the evasion mechanisms for the installationof primary infection in susceptible hosts. We developed a granuloma model in vitro using beads toevaluate the role of isolated mouse peritoneal macrophages and B-1 cells. We also investigatedgranuloma formation in the presence of gp43 which is secreted exocellularly.To determine whether B-1 cells, macrophages, or both, participate in granuloma formation, peritonealcells from Xid mice, were used. Granuloma-like structures were not formed with Xid peritonealcells or with cells from wild type mice that had their peritoneal and pleural cavities irradiated beforethe cultures were established. Granulomas were observed either when total adherent peritoneal cellsor when isolated B-1 cells were added to macrophage cultures. The data strongly suggest that aninteraction of B-1 cells and macrophages plays an important role in granuloma-like formation in thisexperimental model and that the presence of gp43 strongly stimulates this response.Protective immunity in PCM is mainly mediated by cellular immunity nevertheless, the role of Bcells in the process, in particular B-1 cells, is poorly investigated. The participation of B-1 cells in resistanceor susceptibility of BALB/c and BALB/Xid mice to P. brasiliensis (Pb) pulmonary infection wasinvestigated. BALB/Xid, which lacks B-1 cells, exhibited higher resistance to infection when comparedwith BALB/c mice. However, adoptive transfer of B-1 cells to BALB/Xid mice drastically increased thesusceptibility of these animals to Pb infection. The fungal burden in BALB/c and B-1-reconstitutedBALB/Xid was significantly higher as compared to BALB/Xid strain. Compact, well-organized granulomaswere observed in the lungs of BALB/Xid mice, whereas large lesions with necrotic centers witha plethora of fungi developed in BALB/c mice. It was also shown that B-1 cells impair phagocytosisof Pb by macrophages in vitro via secretion of IL-10, which was increased upon stimulation with a Pbantigen, gp43. Finally, in vivo blockade of IL-10 led to a better control of infection by the susceptibleB10.A mice. These findings demonstrate that B-1 cells play a role in the mechanisms of resistance/susceptibly to Pb infection in murine models, most likely via production of IL-10.Extracellular matrix modulation in paracoccidioidal granulomaEva Burger 1 , and Angela Satie Nishikaku 11 Intituto do Ciencias Biomedicas, USP, Sâo Paulo - Brasil.e-mail: evaburger@usp.brIn the isogenic murine model of paracoccidioidomycosis, resistance is associated with sustained immuneresponse, in direct correlation with cure, whereas susceptibility is parallel to depressed immunity,leading to a progressive disease and, ultimately, death. Among other differences that characterizethese profiles, the lesions present in the omentum show remarkable differences between the twostrains: progressive increase of the areas in the lesions where Paracoccidioides brasiliensis (Pb)yeast cells can be identified in the susceptible mouse strain (B10.A) in contrast to the initial increasebut further stabilization of lesional areas containing fungi in the resistant mouse strain (A/J). Thearchitecture of the lesions of susceptible and resistant mice shows, respectively, loose and compactgranulomas with different cell populations, cytokines and estracellular matrix (ECM) components.Animals of either strain inoculated with a low virulence Pb isolate originated residual granulomasafter some period of infection.The components of the ECM participate in the formation and organization of the granulomas andalso of the mechanisms of tissue response developed in the attempt to contain infectious agentssuch as Pb. The balance between synthesis and degradation of ECM components in the granulomascan be influenced by diverse cytokines and enzymes that can be produced in the process of tissuedestruction and repair (necrosis and fibrosis) occurring during the infection. The disbalance of these80

mechanisms can lead to permissiveness for the dissemination of Pb while its efficient modulationcan contain the fungus and contribute for the restoration of the homeostasis of the organism.The fundamental role of gamma-interferon (IFN-γ) in anti-fungal protective immunity as well as its insitu expression and antifibrotic effects in fungal granulomas have been reported. Osteopontin (OPN)is a glycoprotein of great importance in cell migration and activation, and also in the tissue remodelingobserved in granulomatous lesions. Lately OPN has been considered as an important cytokinefor the development of cellular immunity and induction of granulomatous responses. This chronictissue inflammatory process can be associated to a balance between synthesis and degradation ofthe ECM components, This phenomenon involves the participation of proteolytic enzymes, such asmatrix metalloproteinases (MMPs), particularly MMP-9 and MMP-2, and also products secreted bythe fungus, that altogether can regulate the processes of tissue destruction and repair, as well asthe different cellular events occurring during the granulomatous infection with Pb.In the present work, we studied by immunohistochemistry the expression of IFN-γ, OPN andMMP-9 in the granulomatous response developed in susceptible or resistant mice at 15 and at 120days after infection (dai) by the ip route with Pb and compared with the pattern of lesions produced.Gelatinolytic activity of MMP-2 and MMP-9 was evaluated by zymography and nitric oxide (NO) andOPN levels were studied, respectively, by Griess Reaction and ELISA.IFN-γ (+) lymphocytes were detected at the periphery of granulomas in both mouse strains andquantitative analysis showed a significant increase in such cells in compact granulomas of A/J miceat 120 dai compared to 15 dai with the highly virulent Pb isolate. The number of positive cells incompact granulomas of A/J mice at 120 dai was significantly higher than that observed in loose,multifocal granulomas of B10.A at the same time.We found marked OPN expression in macrophage-like and multinucleated giant cells in the centerof the lesions and also in a lesser degree in the extracellular matrix. At 15 dai with the virulentfungal isolate, OPN-positive cells were more numerous and intensely immunostained in the loosegranulomas of B10.A than in the compact granulomas of A/J; at this time high fungal load and lowNO levels were observed in B10.A mice. At 120 dai, increased OPN expression was detected incompact granulomas of resistant mice; at this time point these animals showed higher NO and lowerfungal load than susceptible mice. Residual lesions associated to low OPN expression, high NO andcontrol of fungal dissemination were observed in both mouse strains at 120 days infection with theslightly virulent isolate.At 15 dai, all infected mice with the highly virulent Pb isolate showed disorganized granulomasand particularly three gelatinase bands with molecular weights of 106.2 kDa, 95.2 kDa and 64.2 kDa,probably corresponding respectively to pro-MMP-9 and to the active forms of MMP-9 and MMP-2.The pro-MMP-9 bands were more intense when compared to active-MMP in B10.A, whereas thesame intensity of the two forms was detected in A/J. These bands were not observed in non-infectedcontrol mice. At 120 dai, B10.A showed loose lesions with many viable fungi and had a similar patternof MMP-9 and MMP-2 activity as detected at 15 th day, whereas A/J developed compact lesionsenclosing fungi with altered morphology and also produced MMP-9 and MMP-2 bands similar tothose detected at 15th day, with more pro-MMP-9 activity and less MMP-2 activity. In the infectionwith the slightly virulent Pb, MMP-9 and MMP-2 activity was observed in both mouse strains at 15dai and no gelatinase bands were observed at 120 dai. Addition of metalloproteinase inhibitors,EDTA and 1.10-phenanthroline completely abolished gelatinase activities, confirming the presenceof MMPs. Expression of MMP-9 was observed in mononuclear cells in MGCs and also in fungal cellsin omentum of infected mouse of both strains.The present work suggests that OPN may be associated with more severity in an early phase ofPb infection and with a degree of control of progressive infection and that OPN may exert anti-inflammatoryeffects by inhibiting the expression of NO These data strongly suggest that OPN is involvedin granuloma formation, contributing to its development and organization pattern, depending onthe resistance pattern of the mouse strains and also on the virulence of the P. brasiliensis isolates.Also, this study demonstrates for the first time the presence of MMPs in paracoccidioidomycosis,suggesting their influence in the organization pattern of granulomatous lesions and in the fungaldissemination.81

Pulmonary fibrosis in an experimental model of paracoccidioidomycosis:Modulation through therapeutic interventionDamaris E. Lopera 1 , Tonny W. Naranjo 1 , Andres F. Zuluaga 2 , Jose M. Hidalgo 3 , JairoPatiño 3 , Roberto Jiménez 1,4 , Lucia R. Díaz-Granados 5 , John J. Duque 6 ,Angela Restrepo 1 and Luz Elena Cano 1,71 Grupo Micología Médica y Experimental,Corporación para Investigaciones Biológicas, CIB, Medellín – Colombia.2 Grupo GRIPE, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia.3 Departamento de Radiología, Hospital Universitario San Vicente de Paúl, Medellín-Colombia.4 University of Nebraska, Lincoln, Nebraska, USA.5 Facultad de Medicina, Universidad Pontificia Bolivariana, UPB, Medellín, Colombia.6 Departamento de Patología, Facultad de Medicina,Universidad de Antioquia, Medellín, Colombia.7 Grupo de Microbiología Molecular, Escuela de Microbiología,Universidad de Antioquia, Medellín, Colombia.e-mail: lcano@cib.org.coBackground: Fibrosis, a condition resulting from progressive chronic diseases, does not have an effectivetherapy and, consequently, it gradually diminishes the functionality of the affected organ sometimesreaching an irreversible stage. Patients with paracoccidioidomycosis (PCM) often develop pulmonaryfibrosis and exhibit important respiratory limitations, even if an adequate course of therapy with Itraconazole(ITZ) is given; the latter effectively controls the active stages of this mycosis but does not hinderlung fibrosis. A previous retrospective clinical study evaluated chest radiographs from 47 ITZ-treatedpatients with prolonged post-therapy follow-ups and reported that, at diagnosis, infiltrative lesions wereobserved in 93.6% of patients and fibrosis in 31.8%. Later on, at the end of the observation period, noneof these patients had cleared; on the contrary, fibrosis had developed de novo in 11 patients (25%), sothat by the end of the follow-up period this residual process was seen in 53.2% of patients. Due to thishigh frequency of fibrosis in patients with PCM, it appears desirable to count with new therapies capableof avoiding and/or controling fibrosis.Aims: Based on these clinical findings, we carried out a study in an experimental model of PCM inorder to evaluate by radiologic (tomography), immunologic (cytokines dosing) and histologic analysesthe effects of two combination therapies (ITZ+pentoxifylline or ITZ+prednisolone) on the progression ofthe mycosis and on the development of pulmonary fibrosis.Material and methods: Male BALB/c mice (n=10 animals/group/evaluation period) were intranasally(i.n.) infected with 4x10 6 Paracoccidioides brasiliensis (Pb) conidia and distributed in seven experimentalgroups: (i) non-treated, (ii, iii, iv) treated for 8-weeks with ITZ alone or in combination with Prednisolone(PD) or Pentoxifylline (PTX), initiating the treatment before fibrosis consolidation (4 th week). Groups(v,vi,vii) were treated in the same manner as the last three groups but beginning treatment when fibrosiswas already present (8 th week of infection). Negative controls were inoculated i.n. with PBS. The follow-upbegan before inoculation and continued monthly until the 16 th week. At each time period, n=10 animals/group were subjected to conventional high resolution computed tomography (HRCT), a non-invasivetool for the in vivo monitoring the course of the lung’s parenchymatous lesions, and then sacrificed withhalf of them (n=5) being used for immunological assays and the other half for histologic studies of theirpulmonary tissues. We determined by ELISA the levels of TNF-α, TGF-γ, IL-13, IL-1β, IFN-γ and of PGE2in lung homogenates supernatants. The histopathologic changes occurring in the pulmonary tissue wereevaluated by two independent pathologists who determined the lung area presenting granulomatous,inflammatory response (H&E) and registered the appearance (thin or thick) of collagen and reticulin fibersscoring their relative increase in relation to controls as 0=no changes, 1=minor, 2=moderate or 3=majorchanges. Fibrosis was defined as the observation of thick collagen fibers around inflammatory lesionsin the presence of reticulin fibers whereas incipient fibrosis was taken as presence of only thin fibers.Two-way ANOVA was used to determine differences among groups.Results: Initially, we standardized and adapted the conventional HRCT technique using two groups82

of mice (PBS-untreated as negative controls and Pb-infected untreated as positive controls). For thispurpose we used a multi-slice CT-scanner Lightspeed (General Electric, EU of 16 channels) for humanpatients, to characterize and monitor the course of the lung’s parenchymatous lesions, pulmonary density(pδ, expressed as Hounsfield units, HU) and volume alterations. We observed that the major changesoccurred in the upper lung fields where the Pb-infection induced peri-bronchial and parenchymatousconsolidations that increased significantly (p≤0.001) the pδ in those zones (-263.2±29.6, -191.2±25.1,-269.5±43.5 and -356.5±33.6 HU for 4, 8, 12 and 16 weeks post-infection, respectively) with respectto pδ of negative controls that remained constant around -432.7±24.2HU. Additionally, multiple peripheralnodules were noticed in 60% of the mice at the end of the follow-up. In sharp contrast, the groupstreated with ITZ alone or in combination with PD or PTX normalized the pδ with values similar those ofthe negative controls.Histopathologic analyses showed that Pb-infection induced a significant (p≤0.001) increase of theperi-bronchial granulomatous inflammatory response that involved 10%-35% of the lung’s area, withvalues of 10±7, 22±15, 20±11 and 15±10% at 4, 8, 12 and 16 weeks, respectively. In contrast, after onemonth post therapy, groups (ii) (iii) and (iv) showed a significant decrease (p≤0.01) of the areas exhibitinginflammatory response (9.3±3%, 4±1.5% and 1.8±0.6% for ITZ, ITZ+PD and ITZ+PTX, respectivelyat 8 th week) and also at the end of the follow-up (3.5±1, 2.5±1.2 and 1.3±0.6%). When therapy had beenstarted 8-weeks post-challenge, ITZ alone also reduced significantly (p≤0.05) the inflammatory area afterfour-week of treatment (11±3% Vs 20±11); however, such a reduction was more significant (p≤0.001)with both combination therapies (6.7±3%).Eighty percent of the Pb-infected mice showed incipient pulmonary fibrosis after 4 weeks post-infection;later on (8–12 weeks), this process progressed to well-established fibrosis in 60% of the mice andreached 100% at 16-weeks. In group (ii) at 4-weeks post-treatment, a significant reduction (p≤0.05) inthe score assigned to thin reticulin and collagen fibers deposition was observed along with a score=0for thick fibers (p≤ 0,05). Consequently, at 16-weeks, 80% of these mice had incipient fibrosis, none(0%) had well-established sequelae while 20% had no sings of fibrosis. Notably, ITZ+PD and ITZ+PTXwhen initiated at the 4 th week, were more efficient in diminishing fibrosis than ITZ alone; at the end of thefollow-up period these therapies avoided either form of fibrosis in 60% and 80% of mice, respectively,in contrast with 20% when ITZ was given alone. The remainder mice presented incipient fibrosis. Group(v), presented a significant reduction (p≤0,05) in the score assigned to thin reticulin and collagen fibersdeposition. This effect was observed after 2 months, whereas combination treatment in groups (vi) and(vii) induced the same effect from the first month of treatment. Additionally, ITZ-therapy started at 8weeks, was unable to completely prevent the progress to severe fibrosis and at the end of the followup,40% had incipient fibrosis, 20% had severe fibrosis and 40% of the mice had no apparent fibrosis.Instead, combination treatments, prevented the appearance of severe fibrosis in groups (vi) and (vii). Theimmunological assays showed that Pb-infection induced in the lungs a significantly (p=0.01) increasein pro-inflammatory cytokines (TNF-α, IL-1β) and in PGE2, although each molecule presented differentkinetics. TNF-α levels were higher during all observation periods (p=0.001), IL-1β levels increased untilthe 8-week and then decreased to reach normal levels while and PGE2 increased only at 8 th -week postinfection.Pro-fibrotic cytokines (IL-13, TGF-β also increased but only at 8-weeks post infection. IFN-γlevels did not experience significant changes during the studied periods. All therapies normalized levelsof all cytokines studied and, also of PGE2, except for TGF-β. This molecule, in contrast, increased significantly(p=0.05), especially in groups that received combination treatment.Conclusions: These preliminary results suggest that in Pb-infected mice: 1) the progression to severepulmonary fibrosis can be avoided by early ITZ treatment; 2) ITZ+PD and ITZ+PTX started after 4 weeksof infection, avoided either form of fibrosis in 60% and 80% of mice, respectively and in contrast with20% when ITZ was given alone, and 3) Even when the combination therapies were started during thechronic periods of infection (8 weeks), they also avoided the development of severe pulmonary fibrosis.These experimental results open an important and interesting possibility for avoiding severe fibrosis inthe human patients by the early administration of the combination therapies proposed.Financial support: Colciencias (Project No. 2213-04-16439), Corporación para Investigaciones Biológicas(CIB), University of Antioquia, Radiology Department of Hospital Universitario San Vicente de Paúl.83

Immune reactive cells in skin lesions of patientswith paracoccidioidomycosisCarla Pagliari 1,2 , Naiura V. Pereira 1,2 , Maria I.S. Duarte 1 and Mirian N. Sotto 2 .1 Departamento de Patologia e 2 Departamento de Dermatologia,Faculdade de Medicina da USP, São Paulo - Brasil.e-mail: cpagliari@usp.brThe study of cutaneous lesions in PCM is highly important considering the frequent involvement ofskin in the disease. The skin is considered an immune organ because several of its componentsplay a role in immune response, such as dendritic cells, keratinocytes, macrophages, mast cells andendothelial cells. We studied two populations of dendritic cells in PCM skin lesions. The first onewas the Langerhans cell (disclosed by anti-CD1a antibody) which is known as antigen presentingcell in many cutaneous diseases. The PCM skin lesions were classified, according to characteristicsof granulomatous response, as G1 (well-organized granuloma), G2 (poorly-organized granuloma)and G3 (both kind of granuloma in the same lesion). The Langerhans cells were distributed evenlythroughout the epidermis of all lesions, but displayed an evident decrease in size and number in G1and G2. In G3 this cell population was similar to normal skin control group. These data led to thesupposition that Langerhans cells could be deactivated by products of P. brasiliensis as an escapemechanism, or the cells could suffer phenotypic alterations and not express CD1a antigen in G1 andG2 lesions. The other group of dendritic cells studied was Factor XIIIa+ dermal dendrocytes (FXIIIaDD). These cells were hypertrophic, abundant and distributed around the granuloma in all threegroups. G3 presented the higher number of these cells. Through a double immunostaining techniquewe detected the P. brasiliensis in the cytoplasm of such cells. This result indicates that FXIIIa DD playa role as phagocytic cells in PCM. FXIIIa DD due to their constitutive immunophenotype could act asantigen presenting cells in PCM. For a better understanding of the difference in cutaneous granulomaformation, we studied some cytokines related to Th1 and Th2 pattern. G1 was characterized by thepresence of a high number of IFN-gamma expressing cells. G2 lesions exhibited a high number ofcells expressing IL5 and IL10. G3 presented an equal number of both kinds of cytokines. TNF-alphaexpressing cells were detected in all groups, and in higher number in G3. Some cells expressingthis cytokine were dendritic and observed around the granuloma. This finding suggests that theycould be FXIIIa DD taking part in the granuloma formation. By double immunostaining technique wedemonstrated that mast cells, present in PCM lesions, express IL10. This finding suggests that mastcells could play a role as IL10 producing cells in cutaneous lesions of PCM.84

Symposium6Fungal cellular biology Aspects

Immune recognition of the Candida cell wall: The taste of a fungusNeil A.R.GowSchool of Medical Sciences, University of Aberdeen,Institute of Medical Sciences, Aberdeen AB25 2ZD, UKCandida albicans is the most common agent of life-threatening human disease due to a fungus. Theouter layer of the cell wall of C. albicans is heavily enriched in glycosylated proteins that is the immediatepoint of contact and interaction with the human host. The inner cell wall layer contains thetwo structural polysaccharides, chitin and β-1,3 glucan to which the mannoproteins are attached. Wehave constructed a series of mutant strains with alterations in C. albicans O- and N-linked mannansand used these to explore the role of the glycans on fungal pathogenesis. In a collaborative projectalong with M. Netea and B-J Kullberg in Nijmegen we then used a combination of defined mutantsof the pathogen surface and in pathogen pattern recognition receptors along with receptor-blockingagents to explore how C. albicans is recognised by the innate immune system. Cytokine productionby human mononuclear cells or murine macrophages was markedly reduced when stimulated byC. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediatedby mannose receptor protein binding to N-linked mannosyl residues, and Toll-like receptor 4 bindingto O -linked mannosyl residues. Residual cytokine production was mediated by recognition of β-1,3glucan by the dectin-1/TLR2 receptor complex. In conclusion, recognition of C. albicans by monocytes/macrophagesis mediated by three recognition systems each of which senses a specific layerof the C. albicans cell wall. Recent advances will also be described on how Candida is recognisedby DC cells and macrophages and how differences between fungal species are detected by cellsof the innate immune system. The integrity of the cell wall and the action of cooperative bindingto multiple PAMP molecules are also important in determining the nature of the innate recognitionmechanism. In addition new evidence will also be shown that chitin in the cell wall plays a role ininnate immune interactions.Cell signaling in morphogenesis and virulence of P. brasiliensisLarissa FernandesInstituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil.e-mail: larissaf@unb.brSignalling pathways are commonly used by an extensive array of biological molecules to regulatevarious cell processes including growth, differentiation, and proliferation. Signalling systems in fungialso mediate cell polarity, mating, and pheromone control, and hence play a role in determiningcell morphogenesis. Pathogenic organisms sense and respond to the harsh conditions imposed bythe host, and do so by activating the conserved components of signalling pathways that culminatein the expression of genes responsible for virulence and differentiation of the pathogen in order tosurvive and establish disease. Even though many virulence factors have been reported in severalfungi, all signalling events and cascade components that elicit the activation their virulence factorsremain unknown.Transcriptome analysis and reverse annotation revealed several components of signalling pathwaysin P. brasiliensis known to regulate cell events, such as morphogenesis and virulence. Pathwaysidentified are i) mitogen-activated protein kinases (MAP Kinases) that control cell integrity, cell87

wall construction, mating and osmo-regulation; ii) the cAMP/PKA system, which regulates fungalmorphogenesis and virulence; iii) the Ras protein, a small GTPase involved on the cross-talking ofcascades; iv) calcium-calmodulin-calcineurin, which controls cell survival under oxidative stress, hightemperature, and membrane/cell wall perturbation; and v) TOR – the target of rapamycin pathway,controlling cell growth and proliferation. These components were found to be similar to those of otherpathogenic fungi such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans(Felipe et al., 2005 and Fernandes et al., 2005).Post-transcriptome studies using array technology revealed the transcriptional modulation of manycomponents of signalling cascades of P. brasiliensis. Among 2,583 genes modulated during the differentiationfrom mycelium to yeast, Nunes et al. (2005) identified PKA, G protein α and β-subunits,calmodulin and calcineurin as positively regulated. In addition, Bastos et al. (2007), on evaluatingearly morphogenesis of P. brasiliensis transition, detected a variety of signalling components that aremodulated, such as the histidine kinase (drk1), a conserved dimorphism related kinase that is verywell known in other dimorphic fungi (Nemecek et al., 2006); a MAPKinase; the β-subunit calcineurinand others. Considered as a whole, such studies reveal the ability of P. brasiliensis to modulatesignalling pathways to sense the environment and induce morphological changes accordingly.Experimental studies on components of signalling cascades are still scant on P. brasiliensisliterature. The evidence that cAMP, an intermediate molecule of the PKA system, has a role in thedimorphic transition that is closely related to virulence was first reported in 1985 by Paris and Duran,who observed that exogenous cAMP inhibits rather than induces the process of filamentation in thispathogen. Recently, Chen et al. (2007) cloned and analysed PKA/cAMP signalling components inthe course of the morphological switch from mycelium to yeast. Furthermore, Carvalho et al. (2003)characterized calmodulin, which regulates the protein phosphatase calcineurin. Inhibitors of thecalmodulin pathway impaired the transition from mycelium to yeast cells in P. brasiliensis. In orderto understand the main role of Ras-GTPases in this pathogen, Fernandes et al. (2008) identifiedand characterized two ras genes: ras1 and ras2. As these genes are involved in cell morphogenesisand differentiation, response to heat shock and nutrient deprivation, we decided to evaluate thetranscriptional response of ras1 and ras2 during the temperature-dependent dimorphic switch frommycelium to yeast, during heat shock at 42 ºC and during in vivo macrophage infection. Both geneswere down-regulated inside macrophages upon infection and only ras1 expression decreased at42 ºC. In contrast, ras genes did not show any transcriptional variation during the differentiation.The fact that Ras proteins are attached to the membrane via farnesylation prompted the use of afarnesyltransferase inhibitor to investigate the importance of that process to vegetative growth anddimorphic transition. Farnesylation blockage interfered with vegetative growth of yeast cells, stimulatinggerminative tube production even at 37 ºC. During yeast to mycelium transition the inhibitorincreased filamentation in a dose-dependent manner, indicating that impaired farnesylation favoursthe mycelium form of P. brasiliensis. These data strongly suggest that ras genes are closely involvedin the morphogenesis of P. brasiliensis.The data from in silico analyses and these first experimental studies on signalling components ofP. brasiliensis indicate that this fungal pathogen has co-opted conserved pathways to regulate theappearance of morphotypes required for its virulence, as have the other pathogenic microorganisms.Despite the scarce information about the mechanisms and regulatory connections that controlsignalling cascades, those studies promoted an improvement of our understanding of the plasticityof P. brasiliensis in response to its microenvironment. Certainly, that information will serve to clarifyhow this fungus orchestrates signalling networks to promote morphogenesis, pathogenicity and theonset of disease. Besides, understanding signalling events should enable the development of newdrugs and therapies against this pathogen.88

Paracoccidioides brasiliensis conidia to yeast transition:Analysis of certain genes involved in the processAna M. García 1, 2 , Orville Hernandez 1,3 , Angela Restrepo M 4 , Juan G. McEwen 1,51 Unidad de Biología Celular e Inmunogenética,Corporación para Investigaciones Biológicas (CIB), Medellín, Colombia.2 Escuela de Ciencias de la Salud, Universidad Pontificia Bolivariana (UPB), Medellín, Colombia.3 Departamento de Biología, Universidad de Antioquia. Medellín, Colombia.4 Unidad de Micología Médica y Experimental,Corporación para Investigaciones Biológicas (CIB), Medellín, Colombia.5 Facultad de Medicina, Universidad de Antioquia Medellín, Colombiae-mail: agarcía@cib.org.coBackground: Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM),is a thermodimorphic fungus. The mycelial form produces asexual conidia capable of undergoingthermal transition to either the yeast or the mycelia forms. Dimorphism constitutes not only P. brasiliensismain physiological characteristic but also represents a crucial feature when considering fungaladaptation to its main host, man, since the infective propagules (conidia or mycelia fragments) mustconvert into yeast cells in order to survive and proliferate in tissues for such host.The ability to differentiate morphologically from mould to yeast, or from yeast to mould is one featurethat links many fungal pathogens. In P. brasiliensis diverse virulence factors have been describedwith thermal dimorphism being identified as one of the most important features. At present, thismorphological differentiation has commanded attention for its putative impact on the pathogenesisof invasive fungal infections. Thus, transition appears to be a clue step in the pathogenesis of thedisease and, consequently, constitutes a suitable target for hindering the infectious process.Until recently, little was known about the molecular machinery involved in P. brasiliensis morphogenesisand on the genes specifically involved in fungal transition. In 2005, however, Nunes et al.used broad range approaches to study the M-Y transition process by microarray technology andBastos et al.(2007) constructed a M-Y transition process EST library while Garcia et al (submitted)worked in the C-Y transition process. The later study is the first attempt to correlate P. brasiliensisC-Y morphological changes with concomitant gene expression modifications. These studies havepinpointed genes involved in diverse mechanisms probably essential to this adaptation process.Garcia et al work (submitted) detected certain genes of interest associated to various cellularpathways known to be relevant to transition, such as the following:• Cholestenol delta isomerase (also known as emopamil binding protein) a gene involved in theergosterol pathway and essential for plasma membrane sterol building during growth and transition,as well as representative gene in this process.• Flavoprotein ubiquinone oxidoreductase (ETF-QO) a protein belonging to the electron-transfersystem that plays an important role as an essential component in fatty acid metabolism. Apparently,it is an exclusive protein not detected in other libraries but in the fungus Yeast and C-Y transitionprocess.• Putative response regulator receiver SKN7p a protein belonging to the transmembrane signal-transductionmechanisms most commonly seen in bacteria and fungi, in the “two-componentsystem”. This protein is associated with oxidative stress tolerance and is a transcriptional factor for89

heat shock proteins, processes of huge importance during transition especially when occurring inthe host environment.• Guanine nucleotide binding proteins, important members of the signal-transduction machinery.Alpha and beta subunits have been described before in the Yeast and Mycelia libraries and theirimportance in thermal transition has been described, but the Gamma subunit is being described forthe first time in the C-Y library.Protein tyrosine phosphatase-Pyp, a phosphatase that regulates numerous biological processess byhydrolyzing the protein-associated phosphate monoesters (dephosphorylation), which participatein regulation of cell cycle, growth and proliferation, as well as in cytoskeletal integrity. This gene maybe involved in regulation of the cellular and cell remodeling cycles induced by the stress responseduring the the thermal change to which conidia are submitted during their transition to yeast cells.Recently, the complete genome of three P. brasiliensis isolates has been published in the web andthe annotation of one of them, strain pb03, reveled at least the 90% of all expressed proteins.Aims: Analyze some genes involved in the P. brasiliensis conidia to yeast (C-Y) transition process,based on the knowledge revealed by publication of the fungus’ whole genome, by The BroadInstitute.Methods: We used as probes partial sequences of certain genes integrated to the C-Y EST libraryin a search conducted with the aim of finding complete gene sequences in the Pb03 annotated genome(including introns and exons, as well as expected mRNA products). These sequences weresearched for and found in the data base. PCR primers were designed to amplify in strain ATCC60855 (the same used for construction of the C-Y EST library), the longest fragment possible andthen comparing its sequences with those previously described in the Broad Institute data base, toconfirm the existence of each gene.Results: Search for the complete sequences in P. brasiliensis strain Pb03 annotated genomeresulted in the finding of ORFs (Open Reading Frames) for most of them; complete sequences withintrons and exons were also found. Similarity values were high, between 90 to 100%, with an E valueequal to 0.0 for all of them, so that the probability of at random results was zero, confirming theexistence of these genes in P. brasiliensis. PCR amplification and sequencing of the following geneswas possible:a Cholestenol delta isomerase, a Flavoprotein ubiquinone oxidoreductase (ETF-QO),a putative response regulator receiver SKN7p (annotated as transcription factor prr1), a Guaninenucleotide binding protein gamma 13 (annotated as a predicted protein), a putative protein tyrosinephosphatase (Pyp1). among others, These findings confirm the existence of these genes in P.brasiliensis strain 60855. Clustal W showed the highest similarity between the sequences of thesegenes in strain 60855, as well as in strains with known genomes (Pb01, Pb18 and Pb03).Conclusions: The genes coming from the specific C-Y transition library and their presence in thegenome as confirmed in the Pb03 complete genomic sequence and also, in strain ATCC 60855 PCRamplification, confirm the special connotation of our previous work. As stated, a description of some P.brasiliensis genes obtained from conidia, the infectious propagules causing paracoccidioidomycosis,were shown to correspond to functions related to their transitional process.At the moment and with encouraging results, qPCR is being standardized to estimate the relativeexpression of some of these genes during the C-Y process with emphasis in the differences intheir expression when compared to the Mycelia and Yeast forms; these studies may reveal possiblecandidate genes useful for genetic manipulation of P. brasiliensis. For instance, generation of stablemutant knockouts in transition-related genes, plus modulation of their expression levels using RNAinterference (RNAi), could be fundamental tools in the understanding of their role in the dimorphicprocess itself and/or their relevance as virulence factors.90

CDC42p controls yeast-cell shape and virulencein Paracoccidioides brasiliensisAgostinho J. Almeida 1 , Celia Cunha 1 †, Belem Sampaio-Marques 1 †, Jenny A.Carmona 1 , Agostinho Carvalho 1 , Iran Malavazi 2 , H.Y. Steensma 3 ,D.I. Johnson 4 , E. Logarinho 1 , Cecilia Leão 1 , Gustavo H. Goldman 2 ,A.G. Castro 1 , Paula Ludovico 1 , Fernando Rodrigues 11. Life and Health Sciences Research Institute (ICVS),School of Health Sciences, University of Minho, Braga, Portugal.2. Faculdade de Ciências Farmacêuticas de Ribeirão Preto,Universidade de São Paulo, Brazil.3. Institute of Biology, Leiden University, Leiden, The Netherlands.4. Department of Microbiology and Molecular Genetics,University of Vermont, Burlington, Vermont, USA.† These authors contributed equally for this work.e-mail: ajalmeida@ecsaude.uminho.ptPrevious work in our laboratory The multiple budding nature of Paracoccidioides brasiliensisyeast cells has been suggested to follow alternative control mechanisms during cell growth.As the Rho-like GTPase Cdc42p is a pivotal molecule to establish and maintain polarized cellgrowth, we evaluated the role of this protein in the polymorphic morphology and the virulenceof the yeast-form of this pathogenic fungus.The isolated PbCDC42 gene functionally complements ∆cdc42 S. cerevisiae and triggers aloss of spatiotemporal control of cell division in a temperature-dependent manner.By using antisense technology we knocked-down PbCDC42 in P. brasiliensis yeast cellsshowing that, despite it does not eliminate the multiple budding phenotype, it promotes amore organized and controlled cell growth by decreasing cell size and the typical polymorphismof wild-type cells.Moreover, an 88% decrease in the expression levels of PbCDC42 significantly increasesphagocytosis and abrogates virulence of this dimorphic pathogenic fungus in a micemodel.To the best of our knowledge, we provide for the first time genetic evidences that establish thecentral role of polymorphic morphologies in the pathogenesis of P. brasiliensis, thus openingnew therapeutic targets for the treatment of paracoccidioidomycosis.Acknowledgments:Almeida, A.J., Sampaio-Marques, B. and Carvalho, A. were financially supported by a fellowship(SFRH/BPD/33035/2006, SFRH/BI/15406/2005, and SFRH/BD/11837/2003).The work was mostly supported by a grant from FCT, Portugal (POCTI/ESP/45327/2002)and partially by FAPESP, Brazil.91

Differential analysis of extracellular vesicles and ofpossible cell wall-associated proteins in isolatesof Paracoccidioides brasiliensisRosana Puccia1Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP, São Paulo, Brasile-mail: rpuccia@unifesp.brEvidences accumulate on the existence of a vesicular transport mechanism of molecules from theintra to the extracellular milieu in fungi and parasites. These vesicles are apparently similar to exosomes,which are derived from the exocytic fusion of multivesicular endosomes with the cell surfacein mammalians. Here we describe the isolation of exosome-like vesicular structures from supernatantfluids of Paracoccidioides brasiliensis yeast cells. Pb339, Pb18 and Pb3 were cultivated in definedmedium enriched or not with fetal calf serum and membranous vesicular structures were isolatedfollowing ultracentrifugation (100,000g). We are showing some microscopic, antigenic and enzymaticfeatures of these fractions. We are also presenting preliminary data on the identification of differentiallyexpressed cell wall-associated proteins when comparing Pb18 and Pb3 isolates, yeast phase,cultivated in F12/glucose medium supplemented or not with fetal bovine serum (FBS). This workcounts on groups of collaborators from UNIFESP, USP, UFRJ, in Brazil, and from the University ofTexas in El Paso, USA.Financial support: FAPESP, CNPq, and NIH (grant # 5G12RR008124).92

Symposium7The endemic Mycosesunder considerationby public health Agencies

The evolving public health importance of mycotic diseasesTom ChillerNational Center for Zoonotic, Vectorborne and Enteric DiseasesCenters for Disease Control and Prevention, Atlanta, Georgiae-mail: tnc3@cdc.govThe emergence of new fungal pathogens and the resurgence of mycotic diseases that had previouslybeen uncommon is a serious and growing public health problem. Defining public health’s rolein mycology is a unique challenge.Fungal diseases are generally not notifiable to public health; therefore very few hard data areavailable on incidence and prevalence. Data that exist are fragmentary and there are serious deficienciesin surveillance systems and our ability to detect infections. Given these challenges, it is difficultto understand the true burden of disease which leads to a lack of awareness by the general public.There many other competing public health priorities to consider and in order to get the resourceswe need to address fungal diseases; we need to initiate global efforts to estimate the burden of thesediseases. We must start by improving surveillance of these infections and then work to develop costeffective intervention strategies.Epidemiologic issues in invasive fungal infections:A United States perspectiveMary E. BrandtMycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta GA USAe-mail: mbb4@cdc.govFungal infections are among the most difficult to diagnose, identify, and manage in the immunocompromisedpatient population. Candida is the fourth leading cause of nosocomial bloodstream infectionin the United States, with an estimated national cost of $200-800 million US dollars per year. Althoughits incidence is decreasing in the United States and Europe, Candida albicans remains the leadingcause of candidemia in the United States. Non-albicans species, particularly C. glabrata, are seenincreasingly more frequently in some patient populations. The proportions of these species vary indifferent geographic regions of the United States. In general, Candida albicans, tropicalis, and parapsilosisremain susceptible to most azole antifungal agents. Candida glabrata remains a species ofconcern due to its ability to acquire azole resistance quickly, with about 10% of incident C. glabrataisolates resistant to fluconazole. Candida krusei remains a species of low prevalence and inherentresistance to azole antifungals. Invasive pulmonary aspergillosis (IPA) remains a disease impactingtransplant patients, occurring in approximately 6-11% of allogeneic stem cell transplants, 6-13% oflung transplants, and 1-6% of heart and liver transplants in the US. The TransNet consortium (TransplantAssociated Infection Surveillance Network) is a group of 25 transplant institutions in the USformed to conduct surveillance for invasive fungal infections in transplant patients, to determine riskfactors for these infections, and to assess the impact of prevention programs in these patients. Theincidence and timing of IPA after transplant has been studied in this network. The incidence of IPA95

after stem cell transplant (HCT) is 1.7% for all types of HCTs. The timing of IPA varies as well, withabout half of cases occurring after 90 days after transplant. Aspergillus fumigatus (43%) remains themajor agent of IPA followed by Aspergillus flavus and niger (8% each). Aspergillus terreus (5%) is aspecies of concern due to amphotericin B resistance. The 90-day mortality of IPA in HCT patients is50%. The incidence of IPA in solid organ transplant patients is 0.7% by 12 months post-transplant.Some studies have reported an increase in the incidence of zygomycotic infections after transplant.The TransNet network is studying this question. It is not clear whether this phenomenon is due toincreased survival after transplantation, or due to an increase in prophylaxis with azole drugs, whichhave limited activity against zygomycetes.Paracoccidioidomycosis in Brazil:Surveillance and control ProgramMaria Adelaide MillingtonHead of the Systemic Mycoses ProgramSecretariat of Health Surveillance, Ministry of Health, Brasília,Brasil.e-mail:adelaide.millington@saude.gov.brThe Secretariat of Health Surveillance, Ministry of Health from Brazil is structuring (initiating) theProgram for Surveillance and Control of Systemic Mycoses to determine the magnitude of theseinfections in the country.The Program mainly aims to estimate the prevalence and geographic distribution of Paracoccidioidomycosis(PCM), Histoplasmosis, Coccidioidomycosis and Cryptococcosis. Also other objectivesare: to estimate the mortality rates of each of these diseases, to make early diagnosis and timelyand properly handle (management) of the cases, to detect and control outbreaks, through adoptionof prevention and control measures, to support and promote the scientific-technical development inthe area, providing for the network of health services new diagnostic tools, treatment and control ofthese diseases.The Program will initiate the efforts with Paracoccidioidomycosis (PCM) because it is an indigenousmycoses, endemic in Brazil and represents an important Public Health problem by its high potentialdisability and premature deaths it causes, if not appropriately diagnosed and treated. Studying the period1980-1995, an author showed an annual mortality average rate of 1487 deaths/10 6 of inhabitants,and shows that the PCM is the eighth cause of chronic disease mortality, greater than leishmaniasismortality and the highest rate among the systemic mycoses. Most cases have been reported in thesouth, southeast and center-west of Brazil where It is endemic among the populations of rural area.PCM has been reported in areas of recent colonization most subject to deforestation, as in parts ofthe Amazon Region, where can be considered an emerging systemic mycosis.PCM is caused by the dimorphic fungus Paracoccidioides brasiliensis, that appears to be predominantlyin the chronic form in adults and in the acute or subacute forms when it affects children,adolescents and young adults. The PCM is acquired through inhalation of fungal particles dispersedin the air, from sites with contaminated soil.In Brazil, the systemic fungal infections are not reportable diseases, so they are not object ofroutine epidemiological surveillance and only in the states of Paraná, Rondônia and more recentlyMato Grosso do Sul, the PCM is a reportable disease. In these states the health care is organized,with reference services well defined, and it is subsidizing the Program in other Brazilian states.96

Several difficulties have been identified in the care process, ranging from the clinical and laboratorydiagnosis to thel availability of specific medication, which often makes the adherence to treatmenthard to do .Then, we don’t know the real magnitude of PCM, where are the endemic areas, the prevalencerates. The data of incidence and morbidity available today are based on limited reports of clinicalcases and epidemiological investigations.Since 2005, the Ministry of Health, through the Department of Health Surveillance has invested indesigning and implementing the Program for Surveillance and Control of PCM, developed in partnershipwith experts from Teaching and Research Institutions in health.Among the strategic measures for the structuring the Program, these are highlighted:• Development of a surveillance system to know the epidemiological situation, development ofcase reporting, research, control measures.• Organization of the healthcare network, with basic care units and reference hospitals and standardizationof clinical management and provision of medications.• Organization of the Public health laboratories, with adequate infrastructure and equipment,development of technical standards for collecting, packaging and transportation of samples, standardizationof diagnostic methods and acquisition of laboratory supplies;• Training of human resources - physicians, radiologists, pathologists and laboratory techniciansThe surveillance system will be implemented gradually, in eight (8) of the 27 states, which presentshigh prevalence rates of PCM, and have some organization to attend these patients.The activities already taken were the training of technicians of the Public Health Laboratories inmycological and serological diagnosis, standardization of treatment, and the acquisition and provisionof drugs - Itraconazole, Sulfamethoxazole + Trimethoprim for injection, conventional and liposomalAmphotericin B. It is worth to emphasize that all the standardization were based on Brazilian Consensuson Paracoccidioidomycosis prepared by the Brazilian Society of Infectious Diseases, BrazilianSociety of Tropical Medicine and São Paulo Infectious Diseases Society.The main activities planned for 2008 are highlighted below:• Establishment of a technical advisory group for the various systemic mycoses.• Preparation and publishing of clinical and epidemiological protocols for the deployment of surveillancein the states of Paraná, São Paulo, Rio de Janeiro, Minas Gerais, Mato Grosso do Sul, MatoGrosso, Goiás and Rondônia.• Standardization of epidemiological research and information system• Definition of reference health services at the various states• Training of human resources (doctors and technicians surveillance and Lab.)• Drafting Technical Laboratory Manual for diagnosis of Systemic Mycoses• Distribution of the Antigen of P.brasiliensis to standardize diagnosis• Making available Technical Advisors to the states involved• Facilitating technical cooperation with other countries for improving diagnoses97

Symposium8Clinical and epidemiologicalConsiderations on theendemic Mycoses

The impact of histoplasmosis in HIV-infected patientsJohn R Graybill 1 , Eduardo Arathoon 2 , Blanca Samayoa 2 .1. University of Texas Health Science Center, San Antonio, TX USA,2. Clinica Familiar Luis Angel Garcia, Guatemala Citye-mail: jrgraybill@aol.comHistoplasma, Coccidioides, and Paracoccidioides are genera with pathogenic species sharing manyfeatures, including infection by inhalation, dimorphic morphology, a protective response dependenton T lymphocytes, generation of antibodies which are useful for diagnosis but not protective, andthe propensity to disseminate in situations where T cell mediated immunity is depressed such asin HIV infected patients. However, of these Histoplasma capsulatum has the greatest impact andParaccidioides brasiliensis the least impact in HIV infected patients. Reasons are complex, unclear,and may relate at least partly to the likelihood of exposure to environmental organisms, and to theimmune status of the host.Most patients with histoplasmosis acquire the infection late in the course of HIV disease, whenimmune responses are severely depressed. The illness may take the form of progressive primarydisease with widespread dissemination following on the heels of primary infection. However, subjectsinfected years before are also at risk for H capsulatum to escape immune confinement as their HIVprogresses. In the early years of the HIV pandemic, dramatic increases of histoplasmosis occurred inthe endemic regions of the United States. However, cities like San Antonio, Texas, formerly thoughton the periphery or west of the endemic region, and bothered by very few cases of primary disease,experienced dramatic increases in histoplasmosis. San Antonio (and other cities) is now thought wellwithin an enlarged Histoplasma endemic zone in the USA. In the 1980’s and early 1990’s, hospitalssuch as ours increased from scattered cases to as many as 20-25 cases per year. Histoplasmosisoften appeared late in a difficult course of poorly effective HIV treatment. However, with the adventof HAART in the United States, our case rate decreased by more than half, to 5-7 cases per year.Further, most patients with histoplasmosis now present at their initial evaluation, and are then foundto be HIV positive. Diagnosis is often made by bone marrow aspirate, sometimes also positive bloodor sputum smears, and confirmed by positive urine and/or blood antigen measurements beforecultures turned positive. At present, effective treatment consists of induction with amphotericin B(commonly liposomal amphotericin B) for treatment of the most seriously ill patients, followed by aswitch to itraconazole. In the less seriously ill, itraconazole might be started initially. Fluconazole hasbeen effective in some patients, but the emergence of resistance has made it a second line agent.Perhaps not surprisingly, MIC values, which increased in patients failing fluconazole, also rose tovoriconazole. There have been some patients successfully treated with voriconazole, but this maynot be wise. Posaconazole, much more like itraconazole, appears effective in vitro (including thevoriconazole resistant isolates) and clinically in a limited salvage experience. Histoplasmosis is nowa pathogen of intermediate importance in AIDS patients in the USA.This situation in the United States is not mirrored in the third world, where access to HAART islimited, where it is not possible to perform rapid diagnosis using an antigen test, and where tuberculosisaccompany histoplasmosis in >5% of cases, and where the histoplasmosis remains common.Diagnosis of cryptococcosis is straightforward, whereas diagnosis of histoplasmosis must rely onclinical suspicion confirmed by cultures (which even with the lysis technique which require more thana week, during which the patient may succumb) or identification by histopathology. Optimal culturemethods are not available in many settings, and histopathology and fungal stains may not be usedaggressively. Small countries such as Guatemala (population 13 million) have about 30 cases ofhistoplasmosis diagnosed per year, versus 45 with cryptococosis and 70 with tuberculosis in one101

clinic with a total patient population of 700-800. There few experts in HIV associated diseases, andmany patients likely undiagnosed. Late presentation and delay of diagnosis may account for mortalityover 30%. Latin American constitutes far the greatest reservoir for histoplasmosis, but regions suchas South Africa also have marked increases of patients infected with H capsulatum, which remainsa major pathogen.What can be done to ameliorate the problems of histoplasmosis in Latin America? First, more rapiddiagnosis of HIV infection and initiation of HAART will prevent the deterioration of immunity whichfacilitates histoplasmosis. Second, Histoplasma antigen detection must be readily available in centrallaboratories of countries in endemic regions. A test is under development at the CDC in Atlanta, Georgia.Aggressive biopsies and cytochemistry are also important. Third, every patient with suspectedtuberculosis or histoplasmosis should be investigated for both infections. The use of rifampicin intreatment of concurrent tuberculosis might necessitate extended treatment with amphotericin B inpatients with dual infection. Fourth, in these usually very ill patients, treatment should be initiatedrapidly, sometimes empirically while diagnostics are in process. A short course of amphotericin Bshould be followed by change to a triazole as soon as the patient is stable. Finally, as the patientimproves with reduction of viral load and rise of CD4, sudden worsening may be related to the immuneresponse syndrome rather than progressive disease this happens with both tuberculosis andhistoplasmosis. Therefore not rush to change anti-infectives for documented disease until you knowwhat is transpiring.The coexistence of HIV infection and paracoccidioidomycosisRoberto MartinezFaculdade de Medicina de Ribeirão Preto, Universidade de São Paulo-Brazile-mail: rmartine@fmrp.usp.brThe first cases of paracoccidioidomycosis associated with acquired immunodeficiency syndrome werereported in 1989. Since then, approximately 150 cases of the co-infection Paracoccidioides brasiliensis– HIV were related. The co-infected patients lived mainly in the Southeast and in the Center-Westregions of Brazil, but a few reports described cases in other Latin America countries.A large number of patients acquired both infections in the metropolitan area of Ribeirão Preto, SãoPaulo state, Brazil, where paracoccidioidomycosis is hiperendemic. The 73 patients diagnosed from1989 to 2007 with P. brasiliensis – HIV co-infection, compared with the number of cases of AIDS inthe same period, resulted in a prevalence estimated at 1.7%. Most co-infection patients had a numberof blood T CD 4+lymphocytes lower than 50/µL, and 20/25 (80%) had more than 30,000 copies/mL ofthe HIV1 RNA in the plasma. Only 59% of the 27 patients who were aware of being infected by HIVwere taking antiretroviral drugs, and 41% were in prophylaxis with trimethoprim-sulfamethoxazoleagainst Pneumocystis jiroveci.The P. brasiliensis – HIV co-infected patients were compared with 1000 cases of endemic paracoccidioidomycosiswith respect to epidemiological and clinical data. Mean age was lower in theco-infected patients (33,4 x 41,9 years old), but a nonsignificant difference was observed in relationto gender. Among HIV-infected patients 27% had some type of occupation in the rural area versus53% of the endemic disease cases.HIV co-infected patients had more disseminated and rapidly progressing lesions of P. brasiliensisthan the individuals HIV non-infected. They had more fever, skin lesions, lymphadenopathy, andsplenomegaly. Pulmonary involvement were very common in both cohorts. Lesions of the mucosaof the mouth, nose or larynx were more frequent in patients with endemic paracoccidioidomycosis.102

The conjunct data from 66 cases previously reported of paracoccidioidomycosis associated withHIV/AIDS showed a great frequency of lesions suggestive of fungal hematogenic dissemination, inparticular to lymphnodes (52%), spleen (18%), and skin (41%); pulmonary involvement was observedin 55% of the cases.The diagnosis of the fungal infection in HIV-infected patients was confirmed by histopathologicalexamination (94%) and by isolating P. brasiliensis (42%), particularly from a skin lesion biopsy. Thesera of the co-infected patients had lower reactwity in a test for detection of anti – P. brasiliensisantibodies. The initial treatment of paracoccidioidomycosis – HIV/AIDS patients consisted in generalof HAART and amphotericin B, trimethoprim-sulfamethoxazole or fluconazole. In comparison toendemic disease no difference in response to treatment was observed but late relapses were morecommon in the HIV-infected patients. In some reports the lethality attributed to paracoccidioidomycosisis near to 30%.Paracoccidioidomycosis should be classified as opportunistic disease in HIV/AIDS patients becauseof its clinical and epidemiological characteristics, that are different from clinical forms of theendemic disease.Treatment compliance in paracoccidioidomycosisRinaldo Poncio Mendes 1 , Daniela V. Moris 1 , L.C. Carvalho 2 , E.F. Oliveira 3 ,and Ricardo Cavalcante 1 .1Tropical Diseases Area, Botucatu Medical School,2Biostatistic Department, Biosciences Institute,3Information Technology Service, Botucatu University HospitalSão Paulo State University (UNESP)Introduction. Paracoccidioidomycosis (PCM), a widespread systemic mycosis in Latin America, requireslong treatment in its most frequent clinical forms. Its treatment aims clinical cure, regressionof specific antibodies serum levels to normal and recovery of the specific cell mediated immuneresponse, to reduce long-term sequelae and relapses. Behavioral changes and long-term treatmentcompliance are crucial to reach these goals.Compliance, the act of taking medications as prescribed, is a highly complex clinical behaviorand remains problematic for evaluation. Medication nonadherence can take many different forms,leading to therapeutic failure.Methods. Patients with confirmed PCM by the identification of typical Paracoccidioides brasiliensisyeast forms and/or specific serum antibodies by double agar gel immunodiffusion test, with bothacute/subacute or chronic form, assisted at the Tropical Area Service – Botucatu Medical School– UNESP, were eligible for the study. Overall, 97.4% of the 77 eligible subjects who were contactedagreed to participate and answered a specific questionnaire.The following compliance indices were evaluated: 1) Global attendance compliance (GAC) – percentageof presence in the appointments in a period; 2) Individual clinic attendance compliance(ICAC) – percentage of appointments of each patient in a specific period; only patients with at least10 attendances in the Paracoccidioidomycosis Outpatient Service were evaluated; 3) Antifungalcompliance (AFC) – percentage of evaluations with appropriate sulfonamide serum levels (≥70µg/mL in the initial treatment and ≥50µg/mL in the consolidation therapy); only patients receiving cotrimoxazolewith at least 10 sulfonamide measurements during the follow-up period were included; 4)Serological curve (SC) – evaluation of specific antibodies serum levels by agar gel immunodiffusion103

test during the follow-up period; only patients with at least 10 measurements were included; 5) Oneweekself-report compliance (OWSRC) – percentage of drug intake in the last week of treatment; allinterviewed patients receiving antifungal drugs were included; 6) Last appointment self-report compliance(LASRC) – percentage of drug intake in the period between the last two evaluations. Indices80% or higher were considered patient compliance. ICAC, AFC, OWSRC, and LASRC analyseswere carried out according to this cutoff. SC was evaluated as appropriate when descending, orinappropriate when stable or ascending (dichotomous parameter). Compliance was analysed as toclinical form, education level, family help, and alcohol intake. Adherence measures were evaluatedalone and in combination.Unpaired t test and analysis of variance/Tukey test for continuous variables, x 2 and Fisher’s exacttest for proportions, and Pearson’s test for correlations were used, with level of significance set atp0.05).Individual clinic attendance compliance, studied in 132 patients with at least 10 routine appointments,showed a 90.3% average and revealed that 85.6% came to at least 80.0% of these clinicalevaluations. ICAC presented no variation as to clinical presentation: 90.8%±11.9% in the chronicand 88.2%±9.9% in the acute/subacute form (p>0.05).Antifungal compliance, evaluated in 95 patients treated with trimethoprim-sulfamethoxazolecombination, was 42.1%. A positive correlation was observed between antifungal compliance andindividual clinic attendance compliance (r=0.27; p=0.037).Serological curve, studied in 96 patients, showed descending curves in 90.9% of them and stable/ascending in 9.1%. A direct correlation between antifungal compliance and serological curve, i.e.,higher frequency of adequate sulfonamide serum levels in patients decreasing the antibody serumlevels, was observed in this cohort (x 2 =4.6; p=0.032). Average of AFC was higher (p=0.016) in patientswith descending serological curves (66.6±29.6) than those with ascending ones (42.6±35.2).One-week self-report compliance (28 patients) did not correlate with antifungal compliance (p=0.12).Last appointment self-report compliance did not correlate with antifungal compliance (23 patients;r=0.10; p>0.05) but revealed a linear correlation with individual clinic attendance compliance (30patients; r=0.48; p=0.02).Alcohol intake did not interfere with one-week self-report compliance (28 patients; p>0.05) or withserological curve (70 patients; p>0.05). However, individual clinic attendance compliance was mildlyhigher in patients who denied alcohol intake (75 patients; 97.0% versus 93.3%; p=0.04). Educationlevel was in general low, but individual clinic attendance compliance was lower in patients who hadnever attended school (p=0.02). Family help did not play a role in individual clinic attendance complianceand serological curve (p>0.05).Compliance was evaluated in 96 patients combining individual clinic attendance compliance,antifungal compliance and serological curve indices. Three indices were appropriate in 34 (35.4%)patients, two in 46 (47.9%), only one in 14 (14.6%), and none in 2 (2.1%). Thus, 80 of 96 patients(83.3%) revealed high compliance.The main causes of failure in antifungal intake were blood tests (24.3% of the cases), forgetfulness(16.2%), adverse effects to medication (16.2%), lack of medication (16.2%), and alcohol intake toavoid an alcohol-antifungal compound mixture (13.5%).Discussion. Noncompliance to treatment is a main obstacle to cure and may result in diseaseprogression and resistant microorganisms. Despite the importance of compliance in clinical careand research, rigorous study of measurement techniques is uncommon. Evaluation of complianceto long-term treatment is complex and does not have a gold standard parameter. Patient self-reporthas been used extensively to assess medication-taking, but it tends to overestimate compliance.104

Although drug serum levels are an objective measure of drug intake, they provide only a snapshotof behavior and can be affected by factors other than adherence.Indices obtained by using a questionnaire and laboratory charts, with drug measure (sulfonamideserum level) and antifungal effect (serological curve) were compared. Our study revealed correlationbetween individual clinic attendance compliance and serological curve. However, descending serologicalcurves were observed in patients with lower sulfonamide serum levels. These findings couldbe explained by intake failure in the appointment day, when fasting patients are submitted to bloodtests. Thus, patients are told to take only the medication before having their blood draw. It can also behypothesized that adequate sulfonamide serum level was overestimated. Alcohol intake, family helpand education level played a mild role in treatment compliance. The high values of combined complianceindices provided a more accurate evaluation, and demonstrated high quality of medical care.Guidelines for the treatment of paracoccidioidomycosisFlavio Queiroz-TellesServiço de Infectologia, Hospital de Clinicas, Universidade Federao do Paranáe-mail: queiroz.telles@uol.com.brEvidence-based guidelines for the management of patients with Paracoccidioidomycosis (PCM)were prepared by an Expert Panel of the Brazilian Societies of Infectology and Tropical Diseases.The guideline was published two years from now (Ver. Soc. Bras. Med. Trop. 39(3):297-310, 2006)aiming to help the clinicians to manage most of the clinical forms of the disease. PCM is consideredto be the most important systemic endemic mycosis affecting South America. The prevalence of thisinfection varies among regions of endemicity, and it is estimated that the incidence of PCM rangesfrom 1 to 3 cases per 100,000 inhabitants in regions where the disease is endemic. However, becausethe reporting of PCM is not compulsory, it is difficult to determine accurately the number ofpeople affected by the disease. Without treatment, the natural evolution of the disease is typicallydeath. In patients with immunosuppression, such as AIDS, the infection can progress to full-blowndisseminated disease.P. brasiliensis differs from other pathogenic fungi, because it is a very sensitive organism whenexposed to antifungal drugs; even sulfonamides can inhibit its growth. A large therapeutic armamentariumis available for patients with PCM.Several classes of antimycotic drugs have been used in the treatment of this systemic mycosis,including sulfonamides (sulfadiazine, sulfadoxine, sulfamethoxypyridazine, cotrimazine, and cotrimoxazole),Amphotericin B, azoles (ketoconazole, itraconazole, fluconazole and voriconazole), andterbinafine. The cure rates achieved with these various drugs have ranged between 69% and 100%.To date, no antifungal resistance has been demonstrated in PCM.Itraconazole is considered the standard treatment for mild and moderate clinical forms of PCM.This recommendation is based on the results from a non comparative study of 47 patients, most ofwho had multifocal disease and received itraconazole for a mean duration of 6 months. A scoringsystem indicated a favorable response in 43 patients (89%). In another trial, 90 patients with provedPCM were treated with cotrimoxazole or itraconazole and evaluated for clinical and microbiological;response, safety and tolerability. The final analysis showed that itraconazole is more effective and bettertolerated than cotrimoxazole (trimethoprim-sulfamethoxazole) in the treatment of mild to moderatedPCM. In addition, the mean duration of treatment with itraconazole is significantly shorter (7 monthsversus 24 months, p

and AIDS, as well as on the itraconazole dose (100 mg/day vs. 200 mg/day). Conventional or lipidformulations of amphotericin B are indicated to treat severe disseminated cases in patients who areintolerant to other agents or have refractory infections. Besides its toxicity, the rate of relapse withamphotericin B is generally higher than with itraconazole (20%–30% of cases).After clinical and microbiological diagnosis, all patients must be evaluated for clinical classification(acute/sub acute, chronic or residual form) and graded to severity (mild, moderate and severeclinical form). Itraconazole is used at the daily dose of 200 mg during 6 to 12 months, according tothe clinical, mycological, serological and radiological response. The second therapeutic option iscotrimoxazole (160 to 240 mg of thrimetoprim combined with 800 to 1,200 mg of sulphametoxazoleBID) from 12 to 24 months. Young children can be treated with cotrimoxazole oral solution (08 to 10mg per kg of thrimetoprim combined with 40 to 50 mg of sulphametoxazole BID). For patients withneurologic involvement, cotrimoxazole or fluconazole can be used although the former compoundwas used in most the reported cases.Among the new triazoles, posaconazole and isavuconazole were not investigated in human PCMto date. But voriconazole a second generation triazole was compared to itraconazole in terms ofefficacy and safety in a controlled randomized clinical trial. Fifty tree patients with proved mild tomoderate forms of PCM were included. The response rate among treatment-evaluable patients was100% for both group of patients and no relapses were observed after 8 weeks of follow-up. Bothdrugs were well tolerated but adverse events were slighter more frequent in the voriconazole arm.Because voriconazole is a fluconazole molecular derivative, it presents excellent CNS penetrationand may be an adequate antifungal drug for neuroPCM.106

Historical accountsHistoplasmosisHistoplasmosis: Discovery in PanamaMarion Clarke MartinMicrobiology Department, Faculty of Medicine,University of Panama--Panamae-mail: marionma38@yahoo.comThe discovery of histoplasmosis in Panama is closely linked to the history of the Republic ofPanama, since the first case of this disease, worldwide, was diagnosed by Samuel T. Darling in1905 in a Martinique man employed in the construction of the Panama Canal. Panama becamean independent republic after separation from Colombia in 1903, and in 1904, a second attemptat building the Canal was made by the United States of America, after the French failed at thisendeavor in the nineteenth century. Since the discovery of this disease on the Isthmus of Panamaa little over a hundred years ago, cases spanning the complete spectrum of histoplasmosis -fromthe asymptomatic infection through a mild flu-like illness to full-blown, disseminated disease asdescribed in the first diagnosed case -have been documented in the population of all ages, frominfants to senior citizens, and even in a dog (1945).During the eight months following the first case, Darling diagnosed two additional cases. Twentyyears later (1926), the fourth case worldwide was identified in Minnesota, USA, and in 1934Robert De Monbreun isolated the etiologic agent, the dimorphic fungus Histoplasma capsulatum.Christie and Peterson demonstrated for the first time in 1945 that skin sensitivity to histoplasminwas common in Tennessee in persons with lung calcifications and negative tuberculin reactions;and by the following year, 1946, it was evident that histoplasmosis was most likely a very common,usually asymptomatic infection in endemic areas, and not a rare, deadly illness.In light of the foregoing, investigators in Panama began work in this field, and Mastellari, a specialistin chest diseases, demonstrated (1948) histoplasmin sensitivity prevalence in 48% of childrenbetween 5 and 14 years of age. Various studies published between 1950 and 1964 haveestablished the prevalence of histoplasmin sensitivity at 50 – 85% of the Panamanian population;and later work done by us has generally confirmed these data. However, in the 80s we noticed107

that the prevalence of histoplasmin sensitivity appeared to be dropping, and documented that itwas in the range of 11 to 18% of populations of children and young adults, respectively. Noneof these subjects had a diagnosis of the illness histoplasmosis; their skin reactivity was just afortuitous finding.Having stated that the infection was largely asymptomatic among Panamanians, it is appropriateto point out that an active search for acute, primary cases of the disease was carried out between1954 and 1957 at Gorgas Military Hospital in the former Panama Canal Zone, and based on clinicalhistory, careful physical examination, histoplasmin test and chest x-ray a series of 45 caseswas identified. This work confirmed what we now take for granted -clinical findings and x-raysare not specific for histoplasmosis, since these signs are also found in tuberculosis, influenza,pneumonia due to Mycoplasma or viruses, and other respiratory infections.In addition to the three original cases of disseminated disease diagnosed by Darling in threeWest Indian immigrants contracted to work on the Canal enterprise, this form of the illness hasalso been found in Panamanian mestizos, native Indians and whites, usually the very young andthe aged; and AIDS patients, in whom it is considered to be the reactivation of a dormant infection.Unusual manifestations of histoplasmosis in patients in Panama include perianal abscessin a 40-year-old man, generalized lymphadenopathy, broncholithiasis, bronchial histoplasmosis,histoplasmomas, chronic cavitary pulmonary histoplasmosis, laryngeal histoplasmosis.In Panama, in the 50s and 60s there was a great deal of research relating to the Histoplasmaagent, which gave rise to several isolations of the fungus from soil in bat caves, from the air insuch caves, and from bat tissues. This work was largely carried out by the Middle America ResearchUnit (MARU) located in Ancon on the Canal Zone, with the cooperation of Panamaniancollaborators and scientists.Histoplasmosis in Panamanians most often presents as a benign, flu-like illness, usually diagnosedlong after the initial infection during skin testing surveys with histoplasmin. Skin reactivity to thisantigen has diminished markedly (11 to 18%) from the values obtained in surveys conducted upto the 1970s (in some communities as high as 100%), at least as regards the population residingin the big cities, eg Panama and Colon; probably the prevalence of infection continues to be highin rural areas, which have not experienced the changes found in the large cities. We postulatethat this dramatic reduction in the prevalence of histoplasmin sensitivity may be explained bythe following:1. Bat-proof houses are now being built, so that no guano is available to favor the multiplicationof H. capsulatum in the environment.2. Big-city dwellers have small plots of land, so that patios are reduced in size, therefore.3. No chickens are raised, whose droppings facilitate the survival and reproduction of H. capsulatum, and4. Few or no old trees can be found in which bats can sleep during the day and therefore contaminatethe space with their guano.108

Histoplasmosis in the United StatesJohn R. GraybillUniversity of Texas Health Science Center, San Antonio, TX USAe-mail: jrgraybill@aol.comJust like Coccidioides, Histoplasma was first appreciated in Latin America, and just like Coccidioides,Histoplasma had an initial false identity as a protozoan. And just like Coccidioides, Histoplasma wasinitially considered a very rare pathogen. And just like Coccidioides, initial Latin American discoverieswere followed by discovery in the USA. But not very rapidly…it took more than 30 yearsafter Darlingfor a dedicated pathologist, WA DeMonbreun, to recover H capsulatum from blood and tissue, andto culture the yeast phase using enriched medium. In the next 2 decades investigators at a singleuniversity elucidated most of what we know about the epidemiology of histoplamosis and its clinicalmanifestations. Primary disease and the sequellae of abnormal healing, acute, subacute, or chronicdissemination, chronic pulmonary disease, and hypersensitivity reaction were all relatively rapidlydefined. By the 1960’s histoplasmosis was appreciated as a common infection and a well knowndisease in states bordering the Mississippi River Valley. Major outbreaks in Indianapolis also contributedgreatly to our understanding of primary infection and successful host response. In the 1980’s,HIV/AIDS provided a number of hosts with very depressed immune defenses. AIDS patients, infectedwith either fewer organisms or with less virulent organisms, and extending the appreciated endemicrange of histoplasmosis. With recent control of AIDS in the USA, the relative importance of this diseasehas decreased. However, histopasmosis remains prominent throughout Latin America, whereHistoplasma is still a major pathogen of patients with AIDS, and still carries a high mortality.109

Symposium9New Approaches to theclinical and laboratory Diagnosisof systemic Mycoses

The value and the promise of molecular biological toolsin the diagnosis of aspergillosis and candidiasisBeatriz L. Gómez, Ph.DMycotic Diseases Branch,Centers for Disease Control and Prevention,Atlanta, GA USAe-mail: bgomez@cdc.govImprovements in medical intervention have led to significant increases in cases of invasive fungalinfections (IFIs) in a growing immunocompromised population. Early diagnosis is still a challengefor clinicians, with delays or missed diagnosis increasing mortality. Diagnosis of most IFIs isdifficult, particularly for invasive aspergillosis, with classical culture techniques providing poorsensitivity. Radiological investigations are a useful adjunct but provide nonspecific and transientresults, and histopathology, despite providing definitive evidence of IFI, cannot always identifythe causative organism. Therefore, indirect diagnostic means have been developed.Whereas detection of antigen has gained enough reliability to be included in consensus criteriafor IFI (European Organization for Research and Treatment of Cancer, Mycoses Study Group,EORTC/MSG), PCR has not, despite more than 10 years of molecular-based diagnostics developmentsand publications.The main reason is the absence of consensus technique(s) and of commercial kits. However,this limitation has become less strict as a result of emerging real time PCR assays and thestrong efforts by different groups working together to standardize these methods. Additionally,kits are starting to be commercially available. Well-conducted comparisons should facilitateselection from the various available DNA extraction techniques, targets for amplification withinthe fungal genes and primers, and from the clinical specimens relevant to the given infection.For the more promising choices, sensitivity and specificity of the PCR test can be assessed inwell-designed prospective studies. Efforts are underway towards this important goal. It is alsoimportant to regularly run quality controls to ensure reliability of the tests.Real-time PCR is a quick method for quantifying how much target sequence was present atthe start of the reaction. Second and very important for the routine laboratory, real-time PCRdecreases enormously the risk of false positive results. Indeed, there is no post-amplificationhandling, thus minimizing the risk of contamination from the environment. Another advantagefor routine laboratories is the fast turnaround time of less than 3 h. Additionally, fully automatednucleic acid extraction techniques eliminate the need for vacuum pumps, centrifugation, or othermanual steps that may result in cross-contamination.In this review the benefits and limitations of recently published assays are compared, and therequirement for both international standards and a consensus protocol that is sensitive enoughfor diagnosis of invasive fungal infections will be discussed.113

Molecular diagnostic approaches inparacoccidioidomycosis and histoplasmosisR.M. Zancopé-Oliveira 1 , C.M.A. Soares 2 , T. C.V. Rezende 2 , K.P. Castro 2, , M.A. Almeida1 , A.J.Guimarães 1 , H.L.M Guedes 3 , C.V. Pizzini 1 .1Laboratório de Micologia-Setor de Imunodiagnóstico,Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz.Avenida Brasil 4365, CEP 21045-900 Rio de Janeiro, RJ, Brasil.Tel. (55-21) 3865-9640 e-mail: rosely.zancope@ipec.fiocruz.br2Laboratório de Biologia Molecular, Universidade Federal de Goiás, Goiânia, Goiás, Brasil.3Laboratório de Bioquímica de Proteínas e Peptídeos, Instituto Oswaldo Cruz,Fundação Oswaldo Cruz, Rio de Janeiro, RJ, BrasilEndemic mycoses can be challenging to diagnose, and accurate interpretation laboratory data isimportant to ensure the most appropriate treatment for the patients. Although the definitive diagnosisof paracoccidioidomycosis (PCM), and histoplasmosis (HP) the most frequent endemic mycoses inthe world, is represented by direct diagnosis performed by micro and/or macroscopic observationof the fungi, the isolation of the etiologic agents is time-consuming and lacking in sensitivity. Theaccepted limitations associated with classic techniques for the diagnosis of endemic mycoses havelead to the emergence of many non-culture-based methodologies. Analysis of the humoral immuneresponse to fungal infections has played, and will continue to play, a key role in their diagnosis andimmune surveillance. Although rapid genome detection methodologies, such as PCR, are beginningto replace immune assays for the diagnosis of systemic mycoses, they are not suitable for allapplications, especially the surveillance of the immune status of human populations. Several immunologicalmethods have proven useful for the endemic mycoses diagnosis; however, they are oftentime consuming and many lack sensitivity and specificity, partially attributed to the use of unpurifiedantigenic complexes fromeither whole fungal cells or their culture filtrates, which give cross reactivity.Emphasis has shifted, however, to clinical immunoassays using highly purified and well-characterizedantigens including recombinant antigens. Here we shall review the limitations of current conventionaltools for measuring immune responses and outline principles for the design and production of noveldiagnostic reagents for Paracoccidiodomycosis and Histoplasmosis. Methods for the production ofdiagnostic antigens by recombinant systems are described and their relative merits and disadvantageswill be discussed.Histopathology of paracoccidioidomycosis in the postgenomic era:Reflections and perspectivesHenrique Leonel LenziLaboratory of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.The histopathologic study of Paracoccididodes brasiliensis, considered now as a member of thenew family Ajellomycetaceae (Untereiner et al. Mycology 96:812-861, 2004) is presenting somenew challenges: 1) Different isolates have revealed a great degree of genetic variability in thepathogenesis; 2) The existence of four different phylogenetic species recently demonstrated (S1,PS2, PS3 and Pb01-like) (Matute et al. J Clin Microb 44: 2153-2157, 2006; Mol Biol Evol 23: 65-73, 2006; Carrero et al. Fungal Genet Biol 45: 605-612, 2008); 3) Pb exhibits epigenomic changesdepending on the habitat (Silva et al. Microbes Inf 10: 12-20, 2007; Derengowski et al. Med Mycol46: 125-134, 2008); 4) Limitations of the traditional static granuloma concept, not considered as adynamic lesion, that follows the properties and mechanisms of adaptative complex systems; 5) The114

need to consider the P. brasiliensis-host interactions as determinant of a new system (cohabitome),deeply dependent on the genetic characteristics of the fungi and of the hosts; 6) The importanceof analyzing the tissue in a three-dimensional perspective and not just in two-dimensional plan;7) The inadequacy of the Euclidean geometry as form of measuring most of the complex biologicalphenomena like the tissue fibrosis; 8) The limitations of the reductionist view of the traditionalimmunology, causing excessive “fragmentation” of the biological phenomena . Based on the aboveconsiderations, the pathology of postgenomic era should apply multidisciplinary approaches, showinga profound integration with Cellular and Molecular Biology through the uses of High resolutionphotonic microscopies (laser confocal and two or multiphotons microscopies), laser microdissectiontechniques and tissue microarrays. The high resolution microscopies allow to do virtualtomographic sections of the lesions, providing a three-dimensional analysis of the cellular andextracellular matrix components, obtaining adequate serial sections to further tissue modeling bydifferent technical procedures. These types of microscopies easily enable multichanel fluorescentanalysis with a very sophisticated system of spectra discrimination and can be applied also insamples obtained by tissue microarray, defining histogenomic and histoproteomic characteristics,contextualizing the genomic and proteomic in the cells and tissues (=morphological localizome).Laser microdissections techniques (laser-manipulated microdissection. LMM) will allow verifyingthe epigenomic expression of the P. brasiliensis according to the “metastatic” localization in differentsites of the organism. In fact, LMM constitutes the best bridge between the Pathology and TheMolecular Biology and can be applied in histological specimens, including the fungi, alive cells (exvivo) and in culture cells, using the following technical preparations: fixed and/or frozen specimens,alive or fixed cells, stained or non stained material. The specimens can be analyzed by bright-fieldor multichanel fluorescence microscopies, including confocal and two photons microscopies. Afterthe laser capture, the DNA can be analyzed by PCR, genetic mutation, single nucleotide polymorphism,fingerprinting, loss of heterozygoty, fluorescent in situ hybridization, DNA microarrays. TheRNA can be detected by mRNA isolation, reverse transcriptase-PCR, expression analysis andmicroarrays. The proteins can be distinguished by two-dimensional electrophoresis (2D-PAGE),surface-enhanced desortion–ionization-time of flight (SELDI -TOF), matrix-assisted laser desortionionization-TOF (MALDI-TOF), IMMUNOBLOT, nano-scale liquid chromatography tandem massspectrometry (nLC/MS/MS) and protein microarrays. The tissue microarrays (TMA) offer the greatadvantage of compare simultaneously, in the same assay, a large number of specimens from differentplaces of the same individual or from several individuals (human or experimental animals).Imaging MALDI mass spectrometry has been successfully used to obtain the distribution of proteinsin thin tissue slices (Stoeckli et al. Nat Med 7:493-496, 2001). This procedure can be expanded byadding a third dimension allowing the 3-D mapping of proteins in specific regions of the interestedtissue by imaging serial sections. The 3-D distribution of targeted proteins can be an important toolfor the understanding of disease. The Pb-Host interactions can be now better studied with somegenetic sophisticated details in vivo using genetic engineering of laboratory animals, obtainingtransgenic, knock-out, knock-in, knock-down (iRNA) animals and animals tagged with fluorescentprotein gene (fluorescent animals).It is important to stress out that most of these referred procedures were not yet applied to theparacoccidiodomycosis histopathology due to a clear dissociation between the molecular andthe morphological studies (the “disease” of the specialization) and due to the very expensivecost of these high-throughput modern technologies. This new approach will require a compulsorycooperative work in national and international levels and the change of the way of thinking aboutthe host-parasite relationship, looking under the perspective of Systems Biology and SystemsPathology, considering that the Pb-Host interaction configures the emergence of a new system,the Pb-Host Cohabitome.115

Clinico-immunological and immunopathological correlations indifferent clinical forms of the paracoccidioidomycosisM. A.Shikanai-Yasuda 1,2 , A. Sadahiro 3 , Pagliari, C 4 ., Duarte, M. I. S. 4 , M. Yoshida 21 Departamento de Moléstias Infecciosas e Parasitárias da Faculdade de Medicina da USP,2 Ambulatório de Micoses Sistêmicas da Clínica de Moléstias Infecciosas e Parasitárias e Laboratório deImunologia (LIM 48) do Hospital das Clínicas da Faculdade de Medicina da USP;Av. Enéias de Carvalho Aguiar, 500. Térreo, Sala 4 CEP 05403 0003 Instituto de Ciências Biológicas, Universidade Federal da Amazônia;4. Depto. Patologia da Faculdade de Medicina da USPe-mail: dmip.shikanai@hcnet.usp.br, lim48imuno@yahoo.com.brAfter inhalation of conidia and their phagocytosis by alveolar macrophages, inflammatory infiltrate withneutrophils, followed by mononuclear infiltrate with macrophages and lymphocytes was observed inexperimental model. On dependence of cellular recruitment and organization of granulomas aroundthe yeast, fungus may disseminate through peribronchial lymphatics. In the majority of the cases thisfungus-host interaction is represented by an asymptomatic infection, as the expression of resistantpole of the infection. Lymphocytes from infected individuals secret gamma interferon in the presenceof P. brasiliensis antigens and positive late hipersensitivity skin test to paracoccidioidin and,more specifically to gp43, is usually associated to the presence of mononuclear infiltrate to in theinfected tissues. Fungal dissemination through phagocytic mononuclear system, compromising thelymphnodes, liver, spleen, bone marrow occurs in the acute phase of the disease, as the result ofimbalance of cytokine secretion, increased levels of IL10, IL4 and depressed levels of IFN gammaproduced by patients´ lymphocytes stimulated by fungal antigens concomitantly to high levels ofanti- P. brasiliensis antibodies. IgG4 and IgG2 were more commonly present in high or moderatelevels in some patients with acute and chronic form, respectively. Lower avidity of anti-P. brasiliensisantibodies was associated to recidives of patients whereas higher avidity was associated with controlof the disease.In the infected tissues, high levels of IL10, TGFβ and FAs L and CTLA-4 expression in T cells correlatedwith loose granulomas unable to avoid fungal dissemination. In chronic cases, reactivationoccurs after a long period of asymptomatic infection in different organs and tissues and was expressedby unifocal or multifocal lesions in the lungs, mucosa, skin, lymph nodes, Central Nervous System,adrenal, bones etc. Different levels of anti-P. brasiliensis antibody and different spectrum of cellularimmunity are correlated with extension of the disease; usually increased levels of IL10 and differentlevels of IFN γ were produced by peripheral lymphomononuclear cells. In parallel, local immunitydepends on the organ and on the period of the disease and the duration of the treatment. Differentexpression of cytokines, chemokines or cellular markers was observed in areas containing necrosis,granuloma or perivascular infiltrate. In human infected lymphnodes, fungi germination in smallgranuloma and in the interior of giant Langhans cells, and CD20, CD4 > CD8 lymphocytes, IFγ andsmall amount of IL4 and IL10 were observed in the granulomatous inflammatory infiltrate. In CentralNervous System, great amount of TNFα is observed around necrotic area, CD34, CD68, NK, CD4were observed in perivascular spaces and TGFβ is associated with fibrogenesis. Skin lesion of HIVpatient with paracoccidioidomycosis show intense germination of fungi, presence of macrophage,NK,TNFα, expression of IL10, IL4 and IFγ, and CD8>CD4 associated to low level of peripheral bloodCD4/µl. In human lesions, regulatory T cells exhibit suppressor activity and produce IL1, TGF β. In thetissues, TGF β may represent regeneration of large areas of necrosis. CCR5 chemokine receptorswere also expressed in the cells of the inflammatory infiltrate (Moreira et al, 2008).116

Lymphomononuclear cells of 29 patients with Paracoccidioidomycosis recognizes at least one of fivepeptides selected with the TEPITOPE algorithm), including the peptide from gp43 at the sequenceposition 180-194 (Iwai et al, 2007). The immunogenicity of forty one synthetic peptides of 43 kDaglycoprotein was also tested in “in vitro” lymphoproliferation assay of mononuclear cells from 44cured patients, and 19 controls. Ten GP43 peptides without the sequence position 180-194 wererecognized by 77.3% cured patients; differences among clinical forms were detected on lymphoproliferativeresponse to P15, P16, P22, P28, P29 and P30; higher levels were more commonly seenin acute cases (Sadahiro, 2007a). Interferonγ and TNFα were preferentially induced by G2 groupof peptides (P6 to P10) whereas IL10 was observed after G6 and G7 peptides stimulation (P26 toP30 and P31 to P35, respectively. Regarding HLA frequency, DRB1*11 allele was associated withunifocal form, localized disease, suggesting its possible influence in the outcome of the host-parasiteinteraction in paracoccidioidomycosis (Sadadahiro et al, 2007b). The inclusion of new immunogenicgp43 peptides may represent a strategy for new therapeutical approach associated with antifungaldrugs aiming to control severe disseminated forms of paracoccidioidomycosis.Financial support FAPESP 02/06481-6Clinical diagnosis in paracoccidioidomycosis:Its differentiation from similar entitiesAngela Tobón, MD.Internal Medicine. Hospital La María andCorporación para Investigaciones Biológicas (CIB), Medellín, Colombia.e-mail:atobon@cib.org.coParacoccidioidomycosis (PCM) is characterized by systemic involvement leading to development oflesions in various organs and sites such as lungs, mucous membranes, skin, intestinal tract, adrenalglands, central nervous system, lymph nodes, liver, spleen and larynx, among others. Such an broadrange of lesions demands from the clinician a high level of suspicion concerning this entity so thatappropriate differential diagnosis, supported by pertinent laboratory tests, may be established thusallowing instauration of prompt specific therapy.Pulmonary manifestations (cough, expectoration, hemoptysis, dyspnea) accompanied by constitutionalsymptoms are common to almost all infectious and non-infectious lung pathologies. In mostcases, these manifestations lead to consider tuberculosis in the first place, a disorder that can besuspected by the presence of apical fibrocavitary lesions revealed by the X-rays and diagnosed bysimple acid-fast bacilli stains. In cases like these but when no acid-fast bacilli are found in respiratorysecretions, it would be advisable to consider PCM especially if chest X-rays reveal mixed infiltrates,predominantly alveolar, located in the central and lower lung fields often respecting the apices. Due tothe high frequency of tobacco smoking among the PCM population, cytology of sputum or bronchoalveolarlavage fluids is recommended to discard lung carcinoma, especially of the bronchioalveolartype, as its radiographic representation may resemble that of PCM.Other mycoses such as histoplasmosis in its disseminated, chronic progressive form frequentlyexhibiting nodular interstitial involvement and apical cavitations accompanied by skin and mucosaelesions, represent a challange to the laboratory personnel who should keep up with the suspicionwhile attempting to confirm it through properly chosen tests due to the fact that the clinical presentationof this disorder is much alike that of PCM.117

Mucosal and skin lesions induced by P. brasiliensis are recognized by their ulcerated and crusty appearence,as well as by their thick, well-defined borders often accompanied by important soft tissueedema. Their localization in the lips, oral and nasal mucosae appear to be characteristic of PCM butthey are not patognomonic as long as other pathologies including histoplasmosis, carcinomatousprocessess and the Kaposii syndrome may give rise to similar lesions. They may also resembleleishmaniosis or sporothrichosis especially when lesions appear in the limbs’ surface.One of the clinical problems of great importance due to its severe prognosis is the involvement ofthe adrenal glands which is as frequent in PCM as in tuberculosis. CT studies evidence hypertrophywith or without calcifications but these observations will not allow a differential diagnosis betweenthe two entities; biopsy and anatomopathologic studies, as well as microbiological and mycologicaltests, are required to confirm the etiology. The observation of concurrent lung involvement and/ormuco-cutaneous lesions may avoid the need of the above diagnostic invasive procedure.Involvement of the CNS by P brasiliensis, seldom recognized clinically but frequently observed atautopsy, is another problems when the specific diagnosis is searched for. The single nodular cerebrallesion, the multiple ring lesion revealed by the contrast medium, as well as the perilesional edema,can be found in diseases as varied as toxoplasmosis, cryptococcosis, tuberculomas, lymphoma andischemic cerebral disease.The temporal profile of the neurologic signs and symptoms, the patient’s immune status and theconcommitance of the general clinical manifestations contribute to orient the diagnosis but only thestudy of the tissues obtained through stereotactic biopsy would allow the establishment of the properdiagnosis. Presence of lung and/or mucocutaneous lesions and reactivity of the patient’s serum infront of the fungal antigen, would also constitute important guides to define diagnosis and allow theinstauration of specific therapy.The study ulcers in the larynx causing severe pain, as well as their presence in the intestinal lumen,the latter accompanied by diarrhea and constitutional symptoms in young adults, may surprise thephysician by revealing the characeristic P. brasiliensis multiple budding cells. These abnormalitiesshould be taken into consideration when defining their cause.Lastly, the presence of lymph node hypertrophy of the various chains, especially of the cervical ones,accompanied or not by liver and spleen megalies, should lead to consider disseminated infectiousor neoplastic disorders presenting common general symptoms that do not allow specific diagnosis.Marked hypertrophy of the cervical lymph nodes can be observed in tuberculosis, lymphoma and inthe juvenile, disseminated form of PCM. Only the judicious use of biopsy materials or of secretionsobtained from such lesions would allow to determine the correct diagnosis.This review pretends to alert the clinician about the systemic nature characterizing PCM, an entitythat presents numerous and varied organic pathologies resembling many other entities that althoughmore frequent in their presentation may not have the reserved prognosis of PCM when its diagnosisand treatment are not promptly undertaken, thus allowing for the development of sequelae, speciallung fibrous scarring.FInancial support: Hospital La Maria, Corporación Investigaciones Biológicas (CIB).118

Symposium10Advances on antifungal Antibiotics

Coccidioidomycosis as seen in a nonendemic areaJohn Bennett,M.D.Laboratory of Clinical Investigation,National Institute of Allergy and Infectious Diseases,Bethesda, Maryland USA.The diagnosis of coccidioidomycosis in the USA is rarely considered outside of the southwestern endemicareas, leading to delays in diagnosis. Acute pneumonia in recent travelers is rarely considered.More serious is the delay in the diagnosis of persistent lesions in the lung, bone and central nervoussystem. Absence of a standard serologic diagnostic test has created a plethora of commercial testswith uncertain and often confusing interpretation Cases referred to the National Institutes of Healthin Bethesda, Maryland show the variety of clinical manifestations of coccidioidomycosis seen outsidethe endemic area, including acute pneumonia, chronic solitary cavities, chronic fibrocavitary diseaseand disseminated infection in bone and central nervous system. Development of syrinx in the spinalcord is a uncommon but dreaded complication of coccidioidal meningitis. Obstructive hydrocephalusresponds to CSF shunting but complicates administration of intrathecal amphotericin B. Voriconazolewas effective in one patient with meningitis but the unpredictable blood levels of this drug present achallenge in seriously ill patients.New and experimental antifungal drugsG. San-BlasInstituto Venezolano de Investigaciones Científicas,Centro de Microbiología y Biología Celular,Apartado 20632, Caracas 1020A, Venezuela.e-mail: sanblasg@ivic.vePreferred antifungals for the treatment of paracoccidioidomycosis are sulfamethoxazol-trimethoprim,itraconazole, amphotericin B. Treatment is lengthy, the drugs may have undesirable side effects,and some are costly. Therefore, the search for more selective and efficient antifungals to treat thisand other mycoses continues.1. Lipopeptide derivatives, new antifungals in clinical useThe fungal cell wall is a unique structure with no equivalent in mammalian cells. Because of that,cell wall glucans and chitin should be specific targets for antifungal action, inasmuch as substancesblocking their syntheses may lead to fragile cell walls and ensuing cell death. One such specific groupof substances is the echinocandin class of lipopeptide antifungals [caspofungin acetate (Cancidas ® ,Merck), anidulafungin (Eraxis ® , Pfizer Inc.), micafungin (Mycamine ® , Astellas Pharma US)]. Theytarget the synthesis of β-1,3-D-glucan synthase (GS), the activity of which is essential for the assemblyof a functional cell wall in many fungi. The enzyme is a multisubunit complex, which includesan integral membrane protein and a regulatory subunit, encoded by members of the FKS and RHO1gene families, respectively. Echinocandins such as caspofungin have activity against importantfungal pathogens, including Candida and Aspergillus spp., a result that prompted FDA authorizationin 2001 for clinical use of caspofungin in the treatment of those candidiasis or aspergillosis cases,refractory to treatment with polyenic or azolic antifungal drugs. However, values for Candida showranges of action depending on the species, e.g., Candida albicans (0.03->16 µg/ml), C. krusei (0.03-2 µg/ml), C. glabrata (0.03-2 µg/ml), C. tropicalis (0.06-1 µg/ml), or C. parapsilosis (0.5-16 µg/ml).Limited in vitro studies indicate that echinocandins have high MICs for zygomycetes, Fusarium spp.,121

and Cryptococcus neoformans. MICs for caspofungin in the pathogenic yeastlike phases of severaldimorphic fungi, fluctuate according to species and strains, e.g., Blastomyces dermatitidis (0.5-8µg/ml) or Histoplasma capsulatum ( Pb300 (35%) > Pb381 (27%) > Pb444 (21%) > Pb377 (17%) (values at 1.0 µgcaspofungin/ml). The mycelial phase, as expected, was highly susceptible to caspofungin, inhibitionfluctuating between 74% (Pb381) and 81% (Pb73). In no case, a 100% inhibition was achieved, evenat concentrations as high as 16 µM caspofungin, when 91% inhibition was the observed value forstrain Pb73, M phase. Scanning electron microscopic observations indicated structural modificationsin the cell walls of both phases as a consequence of caspofungin action. Such results may indicatevariability in P. brasiliensis cell wall composition, particularly with regards to the participation of β-1,3-glucan and the related activity of its synthesizing enzyme (GS). Experiments to solve this biologicalquestion are currently under way. But from a clinical point of view, caspofungin does not appear tobe a promising antifungal for the treatment of paracoccidioidomycosis.2. Azasterols and sterol hydrazones, experimental drugsThe sterol biosynthetic pathway has been largely studied for the search of antifungals. Azoles andallilamines act on differents steps of this pathway. However, they may interfere with similar steps inthe host. Hence, we have undertaken the search for drugs that may act on more specific steps in thesterol pathway. While mammals synthesize C27 cholestane-based members of the steroid family,pathogenic fungi, protozoa and plants require the presence of sterols (typically ergosterol and 24-alkyl analogs) which act as irreplaceable essential growth factors for these organisms. The enzymeresponsible for the addition of these alkyl groups to carbon C-24 and for the regulation of carbonflow in the sterol pathway is the ∆ (24) -sterol methyl transferase (SMT). There is no such enzyme inthe pathway of cholesterol biosynthesis, and thus SMT inhibition should selectively block ergosterolbiosynthesis, affecting only fungal cells, and theoretically bypassing any undesirable blockage in thesynthesis of the host’s cholesterol. The crucial and specific role of SMT has stimulated interest on thebiorational design of SMT inhibitors for potential clinical or agrochemical use as antifungal agents.SMT inhibitors such as azasterols and sterol hydrazones (AZA1-AZA3; H1-H3) have proven highlyeffective as antiproliferative agents against protozoa and some fungi, among them, Paracoccidioidesbrasiliensis. In the presence of AZA-1, a dose-dependent inhibition of P. brasiliensis growth (Yphase) was observed from 0.1 to 5 µM, to reach 100% growth arrest at the latter concentration andabove. AZA-2, instead, was only able to inhibit growth by 60% at the highest concentration used inthese experiments (10 µM). AZA-3 was the most powerful drug, as a concentration of 0.5 µM wasable to completely inhibit fungal growth. Lipid analyses indicated that in control cells, main sterolswere brassicasterol (69.1 %), ergosterol (26.8 %) and lanosterol (4.1 %). On exposure to AZA-1,ergosta-5,7,24(28)-trien-3β-ol (17.1%) and lanosterol (11%) accumulated, while AZA-2 led to animportant accumulation of ergosta-5,7,22,24(28)-tetraen-3β−ol (50.5 %). With AZA-3, instead, animportant accumulation of lanosterol (34.5 %) was observed, as a result of SMT inhibition. Concurrentwith the accumulation of these intermediates, final products of the sterol pathway (ergosteroland brassicasterol) decreased substantially. Similarly, sterol hydrazones (H1, H2, and H3) generateda dose-dependent effect in fungal growth, which were active at nanomolar concentrations, alsorelated to SMT inhibition. These drugs were active in the following sequence: AZA3 > AZA1 > H3≥ H1 >> H2 >> AZA2.Acknowledgement: Merck, Sharp & Dohme (Caracas, Venezuela), for partial support in the caspofunginexperiments.122

Nanobiotechnology: Advances in antifungal therapyA.C. Amaral 1, 2 and Felipe M.S. 11Instituto de Ciências Biológicas, Universidade de Brasília, UnB, Brasília, Brazil2Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, UCB, Brasília, Brazile-mail: amaralandre@yahoo.com.brNanotechnology presents enormous potential to prepare new antifungal drugs. This science worksat 10 -9 part of a meter; in this scale it is possible to design a drug-delivery system to drive the drug tothe infection site or to maintain drug levels at a therapeutic concentration over long periods of time(Nat. Rev. Cancer, 6:688-701). Although AMB is a potent drug to combat pathogens, its use is limitedby severe adverse side effects as fever, anorexia and nephrotoxicity. Lipid-based nanopreparationscontaining AMB represent a promise to minimize the adverse side effects caused by this drug, andthree of these formulations are commercially available for clinical use: Abelcet ® , Amphotec ® , andAmbisome ® . These lipid preparations reduce the nephrotoxicity by its link to plasmatic proteins andcan be safely used in patients with various conditions predisposing to renal impairment (Int. J. Antimicrob.Agents, 27S:S12-S16, 2006). Even improving the therapeutic index of AMB, some drawbacksstill remain, as for example, all of these formulations lack the ability to release AMB at controlledrates and, accordingly, it has to be daily administered. Polymeric controlled release systems offerthe flexibility in terms of architecture and drug loading allowing designing different combination toconjugate drugs and targeting molecules to be released at controlled rates. Polymers used to preparethese nanocarriers can be natural (e.g. chitosan and alginate) or synthetic (e.g. PLGA-poly(lactico-glicolicacid)) and they have been choose due to its biocompatibility and biodegradability. PLGAis a co-polymer formed from lactic (PLA) and glycolic (PGA) acids with the ability to control drugrelease due to its polymeric physicochemical properties (molecular weight, hydrophilicity) and theratio PLA:PGA (Indian. J. Exp. Biol. 38:746-52, 2000). PLGA has presented satisfactory results forthe controlled delivering of different classes of drugs, including for the fungistatic drug voriconazole.It has also been shown to improve drug efficacy in the treatment of experimental candidiasis (Int. J.Pharm. 352(1-2):29-35, 2008). In our laboratory we have design, prepared and tested in vivo somestrategies to develop new antifungal drugs to treat mycosis. Based on the principles of nanomedicine,we have prepared and tested a sustained delivery system to D-AMB within PLGA polymeric blendswhich reduced to one third the number of drug administration (patent filled INPI # 0700446-0, Antmicrob.Agents Chemother. paper submitted). This preparation (NanoAnf) was tested in the murinemodel of chronic paracoccidioidomycosis (PCM), which involve mainly the lungs. Nanoparticles werefunctionalized with dimercaptosuccinic acid (DMSA) that presents preferential lung tropism (J. Magn.Magn. Mater. 293:277-282, 2005). The decrease in the number of administrations was possible asthese nanoparticles were able to carry the total D-AMB dose in a single injection. NanoAnf at 6mg/Kgeach 3 days was able to control the clinical signs of PCM and pulmonary infection, as observed bythe CFU decrease in lungs. Therapeutic effects were comparable to those obtained with D-AMB at2mg/Kg administered once a day. Moreover, NanoAnf was able to prevent body weight loss observedin the D-AMB-treated group during the first month. After two months, the animals presented a slightdecrease on body weight, suggesting a delay on the onset of the adverse side effects as observed inother study with lipid nanoparticles containin D-AMB (Transpl. Infect. Dis. 1(4):273-83, 1999). Othersymptomes, such as piloerection and hypotrihosis observed during long time use of this drug wereprevented by our formulation. Yet, even exceed the initial AMB amount extrapolating tolerable limit,NanoAnf have not caused renal and liver toxicities in the animals, probably because of the AMBgradual release from the nanoparticles and by its site-targeting provided by DMSA in the formulation.In addition, we have investigated others polymeric strategies against fungal infections. One consistedin the combination of an immune protector peptide P10 entrapped within PLGA nanoparticles withantifungal chemotherapy against PCM (paper in preparation). The combining therapy started with afour weekly administration of 20µg of the P10 peptide in complete Freud adjuvant (CFA) or 1µg, 5µg,10µg, 20µg, or 40µg of the P10 peptide within PLGA nanoparticles, and 15mg/kg sulfametoxazoleadministrated daily for 30 days. The antifungal therapy efficacy, evaluated by the fungal burden tis-123

sue assay, revelead that P10-PLGA conjugate were 20-times more efficient when compared withadministration of P10 in CFA during the first 30 days of treatment. PLGA nanoparticle containing 1µgof P10 presented the same adjuvancity on therapy than 20µg of the peptide with CFA. It was markedby an increase on IFN-γ level and by the presence of more compact granulomas in lungs. After 60days from the beggining of treatment regimens, that is 30 days without sulfametoxazole therapy,we observed that 10µg of P10 entrapped within PLGA nanoparticle, such as 20µg in CFA, was efficientto avoid the recidive of the disease. This approach proved to be an efficient aid as adjuvantto sulfametoxazol, a drug responsible for several cases of fungal resistance. Besides improving thesulfametoxazol efficacy, this strategy has been shown to be a safe alternative to use its chemotherapy.Other experiments are in progress to evaluate the antifungal potential of conventional drugs in differentpolymeric nanoparticles constructions against vaginal candidiasis. Our findings indicate thatnanotechniques are powerful tools in the formulation of antifungal agents either by decreasing thenumber of drug administration or by improving the potential of the antifungal agents. In conclusion,nanobiotechnology has contributed to the advances in antifungal therapy. The hybrid conjugates thatcombine synthetic polymer with the conventional drugs can help on fight against the fungal diseases byshortening the find of new antifungal molecules. There is no doubt that these ever more sophisticatednano drug delivery systems will provide the improved treatments to combat the infectious diseases,assuring a safely and less painful therapy. Financial support: CNPq and FAP/DF.The epidemiology of Candida and Aspergillus infections:Clinical and molecular aspectsC.C.KibblerCentre of Medical Microbiology, University College London, UKe-mail: c.kibbler@medsch.ucl.ac.ukThe understanding of the epidemiology of these important opportunistic invasive fungal infections(IFIs) has increased considerably over the last decade. Until recently, most of the available datawere from small, often single centre studies, or largely confined to the US. However, increasingnumbers of studies are being conducted at a national level around the world and the largest studyof candidaemia carried out so far has recently been published under the auspices of the EuropeanConfederation of Medical Mycology and took place in seven countries.These studies are expanding our knowledge of risk factors, prognostic factors and mortality andit is becoming possible to compare incidences across the world. Geographical differences in epidemiologymay be explained by differences in patient populations, different interventions in underlyingdisease and differences in measurement and case definitions. The latter has been addressed to aconsiderable extent by the EORTC/MSG criteria for diagnosis of IFIs and these have recently beenrevised and will be discussed in this presentation.Molecular studies have helped uncover new species responsible for invasive disease and sequencingtechniques used in unravelling mould taxonomy are now finding their way the routine laboratory,where they may be used for identification. An international working party lead by the CDC is now inthe process of setting out guidelines for this approach.Typing methodologies such as multi locus sequence typing are beginning to provide an understandingof the global distribution of Candida species, demonstrating geographical clades, providingevidence of microevolution and allowing transmission routes to be more closely elucidated.A number of different molecular typing methods have shown the wide biodiversity of Aspergillusspecies, although some studies have managed to determine the source of invasive isolates from theenvironment or water supplies.Much of what we have learnt will be invaluable in deciding where to target resources, in conductingclinical trials, formulating risk-reduction strategies and investigating outbreaks.124

Symposium11Agriculture andParacoccidioidomycosis

P. brasiliensis growth and infective propagulaproduction in clayey and sandy soilE. Bagagli 1 , Terçarioli G.R. 1 , Barrozo L.V. 21 Departamento de Microbiologia e Imunologia,Instituto de Biociências, Unesp, Botucatu, SP, Brasil.2 Faculdade de Filosofia, Letras e Ciências Humanas, USP, São Paulo – Brasil.e-mail: bagagli@ibb.unesp.brAlthough the existence of some controversies, including the hypothesis that P. brasiliensis occurs inheterothermic animals from fresh water environments (Conti-Diaz, 2007), several pieces of evidencepoint to the soil as its main saprobic habitat. In such environment, the fungus must develop andproduce the infective propagula, the arthrospores or simply conidia. The influence of soil texture andchemical composition in fungus development has been little understood. For example, in BotucatuPCM endemic area, while infected armadillos occur in places containing both sandy and clayey soiltype (Bagagli et al. 2003), the human infection seems to be more prevalent in regions where clayeysoil is more abundant (Simões et al, 2004).We have then observed experimentally that the fungal growth was similar in clayey and sandy soiltextures, and that no growth occur when the soil composition contained high values of ExchangeableAluminum (H+Al) and, consequently, a low Bases Saturation value (V%). The fungal growth washigher in soil saturated with water than in soil with moderate humidity, determined by its field capacity(FC). There was no growth in low humidity soil, such as in the one that contains only half of its FC. Itwas also observed that some isolates did not produce conidia while in others the conidia productionwas relatively high, mainly when the fungus was grown on relatively poor substrates containing soilextracts, and that this feature (conidia production) could be associated with the genetic group of theisolates (abundant in S1 and rare in PS2 genetic group), according to Terçarioli et al, 2007.In conclusion, the major incidence of paracoccidioidomycosis in clayey soil areas may be due tothe widespread use of this soil type in agricultural activity than sandy soil. High humidity is necessaryfor P. brasiliensis develop in soil. Some soil conditions, in particular those that contain elevatedExchangeable Aluminum (H+Al) may inhibit or limit fungus growth. The intensity of conidia productionamong the isolates in soil extract agar appears to be dependent of its genetic group.Financial support: Fapesp (Proc 06/03597-4)On the relationship between land use and paracoccidioidomycosis:Coffee, sugar cane and other culturesL.V. Barrozo 1 , Santana, M.S. 1 , Gonzalez, C.R. 1 , Mendes, R.P. 2 , Marques, S.A. 2 ,Benard, G. 3 , Bagagli, E. 41 Faculdade de Filosofia, Letras e Ciências Humanas, USP, São Paulo – Brasil.2 Faculdade de Medicina de Botucatu, UNESP, São Paulo – Brasil.3 Faculdade de Medicina, USP, São Paulo – Brasil.4 Instituto de Biociências, UNESP, São Paulo – Brasil.e-mail: lija@usp.brIntroduction and Objectives: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thethermal dimorphic fungus Paracoccidioides brasiliensis. It is currently accepted that the portal arethe lungs and the air-borne propagules of the fungus infects the respiratory tract of humans afterinhalation. It is recognized that the disease occurs more frequently in patients involved with soilrelatedactivities, mainly agricultural workers. Land use has been frequently associated with PCM127

since some agricultural activities produce more aerosols, increasing the opportunity of humaninfection. Nevertheless, to date, it is not clear if some specific cropland increases the risk ofacquiring PCM. Thus, the aim of this work is to compare the areas of the most important landuse types and PCM prevalence in a highly endemic area in Botucatu neighboring, São PauloState, Brazil.Methods: The spatial pattern of the series of chronic cases was studied through the applicationof an inference test for the detection of high and low clusters. Chronic PCM datacorresponded to cases seen in the Infectious Diseases and Dermatology Sectors of the UH-UNESP. Only the cases with residence in the study area (n = 280) were selected. A spatialscan statistic was applied. Cases were assumed to be Poisson distributed, adjusted accordingto age and gender, with constant risk over space under the null hypothesis. The analysiswas set to include up to 50% of the total population at risk and to find clusters of excess andlow risks. Data of area for coffee, sugar cane, orange plantation, reforestation (Eucaliptusand Pinus) and pasture (natural and cultivated) were acquired from the Instituto de EconomiaAgrícola de São Paulo, from 1983 to 2006. Population data per municipality by year wereobtained from the SEADE Foundation. The percentages of each land use type in relation tothe area of the municipality were calculated. Exploratory analyses of the evolution of landuse types were made to observe each culture in the studied period. The mean areas of eachland use type for the high and low clusters were calculated and compared through t test and,in the absence of homoskedasticity, through the non-parametric Welch’s test. Also bivariateregression analyses were performed between prevalence and each percentage of land usefor the 44 municipalities.Results: Three important spatial clusters were found. The first high cluster included thefollowing municipalities: Botucatu, São Manuel, Pratânia, Pardinho, Areiópolis, anhembi, Igaraçudo Tietê, Bofete and, Itatinga (P = 0.0001). This cluster presented a mean incidence of2.9 annual cases/100,000 inhabitants. Cerqueira César comprised another high cluster (P =0.0012), with 5.8 annual cases/100,000 inhabitants. The low cluster included: Espírito Santodo Turvo, Paulistânia, Cabrália Paulista, Santa Cruz do Rio Pardo, Lucianópolis, Águas deSanta Bárbara, Agudos, Óleo, Piratininga, Iaras and Bernardino de Campos (P = 0.0001), with0.6 annual cases/100,000 inhabitants. The comparison of the percentage of areas for eachculture between the high and low clusters showed that sugar cane plantation and reforestationare statistically higher (P

Influence of alternating coffee and sugar cane agriculturein the incidence of paracoccidioidomycosis in BrazilFlavio Queiroz-TellesHospital de Clinicas, Universidade Federal do Paraná,Curitiba, Paraná, BrasilParacoccidioidomycosis (PCM) is a chronic or subacute, granulomatous systemic fungal infectioncaused by the thermally dimorphic fungus Paracoccidioides brasiliensis. It occur in most of the LatinAmerica countries, with higher incidence in Brazil, Colombia and Venezuela, followed by Argentina,Peru, Equator, Uruguay, and Paraguay. So far, no cases have been reported in Nicaragua, Chile,and the Antilles. Sporadic cases in the United States, some European countries, and Japan haveoccurred in individuals who came from endemic Latin American areas. PCM is observed as a naturalinfection mostly in humans but sporadic infections have been reported in wild as in domestic animals.There is uncertainty about the natural habitat of P. brasiliensis but is assumed that the fungus is asoil saprobe. This evidence comes from sporadic fungal isolations from soil samples and armadillosinternal organs.Several investigators have indicated that PCM is more prevalent among rural workers engaged inintensive agriculture. The higher frequency of the disease in men can be attributed to their exposureto the fungus’ habitat (soil) through agricultural work. The predominance of sugar cane, cotton, andcoffee plantations in the endemic regions favors contact between the rural worker and soil particlesand plants, in which the fungus is found. There are some data supporting that coffee and tobaccoplantations may constitute ecological variables to PCM infection. Records of Colombian PCM patients,collected by Calle et al, suggest that beside geo-climatic factors, coffee and tobacco plantations maybe linked to PCM infection. In addition, P. brasiliensis has been isolated from the soil of coffee cropsin Venezuela and Brazil, but attempts to re isolate it from the same sources have failed. In the past,the coffee monoculture usually occurred in several South America countries preceded by a progressivedeforestation and this was associated with human PCM by several authors.In Brazil, with time, soil depletion led to gradual substitution of coffee plantations for pastures, corn,wheat, soybean, vegetable gardening, fruit growing and others. Historically, the sugar cane cultivationin Brazil have been an important economic source but mainly associated to sugar production. Thisscenario has changed during the last and actual decades due to the energetic requirements, mainlybecause of petrol prices. Nowadays, the sugar cultivation area has significantly increased, especiallyin some areas of the hinterland of the state of Sao Paulo, located in Brazil Southern Region. Sugarcane plantation is usually related to wide pesticide aspersion and plant burning. The use of fire maysignificantly elevate the soil temperature. In addition, agricultural insecticides, herbicides and fungicidesare widely used in most crops, including sugar cane. The combination of these agriculturalpractices in sugar cane cultivation may affect the saprobiotic life of several soil micro organisms,including P. brasilieinsis.In a recent article, Ono et al demonstrated that most of the agricultural pesticides may inhibit thein vitro growth of P. brasiliensis, what may explain, at least in part, the difficulty in its isolation fromagricultural soil. Although new PCM cases are still reported in agricultural areas, data of contemporaryincidence and prevalence of this disease is lacking, since the reporting of PCM is not compulsory.In the future, the epidemiological data related to new cases of PCM, especially from acute/subacuteforms will help to define if PCM is declining in areas where sugar cane cultivation predominates.129

Analysing coffee’s chemical composition andeffects upon human healthManuel Elkin PatarroyoCoffee is one of the most widely know beverages, drunk by people all around the world and know foracting as a stimulant of people’s psycogenic activity. Coffee is one of the most important agriculturalproducts for the social and economical growth of producing countries. Due to this psycogenic activityand its economical, political and social relevance, coffee has been the subject of extensive analysis(chemical and biological concerning its psycogenic activity) aimed at understanding the mechanismsunderlying such effect, its action or relation to the nervous system, as well as the positive or harmfuleffects that its consumption might have on human health. It has become need thus, to gain a deeperknowledge of its chemical components in order to understand its biological activity.Coffee, same as any other biological product, has a huge biological and chemical complexity, withmore than 100 substances and chemical components being identified to date, some of which havebeen clearly identified and their biological function has been defined.When studying the chemical characteristics of human beings, several factors must be taken intoaccount, including their genetical origin, degree of biological development, environmental framework,type of analysis being applied and such method’s ability to reach valid conclusions etc. Such samerestrictions applied to coffee.Regarding coffee thereby, variables such as the variety’s degree of maturity (Robusta, Arábica,etc.), the way they are prepared, analysis methods being employed and their intrinsic limitation, etc.;may account for different between interpretations (another variable) and may even contract be contradictoryin some occasions.Soluble coffee contains a larger amount (expressed as a percentage of dried weight) of minerals(2 to 3 times) and caffeine (4 or 5 times), compared to other substances susceptible to degradation(processed products) where such components disappear or diminish drastically their concentration,as occurs for instance with trigonelline, aliphatic lipids (10%) and oligosaccharides.Such variation in composition is not only important in terms of the products’ physicochemical characteristics,but also concerning its organoleptic features such aroma, taste, stimulating activity, etc.Differences between the Arábica and Robusta varieties, mainly regarding the of carbohydratescontent of green coffee beans (in relation to the variation in dried weight), and most of all in the contentof sucrose; regarding this later aspect, Arábica content of sucrose is found to be 2 or 3 times largerthan the one in Robusta, which has a key effect on taste related characteristics (2).Roasted coffees have a higher level of lipid content (around 16% in Arábica and 11% in Robusta),associated in two coffee specific diterpenes (cafestol and kahweol), which are the ones releasingtheir most volatile products along the roasting process and whose level might be help determining theproportion of Arábica and Robusta coffee chosen for preparing blended coffees, given that kahweolis present in Robusta but not in Arábica coffee (4).The protein content of both varieties is practically the same, being between 8.8% to 12.2% in greencoffee beans, but the content of free amino acids is highly scarce, ranging between 0.2% to 8%. Bythe same token, around 20% to 40% is lost during the roasting process (depending on its intensity),as result of protein’s denaturation. Some amino acids’ proportion might increase during roasting,whilst some other amino acids, as arginine, cysteine, serine and threonine, might disappear as aconsequence of this process (5,6).Caffeine is the major alkaloid substance contained in coffee. Its concentration (in milligrams/Kg/driedweight) varies; being Arábica’s (9.000-14.000) practically half of Robusta’s concentration (15.000-26.000). All other alkaloids in coffee, such as theobromine and theophylline, are found in much lesserproportions.Nitrogenous bases are divided in two large groups: those remaining stable after raosting as ammoniac,betaine and choline, and some instable, such as trigolline (present in around 0.6% to 1.2%130

in Arábica and 0.9% Robusta varieties), which are decompose and turn into nicotinic acid or niacin.The content of free acid is relevant for coffee’s organoleptic properties, mainly when it results inthe formation of acetic, citric or phosphoric acid. Phosphoric acid content (same as pyroglutamic acidderived from glutamic acid) becomes considerably higher during the roasting process (9).Quinic and vinidinic acids’ content, which varies between 0.3% and 0.5% in green beans andincreases during treatment, but from a biological point of view its byproducts are more important.One of them is n-chlorogenic acid, whose proportion and byproduct depend on the bean’s degree ofroasting (10,11).Coffee quality depends largely on the relative proportion of monochlorogenic and dichlorogenicacid. To much dichlorogenic acid might result in a metallic and bitter taste associated with a coffeethat has been heated for to long. Such flavor results from an increase in quinic acid, lactone formationand diminishes of pyridine concentration (11).Regarding coffee’s mineral content, the one found in large proportion is potassium (around 80 milligramsin a cup of instant coffee). Although cupper concentration tends to be low, its content is muchmore higher in Robusta coffee, which led to argue that such content might explain this variety’s higherresistance to fungi due to copper’s fungicidal activity.It can be say then:• Coffee’s composition is very complex, including more than a thousand substances;• Not all coffee components that have been identified possess physiological effects, being caffeinethe most widely known among them.• In order to validate experimental results of studies done in animals, the following criteria must beconsidered:• Dose used• Length of coffee’s administration;• Metabolic characteristic of each animal species• Caffeine is the most widely-known alkaloid amongst those known to produce significant physiologicaleffects; others such as theophylline and theobromine cause also significant biological effects.• Coffee’s metabolism is complex. Its physiological effects may in part be explained by three mechanisms:• Antagonism of adenosine receptors;• Phophorodiesterase inhibition; and• Intracellular calcium mobilization• The only neurological effects being clearly demonstrated so far are increased mental alertnessand delayed of onset of sleep. Coffee content produces vasoconstriction of brain vessels, whichis why it is present in several pharmaceutical products used for migraine treatment. Caffeine alsoboosts the analgesic effect of some drugs,• Coffee enlarges short-term memory, alertness and mental sharpness;• Coffee regular consumption in moderate quantities does not affect normal cardiovascular functioingor systolic and diastolic arterial pressure• The gastric or intestinal intolerance regularly attributed to coffee is usually linked to somepeople’ssensibility to coffee and has not been successfully reproduced in any experimentalstudy;• Coffee exerts a cholecystokinetic action and increases pancreatic secretion;• Coffee regular consumption does not cause respiratory apparatus associated diseases;• Endocrine functions are not modified by consuming coffee;• Consuming coffee does not have any important effect on muscle function;• It has not been demonstrated that consuming coffee increases the risk of bone fractures;• Coffee is a good source of potassium, magnesium and fluoride;• Coffee consumption increases energy metabolism in the hours following its ingestion, but does notmodify the total energy expenditure;• Coffee consumption has no harmful effects on reproduction or fertility;• Coffee (in the regular amounts consumed by human) has no teratogenic effect; and• In the habitual amounts consumed by humans, has no genotoxic, mutagenic or carcinogenic potential.131

Impact of regular coffee consumption on alcohol intakeand depressive feelings among studentsSantos, Roseane Maria M 1 , Le, Thaovy 2 and Lima, Darcy Roberto A 31Assistant Professor, Department of Pharmaceutical Sciences,South University School of Pharmacy, Savannah, Georgia2Pharm. D Candidate, South University School of Pharmacy, Savannah, Georgia3Professor, Instituto de Neurologia, Universidade Federal do Rio de Janeiro, Brazile-mail: drlima@cafeesaude.com.brRecent studies have associated regular coffee intake with beneficial effects in diseases such as adultdiabetes, Parkinson’s disease, cancer and cirrhosis as well as on suicidal tendencies. Results of ourrecent pilot study have led us to hypothesize that compounds known to possess opioid antagonistproperties occur naturally in coffee and could be beneficial helpful in the control alcoholism.Objective: Evaluate the relationship between the consumption of coffee and alcoholic beverageswith the development of signs and symptoms of apathy and depression within student population.Method: A cross-sectional study utilizing a standardized questionnaire regarding coffee intake,depressive feelings and alcohol consumption was completed anonymously. The population studiedwas somewhat heterogeneous, composed of students from different parts of the country and backgroundsand of different ethnicity.Results: The incidence of apathy and/or depressive feelings associated with regular alcohol intakerelationship was found to range from 0 % to 9 % and 0 % to 31 % on a daily and weekly basisrespectively. Moderate daily coffee intake varied widely (from 10 to 64%) and was age-related. Ourdata suggests a strong inverse association between regular to moderate coffee intake and depressivefeelings and to alcohol intake compared with non-coffee drinkers.Implications: Coffee and its health related effects do not seem to be exclusively dependent oncaffeine. Properly roasted coffee has antioxidant poliphenolic chlorogenic acids and lactones withopioid antagonist activity. This might well explain the diverse and controversial data published sofar, either positive or negative, about coffee and health.132

Symposium12Evolutive Biology of P. brasiliensis

What can we know about speciation in P. brasiliensis?Daniel R. MatuteDepartment of Ecology and Evolution, The University of Chicago1101 E. 57 Street, Chicago, IL 60637e-mail: dmatute@uchicago.eduThe definition of a species has been a major impediment to studies of speciation in organisms otherthan animals. In some cases botanists and microbiologists have often expressed doubt that specieseven exist, because of frequent reports of interspecific hybrids, phenotypic variation that does notassort into discrete categories and because of the inherent philosophical difficulties to define speciesin these clades. Nevertheless, like the formation of animal species, speciation in other organismsincluding fungi is characterized by the evolution of barriers to genetic exchange between previouslyinterbreeding populations. Defining species boundaries (and taxonomy in general) in fungi has manydifficulties. The main issue is that morphological characters are still the most common way to assessthe existence of different species without taking into account that species may become geneticallyisolated before the development of morphological autopomorphies. A novel approach that has beenused recently used is the use of phylogenetic methods to estimate the genetic diversity and determinethe existence of groups reproductively isolated by assessing the degree of gene flow between crypticgroups. By this approach it has been estimated that the use of phenotype as the sole parameter todefine fungal species highly underestimates their number when compared to species recognized byphylogenetics. Phylogenetic species recognition using concordance of multiple nuclear genes hasenjoyed widespread use with fungi earlier than other groups like plants and animals, mainly becauseof their lack of phenotypic characters, making nucleic acid characters attractive. However, in animals,the use of multiple nuclear markers is becoming increasingly popular. Dettman et al. (2003) (Dettman,Jacobson, and Taylor 2003) proposed a simple approach to identify independent evolutionarylineages and phylogenetic species from multilocus genealogies. Species boundaries in Neurosporasp. that were recognized by this approach have been shown to be in good agreement with thoseidentified by mating tests (Dettman et al. 2003).P. brasiliensis has shown extensive genetic variability when analyzed by molecular tools suchas random amplified polymorphic DNA, restriction fragment length polymorphism (Soares et al.1995; Nino-Vega et al. 2000), and electrophoretic karyotyping (Montoya et al. 1997). These resultshave been the ground to study and characterize natural genetic variation in this fungus. Recently,the existence of three genetically distinct evolutionary lineages in P. brasiliensis was demonstratedthrough analysis of DNA sequence data for multiple genes (Matute et al. 2006). These groups arecurrently designated S1 (species 1), PS2 (phylogenetic species 2), and PS3 (phylogenetic species3). Additional support for these lineages comes from variation in virulence and expression levels ofantigenic proteins previously found between P. brasiliensis isolates which are now known to belongto S1 and PS2 groups (Carvalho et al. 2005). Moreover, other isolate with a considerable genomicdivergence has also been detected by the use of the genealogical phylogenetic species recognition(Carrero et al. 2008).So far, all the studies on genetic diversity in P. brasiliensis have focused on the phylogenetic reconstructionof genealogies but certainly none of them has tried to use the biological species conceptthat is considered the gold standard for speciation and diversity inventories. This issue is not restrictedto P. brasiliensis but to all those fungi that have not known sexual stage what makes difficult to applycanonical approaches to the study of speciation. Yet, inability to demonstrate the sexual stage doesnot mean that it does not exist. Many fungi, P. brasiliensis amongst them, have been concluded tohave a sexual stage by population genetic methods although we have no morphological evidence fortheir sexual reproduction. In this talk I want to stress out the need for reconciling the biological speciesconcept and the phylogenetic concepts in fungi in order to understand speciation processes.135

Is Paracoccidioides brasiliensis an unique specie?M.S. Felipe and Teixeira M.M.Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil.e-mail: msueli@unb.br or marcus.teixeira@gmail.comParacoccidioidomycosis is a systemic illness with an area of occurrence covering most of LatinAmerica, with highest prevalence in Brazil, Venezuela and Colombia, and that affects mainlyindividuals living in the countryside. Studies on difference isolates of the causative agent, Paracoccidoidesbrasiliensis, have revealed a great degree of genetic variability in this pathogen. ThePb01 isolate, in particular, differs from the three previously described phylogenetic species by thegenealogical concordance (S1, PS2 and PS3; Matute et al., 2006) and its taxonomic classificationin the genus Paracoccidioides remains undefined. In the present work, sixteen isolates genotypicallysimilar to Pb01 have been identified by analysis of a molecular marker from the first intronof the hsp70 gene; these isolates have been named “Pb01-like”.Using the method of recognition of phylogenetic species by genealogic concordance (GCPSR),this work aimed at looking into the existence of a systematically significant group of “Pb01-like”isolates from a total of 88 samples. For phylogenetic analysis the methods of Maximum Parsimonyand Bayesian analysis were applied to thirteen (13) polymorphic regions, including individual andconcatenated loci. This enabled the grouping of the “Pb01-like” isolates in a distant phylogeneticgroup distinct from S1, PS2 and PS3. A high number of fixed polymorphisms were identifiedbetween the “Pb01-like” cluster and that composed of the other three species, which suggests ablockage of all genetic flow between them.The speciation event that defined the new phylogenetic group is sympatric relative to S1 and PS2.The “Pb01-like” cluster and the group that includes S1, PS2 and PS3 are highly divergent andhave been genetically isolated for about 19 million years. Recombination analysis revealed thatrecombination events occur independently inside the two groups, which suggests reproductiveisolation. Two isolates from the “Pb01-like” group, 769 and 133, have quite distinctive genotypes;in the phylograms and in the recombination analysis these isolates share alleles with isolates fromthe S1 and PS2 phylogenetic species respectively.In keeping with GCPSR, the “Pb01-like” clade can be considered a new phylogenetic speciesdistinct from the other three, since the clade corresponding to “Pb01-like” isolates is strongly supportedby all phylogenetic trees independently and concatenated generated from polymorphismdata of the thirteen loci, with values of posterior probability (1.0) and of bootstrap agreement(100%) highly significant.Exclusive characteristics were identified for the “Pb01-like” group, such as the morphology of yeastcells and conidia (Bagagli et al., personal communication). In conjunction with molecular phylogenydata, the formal description of a new species for the genus Paracoccidioides should be accompaniedby a renaming of the group, in the light of the medical relevance of this pathogen in LatinAmerica. A new nomenclature would obviously ease information exchange among pathologistsand researchers. We suggest formally that the phylogenetic species encompassing the “Pb01-like”clade should be named Paracoccidioides lutzii, as a tribute to medical mycologist Adolpho Lutz,who first described human pathogen P. brasiliensis exactly one hundred years ago.Financial support – CNPq, FAP-DF, FUB.136

Climate variability and acute / subacute paracoccidioidomycosisin a hyperendemic area in BrazilL.V. Barrozo 1 , Mendes, R.P. 2 , Marques, S.A. 2 , Benard, G. 3 , Silva, M.E.S. 1 , Bagagli, E. 41 Faculdade de Filosofia, Letras e Ciências Humanas, USP, São Paulo – Brasil.2 Faculdade de Medicina de Botucatu, UNESP, São Paulo – Brasil.3 Faculdade de Medicina, USP, São Paulo – Brasil.4 Instituto de Biociências, UNESP, São Paulo – Brasil.e-mail: lija@usp.brIntroduction and Objectives: Paracoccidioides brasiliensis has rarely been identified in the naturalenvironment, which makes difficult to characterize its ecological niche. The geographical distributionof the disease is associated with specific environmental conditions such as mild temperatures, fertilesoils and high humidity. Many studies have focused on defining the niche of the fungus based on thelocation of residence of the patient. However, since acute/subacute cases present shorter latencyperiods, they may allow spatial and temporal analyses. To date, variations in incidence have beenobserved in Colombia, among the Suruí Amerindian population in the Amazonian region of Braziland in Argentina after the built of a hydroelectric power. Besides land use changes, which causessoil disturbance and the exposure of larger numbers of individuals, P. brasiliensis responds to themoisture content and temperature; therefore, a relationship between climate conditions and PCM inits acute/subacute form is suggested. To date, studies that characterize the space-time pattern of theseries of acute PCM and its relationship with climate variables are not yet available. The aim of thiswork was to analyze the series of acute/subacute cases admitted to the University Hospital of the SãoPaulo State University (UH-UNESP), located in a highly endemic area in Botucatu neighboring, SãoPaulo State, Brazil, from 1966 to 1999 and the possible associations between the acute/subacutePCM incidence and climate factors.Methods: Acute/subacute PCM data corresponded to the cases seen in the Infectious Diseasesand Dermatology Sectors of the UH-UNESP. Stepwise regression of the 1966-1999 annual data wasused to model acute/subacute PCM incidence from lagged variables: antecedent precipitation, airtemperature, soil water storage, absolute and relative air humidity and, Southern Oscillation Index(SOI). Previously, all data were linearly detrended.Results: Exploratory bivariate analyses showed that incidence correlates positive and linearly withair temperature (r = 0.45, P < 0.05) and with absolute air humidity (r = 0.50, P < 0.01) in the yearpreceding diagnosis. Backward and forward stepwise analyses aided to select the most importantvariables for explaining the acute/subacute PCM incidence. Multiple regression analyses resultedin two models: the first explains 44% (P < 0.001) of the incidence in this period, taking into accountonly the 1-year lagged absolute air humidity and 3-year lagged SOI and, the second additionallyincludes the soil water storage of 3 years before incidence, explaining 49% (P < 0.0001) of the PCMvariance in the yearly series.Discussion: The higher incidence could then be understood as resulting from positive absoluteair humidity of the year of exposure, and negative SOI and positive soil water storage of the 2 yearsbefore exposure. Although it is recognized that PCM distribution is driven by environmental variablesand human activities, to date any data based attempt has been made to verify the links betweenfluctuations in PCM incidence and climate. This first approach of explaining acute/subacute PCMincidence based on climate variability suggests some relevant implications to the biology of P. brasiliensis.Greater absolute humidity is found to be significant in the year before increase in incidence,or according to our assumptions, in the probable year of infection. Although laboratory studies havenot been carried out to observe the influence of humidity on conidial liberation of P. brasiliensis, aparallelism can be made with the two other closest pathogenic fungi (e.g., Blastomyces dermatitidis,Histoplasma capsulatum) due to some similarities among them. Higher soil water storage two yearsbefore exposure may explain the already suggested relationship between P. brasiliensis and rainfall/humidity favoring fungus growth in this period. How SOI may affect the growth of P. brasiliensis is a137

matter of conjecture. As a global index, SO may be a proxy for others local environmental variablesnot measured (e.g., dew, fog drip), which could also increase humidity in soil surface two years beforeexposure. Teleconection patterns defined by ENSO events throughout the globe establish differentconditions of atmospheric circulation, influencing regional humidity convergence and divergence. Byits turn, it defines preferable regions for development of precipitation. This result suggests that thereis fungal growth after increase in precipitation in the longer term, as has been noted for Coccidioidessp (Comrie 2005), and probably greater spore release with increase in absolute humidity in the shortterm. We can hypothesize that an antecedent period of high moisture is necessary to the fungus toestablish itself in the soil. Human activity then dislodge the superficial portion of the soil, exposingthe filamentous, spore producing, form of the fungus. If the absolute humidity is high enough at thatmoment, the spores are wet and then freed and aerolized.Supported by FAPESP and CNPq.Overview of the biogeography of P. brasiliensisE. Bagagli, Teodoro R.C, and Gimenes Bosco S.M.Departamento de Microbiologia e Imunologia,Instituto de Biociências, Unesp, Botucatu, SP, Brasil.e-mail: bagagli@ibb.unesp.brThe geographic distribution of P.brasiliensis is located mainly in South America, where the fungus hasevolved in the latest millions of years, as others Ajellomycetaceae family members. Although detailsabout the fungus ecology are not yet completely revealed, several pieces of evidence indicate that P.brasiliensis presents two distinct ecological niches, one inside hosts as individual yeast cells, and onein the saprobe environment as mycelia that produces the infective propagula. While P. brasiliensishas been rarely isolated from saprobe sources, it has been frequently recovered from clinical samplesand from tissues of armadillos, a dwelling soil mammal. Human cases of Paracoccidioidomycosis stillrepresent the most important sources of information about the spatial distribution of the pathogen inthe endemic areas, and have been described in patients from distinct biomes, such as in rain- andsemideciduos forest, open fields, and savannas, in a wide range of biotic and abiotic factors.In the latest years, it becomes clear that P. brasiliensis does not represent a unique biological entity,but a complex of different genetic groups that has diverged separately as cryptic species. Fourdistinct genetic groups have already been detected (S1, PS2, PS3 and Pb01-like) and the divergencetime among the groups was estimated in 8 to 19 millions of years, according to Matute et al, 2007 andTeixeira et al 2008, respectively. While S1, a paraphyletic group, appears to be the most ubiquous,occurring in different regions and countries of Latin America, the remaining ones, apparently monophyleticgroups, seem to be geographically more localized: PS2 has been detected sympatrically withS1 in Southeast region of Brazil, PS3 as clonal in Colombia, and Pb01-like as a well diverged group inCentral-west region of Brazil. Some mycological features, such as the intensity of conidia production,appear to be associated with the genetic group and, in this manner, contributing to the different infectionrates. For example, conidia production tend to be high in S1 and very low in PS2 group isolates, andthe infection incidence rates of S1 and PS2 genotypes in human and armadillos, in Botucatu endemicarea, is nearly 5:1, respectively.Important points to be addressed: In which condition and circumstances sexual reproduction is occurringin S1, PS2 and Pb01-like groups? What kinds of biological barriers could be acting to avoidinterbreeding between S1 and PS2, the sympatric groups? Considering that each species has its ownecological niche, what is the role of parasitism for the species divergence and also for defining the effectiveecological niche? The evaluation of representative numbers of isolates from different regions ofLatin America, both by molecular and mycological approaches, must be carried out in order to providea more realistic picture of P. brasiliensis biogeography.Financial support: Fapesp (Proc 06/03597-4)138

Poster Session1Clinical Aspects / Case Reports

1-01Clinical epidemiology of paracoccidioidomycosis patients in the region of Botucatu (SãoPaulo state, Brazil)D.F. Lopes 1 , L.R. Carvalho 2 , R.P. Mendes 1 .1Tropical Diseases Area, Botucatu Medical School; São Paulo State University – UNESP. 2 Department of Biostatistics, BiosciencesInstitute. São Paulo State University – UNESP. faccioli_lopes@yahoo.com.brIntroduction: This study was carried out in order to clearly differentiate acute/subacute-AF from chronic-CFform and to evaluate aspects of medical attention to paracoccidioidomycosis patients.Methods: Clinical records of 100 confirmed paracoccidioidomycosis patients with AF or CF attended atthe Tropical Diseases Area – Botucatu Medical School were reviewed. Information collected on standardizedquestionnaire included age, sex, occupation, smoking history, clinical manifestations, previous treatment, andhospitalizations. Chi-square and Fisher’s exact test were used in the statistical analysis; significance was set upat p

1-03Paracoccidioidomycosis in Mexico1R López-Martínez, 2 O Velasco-Castrejón, 3 A Bonifaz, 3 J Cazarín-Barrientos, 3 A Saul, 4 R Arenas, 5 MC PadillaDesgarennes1- Departamento de Microbiología y Parasitología. Facultad de Medicina, Universidad Nacional Autónoma de México. México D.F.México. 2.- Unidad de Medicina Experimental. Facultad de Medicina. Universidad Nacional Autónoma de México. México D.F. México..3.- Servicio de Dermatología. Hospital General de México, S.S.a. México D. F. México. 4.- Servicio de Dermatología y Micología.Hospital General “Manuel Gea González”, S.S.a. México DF. México. 5.- Laboratorio de Micología. Centro Dermatológico “Ladislao dela Pascua”. S.S.a. México DF. México.e-mail: rlm@servidor.unam.mx, rlmmex@hotmail.comParacoccidioidomycosis is an endemic infection in Latin America. In the south countries most cases isobserved in Brazil, Colombia and Venezuela, with fewer cases in Central America and Mexico. Spite of inMexico the paracoccidioidomycosis cases are sporadic, disease is important due to both its severity anddiagnosis difficulty. Here 109 cases reported in a 57 years period within five dermatological hospitals arepresented. The disease evolution time before clinical diagnosis was between one and nine years, but inmost cases was of two years.The most affected age group was that from 41 to 50 years; cases in children of 11 years and in olderthan 72 years were also observed. The man/woman rate was 20/1. By occupation, the distribution wasfarmers 88.7 %, housewives 4.8 % and others activities 6.5 %. In the Veracruz state, situated in the Gulfof Mexico, 63 cases were observed. The most northern case was originated from San Luis Potosí state(Huasteca potosina) at 23° North latitude. The primary pulmonary form was followed by skin disseminationin 36.6 % and mucosal dissemination in 37.6%. The treatment of first observed cases was based on SMX/TMP and amphotericin B. In the last years itraconazole only or in combination with other antifungal drugwas used obtaining a faster disease resolution.1-04Paracoccidioidomycosis in BoliviaJ. Vargas F.Unidad de Micología, CENETROP, Santa Cruz-Bolivia. e-mail: drjvargasf@hotmail.comParacoccidioidomycosis is the most important systemic fungal infection in Bolivia not merely because itis the most common, but because of its high potential to cause disability of the severity of its clinical formsand its severe sequelae.History: There is no medical literature sufficient to determine from where the Paracoccidioidomycosis isknown in Bolivia. The works of Morales Villazon (1916) and Veintemillas (1935) confuse this mycosis withleishmaniasis. The first cases published and confirmed by laboratory testing belong to Sangueza (1965),Galindo Decker (1969) and Rios Dalenz (1979) The creation of CENETROP in 1974 and the start of theactivities of the Mycology Unit in 1979 are two important milestones in the history of this disease in Bolivia,to become the national focal point of Paracoccidioidomycosis.Geographical distribution and frequency: Based on the origin of cases, it is considered that tropicalregions and valleys are the endemic zones of Paracoccidioidomycosis, covering 75% of Bolivian territory.There are diagnosed 30 to 40 cases per year.Clinical forms: Chronic-Multifocal: 80% Chronic-Unifocal: 16% Acute (child-young): 4%Site of infection (chronic multifocal form): Lungs: 80% Lymph nodes: 78% Pharingeal mucosa: 60%Skin: 58% Adrenal glands: 3% other sites 3%Therapeutic experience: In 1988, a work was published about 30 cases treated with ketoconazole with93% of good results (6), in 1992 at the V International Meeting of Paracoccidioidomycosis in Buenos Aireswere presented the results of 40 cases treated with itraconazole, with an efficacity of 95% Actually, thedrug itraconazole is the treatment of choice. Clinical pictures with the results obtained are shown.142

1-05Epidemiological and clinical features of patients with paracoccidiodomycosis attending ata University Hospital from Huila - Colombia, between 1999-2007Molano VM 1 , Gualtero S 2 , Duran LF 1 , Gómez CA 2 , Diaz G. 11. Departamento Medicina Interna, Hospital Universitario Hernando Moncaleano Perdomo, Neiva-Colombia. 2. Unidad de Infectologia, HospitalUniversitario Hernando Moncaleano Perdomo, Neiva-Colombia. e-mail: victormolano2007@gmail.com - sandra.gualtero@gmail.comIntroduction: Paracoccidioidomicosis (Suramerican Blastomicosis or Lutz Splendore de Almeida disease) is aendemic mycosis with highly concern in latinamerica countries. In Huila-Colombia there are not studies that describeits behavior.Objetive: describe clinical and epidemiological features from patients with Paracoccidioidomicosis brasiliensis thatrecieve medical care at Hospital Universitario Hernando Moncaleano Perdomo, between january 1999 to september2007.Materials and methods: retrospective study starting from medical records of patients with Paracoccidiodomicosisbrasiliensis histological confirmed. A personal phone call was used to establish survival. Reservarea area and endemicarea concepts was used according to Borelli´s definition. EPI INFO 3.4 programs was used for statistical analysis.Results: Ten (10) patients was identified. All of them were man with histopathological confirmedParacoccidiodomicosis. Chronic presentation was observed in 9 patients and only one had the juvenil type. 6 hadbetween 30 and 60 years old. 7 were farmers dedicated to coffee cultivation. 8 patients were heavy smokers and 1had HIV infection. 2 refered armadillo intake at the past. 3 had lymphoid nodes and mucous compromised. 8 hadlung, skin and central nervous systems concomitant involment and 1 had ileal compromise. Four received treatmentwith anfotericine B followed by itraconazol, 3 with ketoconazol and 1 with fluconazol. Seven patients was alive atseptember 2007, 2 was dead in one it could not settle down.Neiva city and the municipalities of Gigante, Acevedo, Argentina, Garzon, Oporapa, Rivera and Yaguara wereidentified like probable reservarea area. Neiva city (4 cases), Gigante (2 cases), Acevedo, Garzón, Paico y Rivera (1 case each one) were considered endemic area.Conclusions: the factors associated with the patients are similar to those described in the literature. Importantregions was identified likely Endemic and reservarea area. This would allow to carry out active epidemiologicalsurveillance.1-06Paracoccidioidomycosis: Frequency, distribution, and hospitalization flows due to theendemia in Brazil (1998-2007)Coutinho, Z.F.(1), Travassos,C.M.R.(2), Wanke,B.(3), Oliveira, E.X.G.(4), Valle, A.C.F. (3), Coimbra Junior, C.E.A. (1).(1)National School of Public Health/FIOCRUZ, Brazil. (2) Institute of Scientific and Technological Communication and Information in Health/FIOCRUZ. (3) Evandro Chagas Clinical Research Institute /FIOCRUZ. (4) Brazilian Institute of Geography and Statistics – IBGE.e-mail:ziadir@centroin.com.br (presenting Author)Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis,an endemic exclusive to the Americas. In Brazil, the mean annual PCM mortality rate was 1.49/million inhabitants from1980 to 1995 (COUTINHO, 2002). This study analyzes the frequency, distribution, and case flow between the municipalityof residence and that of hospitalization for PCM patients. Hospitalization data were obtained from the Health Ministrythrough the Hospital Information System of the National Health System (SIH-SUS) for January 1998 to May 2007.Hospitalizations were selected with a principal diagnosis of blastomycosis (B40-ICD10) or paracoccidioidomycosis (B41-ICD10), with blastomycosis considered equivalent to PCM. The period totaled 7,017 hospitalizations for PCM, of which48.98% were concentrated in 21 hospitals and 82% were male patients. Spatial distribution of hospitalizations showeddistinct patterns between the different States and regions of Brazil: (a) the Southeast had 57.90% of hospitalizations,followed by the Central-West with 13.34%; (b) in Piauí, 22.36% of the 626 hospitalized cases were residents of thatState, while 49.52% were from Maranhão and 24.92% from Pará. The States of Piauí, Maranhão, Pará, and Tocantinshad 12.64% of all hospital admissions; (c) Rondônia (257 admissions) and Mato Grosso (612) had 12.38% of allhospitalizations; (d) Among 88 hospitalizations of residents from the Federal District, 56.81% were admitted there and25% in the State of Goiás; (e) Santa Catarina (209 admissions) had more hospitalizations than Rio Grande do Sul (93);(f) Most States in the North and Northeast had small caseloads. PCM hospitalization flows display places with largedisplacement by residents to obtain hospital care. The North of Brazil exported nearly 30% of its PCM patients to otherregions. The pattern of States cited in items (b) and (c) appears to correspond to expansion of the agricultural frontier inAmazonia. The current analysis shows the inadequacy of public policy for PCM care, identifies the healthcare system’slimitations for adequately treating patients, and reflects the trend in the ecodynamics of the PCM endemic in light ofeconomic, environmental, and migratory changes.Key words: paracoccidioidomycosis, epidemiology, mycosis, health policy143

1-07Epidemiological and clinical profile of 137 pacients with paracoccidioidomycosis in Uberaba,Minas Gerais, BrazilNeves, FF; Gerolin, GP;Tavares, MG ; Castro-Silva, MH; Lopes, GP;Michelin, MA;SilvA; Vergara, MLDoenças Infecciosas e Parasitárias, 2. Pneumologia, 3. Imunologia e 4. Radiologia, Universidade Federal do Triângulo Mineiro – Uberaba,MG, Brasil. E-mail: ffneves@bol.com.brIntroduction : After hundred years of the first report, paracoccidioidomycosis is still one of the most prevalentsystemic mycosis in Latin America countries. The follow-up of these patients commonly is difficult and the relapsesnumber cases is frequently unknown. The aim of this study is to present preliminar information of a cases serie ofpatients diagnosed between 1988-2007.Methods: Medical records of patients with paracoccidioidomycosis diagnosis performed in the teachinghospital were reviewed and after this, patients were contacted and invited to perform a new clinical and laboratorialassessment.Results: During 19 years, 137 patients with paracoccidioidomycosis were diagnosed. From these, 108(78,8%) were male, median age of 39,2 years. Between them, 45 (35,6%) were farmers and the median timeof evolution of their symptoms was of 4 months. The main clinical features were related to respiratory, lymphaticand mucocutaneous involvement in 79 (56,6%), 84 (61,3%) and 63 (46%) cases respectively. Clinical pictureaccording to the present classification was: acute/subacute 29 (21,2%) cases localized chronic form 35 (25,5%),disseminated chronic form 72 (52,5%) and mixed two (1,4%) cases. Twenty (14,5%) patients presented coinfectionwith HIV. Most of cases were diagnosed by cytological or histopathologic exam of secretions and/or fragmenttissues. Lung involvement by X-ray plain was evidenced in 76 (55,1%) cases. Amphotericin B and/or itraconazoleor Trimethoprim-Sulfamethoxazole were administered to these patients. Twenty five (18,1%) cases presentedclinical relapse. At present, 76 (55,4%) of 137, are alive and seven of them are receiving therapy and 29 (21,1%)died. No definitive information about the other 32 was yet obtained.Comments: The clinical and epidemiological features of 137 patients with paracoccidioidomycosis described,in this study are similar to other reports from Brazil and the high rate of relapse observed among them can beexplained by the poor adderence registered during the follow-up. Otherwise, 25 (14,5%) patients with HIV/AIDSassociation were diagnosed and most them presented a low CD4 count, supporting that immunosuppresion isa important risk factor to develop this mycosis. Otherwise 50% of the deaths occured in HIV patients and nodefinitive cause for the others was obtained at present functional and laboratory assessment of patients alive iscurrently performed.1-08Paracoccidioidomycosis associated to HIV/AIDS infection in Uberaba, Minas Gerais,BrazilGerolin, GP1; Tavares, MG2; Castro-Silva, MH3; Lopes, GP4;Michelin, MA5; Silva-Vergara, ML, Neves, FF.1.Doenças Infecciosas e Parasitárias, 2. Pneumologia, 3. ImunologiaE 4. Radiologia Universidade Federal do Triângulo Mineiro – Uberaba,MG, Brasil.E-mail: ffneves@bol.com.brIntroduction: The coinfection HIV/Paracoccidioides brasiliensis was described for the first time in 1989 inBrazil. After this, near of two hundred cases has been reported and most severe clinical forms in. The aim ofthis study is to present the main epidemiological and clinical features of this cases has been already describedpatients with both infections. Methods: Medical records of HIV/AIDS patients with Paracoccidioidomycosisdiagnosed at teaching hospital from 1988 to 2007 were reviewed. Results: Twenty (14,5%) patients among137 with Paracoccidioidomycosis presented the HIV infection. From these, 16 (80%) were male, median age of34,7 years. Illicit drugs use and multiple partners were reported by most of them. The median time of symptomsuntil mycological diagnosis was 2,9 months. The main clinical features were related to respiratory, lymphatic andmucocutaneous involvement, in 13 (65%), 13 (65%) and 5 (25%) cases respectively. Clinical form was characterizedaccording to the current classification as acute/subacute in nine (45%) cases, chronic localizated in five (25%),chronic disseminated in four (20%) and mixed in two (10%) patients. Lung involvement was evidencied by X-rayplain in 10 (50%) cases. CD4 count values varieted from 5 to 349 cells/mm with mediacy of 88 cells. Trimethoprimsulfamethoxazoleand itraconazole were the drugs more frequently prescribed. At present six patients (30%) arealive. Comments : Despite the epidemiological overlap between HIV/AIDS and Paracoccidioidomycosis patients,no substancial increase of number cases of this mycosis were observed, although it’s opportunistic character inAIDS patients is to be determined yet. However, severe disseminated acute/subacute clinical form or mixed offeatures of the two classical clinical forms were frequently observed. The epidemiological and clinical profile of thepatients described is according to the other series already reported.144

1-09Acute severe paracoccidioidomycosis in an urban-living boy with a single exposure episode:Evidence for a long subclinical evolution before full-blown diseaseG. Benard 1,2 & Barata. L. C. B. 31Laboratory of Dermatology and Immunodeficiencies, Medical School of the University of São Paulo; 2 Systemic Mycoses Outpatient Clinic,Division of Infectious Diseases, Hospital das Clínicas da Faculdade de Medicina da USP (HC FMUSP), Brazil 3 Hospital e MaternidadeSanta Catarina, São Paulo, Brazil mahong@usp.brWe describe a 16 year-old boy born who always lived in downtown São Paulo and developed a severe acuteparacoccidioidmycosis. He had no history of exposure in the previous ~4 years except from having gone to a oneday“ecological” hiking (January 25, 2007) in a riverside at the Vale do Paraíba, a well-known reservarea describedpreviously by Gonçalves and Londero (1998). He walked trough remnants of rain forests and along the borderof plantations. A sorethroat led him to a medical visit on March 9, where a blood film revealed an eosinophilia(20%, 2499 eosinophils/mm3) and chest X-rays showed a left peri-hilar image compatible with a lymph nodeenlargement; pulmonary parenchyma was unnoticeable. Neither alteration was investigated. By June, he referreddevelopment of discrete acneiform lesions on the face. These lesions progressed, and by mid September hewas then evaluated by a dermatologist who diagnosed acne. He was treated accordingly. At this time he was stilldoing good and following his studies regularly. By December, skin lesions were more pronounced and spread tothe upper trunk; fever and cervical, axilar, and retroauricular adenopathies and hepatosplenomegaly were noted,together with weight loss, weakness, persistent eosinophilia, discrete anemia and hypergammaglobulinemia. Hewas investigated for several hypotheses other then paracoccidioidomycosis. This diagnosis finally came out onJanuary through a cutaneous lesion biopsy. He received itraconazole, 300 then 400 mg/day, but after 3 weeks hisgeneral condition continued to deteriorate, the inflamed lymph nodes were even more enlarged, some fistulizated,and the hemoglobin dropped to 8.5 g/dL. He was then hospitalized and put on lipossomal amphotericin. He wasdischarged 15 days later, somewhat improved, and was punt on sulfadiazine (100 mg/kg/day). He continues toimprove gradually. His hemoglobin is now 13.5 g/dL and he is reassuming his normal activities.This case allows to estimate precisely the incubation time (or subclinical evolution) before the full blown acuteform disease manifests: 5 months.Other recent travels referred by the patient were to USA and to mountain counties within São Paulo state(altitude >1500 m above see level), which do not correspond to reservareas in our state.1-10First description of a fatal adult respiratory distress syndrome in a severe acuteparacoccidioidmycosis patientRavanini. J.N., 1 Silva L.F.F. 1 , Goulart S., 2 Benard G. 2,31Department of Pathology, Medical School of the University of São Paulo, Brazil, 2 Systemic Mycoses Outpatient Clinic, Division ofInfectious Diseases, Hospital das Clínicas da Faculdade de Medicina da USP (HC FMUSP), Brazil, 3 Division of Clinical Dermatology,Clinics Hospital, Medical School of the University of São Paulo, Brazil, mahong@usp.brWe describe a 22 years-old patient who was born and always lived in São Paulo downtown, with a 5-monthshistory of progressive weakness and malaise and generalized adenophaties. During this period he lost 7 kg andalso complained of an initially non-productive cough that lately produced some sputum. He denied any drugabuse and tobacco smoking habit. He used to go on week ends to a county near São Paulo from where somepatients with acute PCM have been referred in the last years. On admission he was in a good general condition,eupneic, presented an hepatosplenomegaly and a generalized adenopathy involving the cervical, inguinal, axilar,and occipital chains. Laboratory data showed marked leukocytosis, eosinophilia, abnormal liver function tests. Hewas HIV negative. A thoracic CT scan mediastinal adenomegalies e several lytic lesions on ribs. A biopsy of acervical lymph node done previously at an outside service was inconclusive, but P. brasiliensis yeast forms werevisualized in sputum. Specific serology tests were also positive. When receiving the first dose of amphotericin Bhe developed respiratory insufficiency, tachycardia hypotension. He was moved to the ICU immediately, wherehe received antibiotics to an eventual hospital-acquired infection. His respiratory insufficiency worsened, hewas intubated, put of assisted mechanical ventilation. Chest X-rays showed the typical pattern of ARDS. Hisrespiratory insufficiency was refractory to the ventilatory support and he died after 4 days at the ICU. Autopsyshowed disseminated paracoccidiodidomycosis and histopathological findings in the lungs compatible with theARDS. All cultures were negative for bacterial growth and the autopsy did not disclose any finding suggestive ofother bacterial or viral infection.145

1-11Sub-acute paracoccidioidomycosis in a 49 years-old womanD. Myiamoto 1 , Kono Adriana. S. G . 1 , Benard. G. 1,2 , Yoshida. M. 1 , Giarolla. I. 1 , Shikanai-Yasuda. M. A. 1,31Systemic Mycoses Outpatient Clinic, Division of Infectious Diseases, Hospital das Clínicas da Faculdade de Medicina da USP(HCFMUSP),Brazil.2 Division of Clinical Dermatology, HCFMUSP, Brazil . 3 Department of Infectious and ParasiticDiseases, FMUSP, Brazilmasyasuda@yahoo.com.brA 49 year-old patient complained since 2004 of occasional upper abdominal pain irradiating to flanks. Thissymptom became more intense by 2006, and was then accompanied by fever and abdominal distension. Anultrasound imaging revealed peri-pancreatic and peri-aortic nodules, as well as gallbladder microcalculi. Thepatient was submitted to videolaparoscopic cholecistectomy during a peri-gallbladder lymph node was biopsed.This biopsy revealed paracoccidioidomycosis. She had a good recovery of the surgical procedure but receivedno treatment for the mycosis at this time. In the following two months she lost 20 kg, and then was referred to ourservice.The patients was born in São Paulo, where she lived most time, except for a short period between 1998 and2000, during which she went on the weekends to a rural area close to São Paulo for resting purposes. This areamay be considered a reservarea. She developed her menopause at the age of 42 (thus before developing thesymptoms), and she had been diagnosed as having hypothyroidism at 46, to which she has been adequatelytreated since then with levotyroxin .On admission she presented hepatosplenomegaly and cervical lymph node enlargement. Acute phasereactants and liver function tests were also abnormal. A thoracic CT scan showed mediastinal and supraclavicularlymph node enlargements, while an abdominal TC scan revealed generalized and remarkable lymph nodeenlargement and hepatosplenomegaly. She was treated with trimethoprim-sulphamethoxazole 160/800 mg twicea day with a good response and since then has been on regular follow-up at outpatient service.Conclusion: This case describes a 49 year old women who developed a typical acute/subacute form of thedisease, soon after her menopause. Paracoccidoidomycosis is rare among women, with cases being describedeither before puberty, when the clinical presentation is the acute form of the disease, or after menopause, whenthe clinical presentation is the chronic form of the disease. From the epidemiological point of view, it is possible toadmit the she acquired the disease recently, when frequenting a reservarea, and in a period when she was alreadypresenting estrogen levels reduction.1-12Paracoccidioidomycosis presenting as evanescent pleural effusion associated to focalconsolidation: An uncommon presentationCosta AN 1 ; Junior SAD 1 ; Takagaki TY 1 ; Kairalla RA 1 ; Carvalho CRR 1.1.Pulmonary Division – Hospital da Clinicas - University of São Paulo Medical School, SP, BRASIL. e-mail: nathan.andre@gmail.com(presenting Author).Men, 72 years old, born and living in São Paulo, ex-smoker (15 pack/years; 30 years ago), presented with leftthoracic discomfort while exercises that began 2 months before, without associated systemic symptoms. Whileexcluding cardiologic and coronary causes of the pain, a left inferior lobe infiltrate associated to a moderate pleuraleffusion was found, and he was then sent to a pneumologist. The computed tomography of the chest showedparenquimal consolidation associated to moderate pleural effusion, without lymphadenopathy. One week later,when he came back for a thoracocentesis, there was no more pleural effusion, neither in the physical exam nor inthe chest ultrasound. A bronchoscopic lavage was then performed, which revealed the typical yeast form of theParacoccidioides brasiliensis. Although the serology was negative, he was treated with Itraconazol 200mg/day,with total remission of the symptoms after 2 months of treatment. We performed a new computed tomography twomonths after the beginning of the treatment that showed only mild residual reticular infiltrates, no pleural effusionnor consolidations. Discussion: Pulmonary paracoccidioidomycosis is one of the most important fungal infectionsin Brasil, being responsible for 200 deaths/year and concentrating 80% of the cases in the world. Primary infectionoccurs in the infant and the chronic form affects adults in the third to the fifth decades of life. Radiologically, thefindings are typically bilateral and symmetrical, with interlobular septal thickening, nodules, peribronchovascularinterstitial thickening, centrilobular opacities, ground-glass opacities, cavitations, consolidations, tractionbronchiectasis and paracicatricial emphysema. In this case, de presentation was atypical because of the focalair space consolidation, absence of lymphadenopathy and the evanescent pleural effusion, besides the negativeserology. The microbiological confirmation and resolution of the clinical and radiological findings after imidazolicuse support the diagnosis.146

1-13Nested-PCR, immunoblotting and double immunodiffusion in meningoencephalitis byP. brasiliensis without radiological alterations of the central nervous systemR.A.M.B. Almeida 1 , S.A. Marques 1 , R.P. Mendes 1 , D.V. Moris 1 , S.M.G. Bosco 2 , S.A.G. Macoris 2 , E. Bagagli 2 , A.P. VicentiniMoreira 3 , V.S. Kohara 3 , R.S. Cavalcante 1 , C.R. Chaves 1 e S.A. Calvi 1 .1. Botucatu Medical School, UNESP, São Paulo State University, Brazil. 2. Botucatu Biosciences Institute, UNESP, São Paulo StateUniversity, Brazil. 3. Adolfo Lutz Institute, São Paulo, Brazil. e-mail: mip.ricardo@gmail.com.Background: Meningoencephalitis by P. brasiliensis (Pb) affects 10 to 27% of paracoccidioidomycosis (PCM)patients and its diagnosis is generally made by serological, radiological as well as clinical evidence of PCM in differentsites. Aim: To report an unprecedented case of meningoencephalitis by Pb, without CNS radiological alterations,diagnosed by Nested-PCR. Case report: A man, 51 years old, presented PCM lesions in the oral mucosa, confirmedby biopsy, DID = 1/32, paracoccidioidin = 20x18 mm and oral candidiasis, without additional symptoms. After oneweek of cotrimoxazole, he started presenting dizziness, epigastralgia, anorexia and adynamia, followed by fever andbehavioral alteration 14 days later. The patient used depot corticosteroids for 11 months, due to contact dermatitis.He was feverish, with meningeal signs and Glasgow = 8. There was oral candidiasis, without active PCM lesions.Cerebrospinal fluid (CSF) showed 13 cells, predominantly lymphomononuclear, hyperproteinorrhachia (123 mg%) andhypoglycorrhachia (34 mg%). Cytology for fungi, bacteria, mycobacteria, viruses, protozoa and neoplasias showednegative results and there was no fungal, bacterial and mycobacterial growth. EEG demonstrated diffuse slowness,without herpetic encephalitis pattern, and cranium CT was normal. Aciclovir was initiated and cotrimoxazole was stoppeddue to suspected medication-associated meningoencephalitis and hepatotoxicity. Two days later, the clinical signs andsymptoms completely resolved. Serology for Epstein-Barr, Chagas’ disease, HIV, hepatitis B/C, and syphilis, as wellas the tuberculin test, was negative; IgG antibodies to CMV were positive and brain MRI was normal. DID resulted in½. Serological tests for toxoplasmosis indicated recent infection contracted, however, more than six months before.Nested-PCR reaction was performed using primers derived from the rDNA region of Pb, being positive in CSF andbiopsy of the initial lesion, and DID and immunoblotting in CSF were negative. The levels of TNF-alpha, INF-gammaand IL-10 in the serum and CSF were high (TH0). Conclusions: The clinical evolution and the cytokines profile maysuggest hypersensitivity to the antigens released after the initiation of cotrimoxazole, corroborated by the immediateimprovement after the medication suppression. Meningoencephalitis by Pb without CNS radiological images was notidentified in literature, which demonstrates the importance of using Nested-PCR in etiologic diagnosis.1-14Cutaneous sarcoidic lesions and moriform stomatitis lesions in a young patient: Two sidesbut not the same coinN. Fairbanks 1 , A. Kono 1 , M. Yoshida 1 , N.Y. S. Valente 2 , M. A. Shikanai-Yasuda 1,3 , G. Benard 1, 21Systemic Mycoses Outpatient Clinic, Division of Infectious Diseases, Hospital das Clínicas da Faculdade de Medicina da USP (HCFMUSP), Brazil,2Division of Clinical Dermatology, HCFMUSP, Brazil, 3Department of Infectious and Parasitic Diseases, FMUSP, Brazil mahong@usp.brA 24-year-old man from Barueri, a town nearby São Paulo city, was admitted for investigation of a relapsing,ulcerated oral lesions and development of disseminated cutaneous plaques. He referred to have played in remnantsof rain forests around his birthplace since childhood. Paracoccidioidomycosis has been diagnosed by identification ofParacoccidioides brasiliensis on direct examination of an oral lesion in 2001 (when 17 y-old). Since then he has beenusing short courses (~15 days) of sulfametoxazol-trimetoprim on his on, with partial improvement. In 2006 he developedskin lesions that also partially subsided with sulfa treatment to reappear soon after. On the present admission therewere multiples, infiltrated, sarcoid-type, erythematous plaques lesions on face, neck, thorax, arms and buttocks and atwo typical mulberry-like lesion on the palate. The aspect of the skin lesions suggested tuberculoid leprosy in reaction.Evaluated by a hansenologist, a partially (although inconclusive) altered pattern of skin sensitivity tests and suspectedthickening of ulnar nerves pointed to this diagnosis. However, ultrasound imaging showed sural nerves within thenormal range. Lymphadenopathy and hepatosplenomegaly were absent. CT scan revealed normal lungs. Biopsy ofa skin lesion revealed sarcoidic paracoccidioidomycosis: compact granulomas with rare yeast cells. Simultaneously,biopsy of the palate lesion revealed a granulomatous reaction with numerous eosinophils and plasmocytes, epidermicmicroabcesses and numerous yeast-like cells. The suspected leprosy coinfection was discarded. This case highlightstwo main points: a) the occasional difficulty in classifying the clinical presentation either as the acute/subacute orchronic form. This patient presented oral lesions suggestive of the chronic form, but not pulmonary involvement, and askin involvement more commonly seen in young patients with good general condition but no systemic involvement. Henot only did not present the typical features of the acute form, but also had a 7-y history of relapsing manifestations; b)the compartmentalization of the immune response; at the same time the oral lesion depicted a less organized reactionwith abundant yeast and eosinophils, the cutaneous lesions were more organized and compact, sometimes sarcoidiclike,denoting two different outcomes of the host-parasite relationship in the same patient depending on the site ofinvolvement.147

1-15Adrenal insufficiency by paracoccidioidomicosys (PCM) - series of casesC.S.O. Bredt 1 ; G.L.Bredt Jr 1 ; P.A.D.Duarte 1 ; F. Pedott 2 ; T. Pereira 2 ; L. De Campos 2 . Myiamoto 2 ; A.A.Ribeiro 2 ; E.Soliva Jr 2 ; L.C.Volpolini 21 Medicine Associated Professor. Universidade Estadual do Oeste do Paraná-Cascavel-PR-Brazil, 2 Medicine Students- UniversidadeEstadual do Oeste do Paraná- Paraná -Cascavel - Brazil. email: csakuma21@yahoo.com.brBackground: In endemic areas, PCM is the most frequent etiology of Addison’s disease. Paracoccidioides brasiliensis,the causative agent of PCM, exhibits high tropism will be the adrenal glands, which results in low hormone reserves and inmore severe cases, in symptoms of primary adrenal insufficiency. Adrenal insufficiency in symptomatic you marry is describedin 10-15%. Methods: report cases of three patients with adrenal insufficiency caused by PCM. Results: 1 st case: a 75-years-old-woman, with fever, malaise, anorexia 10-kg weight lost. She was presented with hypothension and dehydrated.Clinical examination disclosed abdominal masses in bilateral renal topography. CT scan of abdome demonstrated solidinjuries in the adrenal glands. Biopsy disclosed abscess due PCM. She was submitted to a bilateral adrenalectomia.The patient was placed on antifungal therapy with B anfothericin and glucocorticoid replacement and made an excellentsymptomatic recovery. 2 nd case: a 43-years-old male had a 2-years history of progressive darkening of the skin and a 20-kg weight loss, in addition to lethargy, anorexia, nausea and hypothension. X-ray of thorax demosntrated hilar infiltratedbilateral. Sorologia for PCM 1:8 (IDD) and plasma cortisol levels was determined by radioimmunoassay of baseline bloodsamples 1,3 ug/dL. Initiate treatment with Itraconazol and prednisona with important improvement. 3 rd case: a 47-yerasold-woman, presented weakness, cutaneous darkening and 12-kg weigth lost in 1 year beyond complaint of intense avidezfor salt. She looked attendance with persistent and dispnéia cough. X-ray of thorax presented multiple bilateral areas andthe lung biopsy disclosed PCM. Plasma cortisol levels was 1,72 ug/dL and 89,2 ACTH of pg/mL. Initiate treatment withitraconazol and prednisona with good evolution. In all the cases described above there were significant improvement of thesymptoms and the quality of life. Conclusions: The authors conclude that an early detection of hipoadrenalism in patientsliving in the endemic areas is necessary and very important to minimize further adrenal damage and consequently permitsto shorter hormonal treatment in these patients.1-16Adrenal insuficiency associated to paracoccidioidomycosis: Report of three autopsy casesMantilla J.C. 1 , and Serrano A. 21 Hospital Universitario de Santander, HUS, Bucaramanga – Colombia. 2Universidad Industrial de Santander, UIS, Bucaramanga –Colombia. e-mail: eterna888@hotmail.comParacoccidioidomycosis or South American Blastomycosis is an endemic fungal infection prevalente in LatinAmerica, which is caused by a dimorphic fungus, Paracoccidioides brasiliensis. This systemic infection, primarilyinvolves lungs, from where it spreads via lymphatic or blood to other organs such as the skin, mucous membranes,lymph nodes, central nervous system and adrenal glands, among others. Objective: Describe the main pathologicfindings in three older adults with chronic progressive disease, who died at Hospital Universitario de Santander (HUS),and who were diagnosed with paracoccidioidomycosis boy medical-scientific autopsy, performed in the Departmentof Pathology, Medical School of Universidad Industrial de Santander (UIS). Materials and methods: It is presentedthe clinical-pathological report or three autopsies performed in the Department or Pathology or UIS, Bucaramanga,Colombia, during the years 2005 and 2006. Results: case: The first of these relates to a patient of 52 years of age, malegender, with clinical symptoms of a month of evolution characterized by progressive loss of weight, chills, diaphoresis,appearance of vesicles in the oral cavity, accompanied by bleeding, odynophagia, cough with white expectoration andpints of blood and progressive dysphagia, who died as a result of multiple organic failure. The second, is a patient of 53years of age, male gender, with clinical symptoms of is months of evolution characterized by occurrence of mass in theleft eye of progressive growing, who died due to a complex hydroelectrolitic imbalance characterized by dehydrationunwieldy and sustained hyperkalemia. The third case is about a man of 59 years of age, male gender, consulting porclinicals symptoms of three months of evolution represented by aletered state of consciousness with subsequent inabilityto walk, loss of sphincters control and hydroelectrolitic imbalance with hiperkalemia and dehydration unwieldy, whichwill eventually produce death. Findings of necropsy: The first case shows corpse of an elderly, male gender, withcervical, thoracic, abdominal and groin lymphadenopathy and multiple white-grayish nodules of firm consistency andappearance fibrotic in soft tissue of the thorax, pleura, lungs, pericardium, kidneys, adrenal glands and right testicle. Themicroscopic study shows inflammatory lesion characterized by multiple granulomas with abundant multinucleated giantcells type langhans, in which cytoplasm are rounded birrefringent structures with multiple budding, which correspond toParacoccidioides brasiliensis. The second patient was an elderly, male, with small curvature of the stomach and aroundthe pancreas, fibrous adhesions in left hemithorax and yellowish-white nodules in lungs and adrenal glands, whichsere observed increased of size, histopathological examination revealed chronic inflammatory process with countlessgranulomas with multinucleated giant cells type Langhans and fungal structures of Paracoccidioides brasiliensis inits cytoplasm. The third case refers to an elderly, male gender, with findings of cervical lymphadenopathy and whitegrayishnodules of firm consistency and various sizes in meninges, lungs, liver and adrenal glands, microscopicexamination revealed a chronic granulomatous inflammatory lesion widely distributed in those organs, with multiplebudding yeast of Paracoccidioides brasiliensis inside the giant cell type Langhans. Conclusion: Three autopsy casesof patients who died at HUS as a result of widespread infection by Paracoccidioides brasiliensis are presented. Thedeep hydroelectrolitic imbalance due to an adrenal insufficiency secondary to granulomatous diffuse infiltration was thedirect cause of death in these cases.148

1-17Scintigraphic evaluation of enteric protein loss in patients with activeparacoccidioidomicosisB.L. Griva 1 , R.P. Mendes 2 .1 Nuclear Medicine Service, University Hospital; 2 Tropical Diseases Área. Botucatu Medical School – São Paulo State University.e-mail: tietemendes@terra.com.br (presenting Author)Introduction: The acute subacute form of paracoccidioidomycosis is characterized by intense involvementof organs rich in the phagocytic mononuclear system, such as lymph nodes, spleen, liver and bone marrow.Some of these patients present intense involvement of the abdominal lymphatic system, with protein loss. Thesepatients can also reveal a malabsorption syndrome, becoming severe their prognostic. Methods: Ten patientswith the acute/subacute paracoccidioidomycosis form, confirmed by the identification of typical Paracoccidioidesbrasiliensis yeast forms, were included in this study. Technetium-99m labeled human serum albumin abdominalscintigraphy was performed in all patients. The radiopharmaceutical was prepared according to the manufacturer’sspecifications and the dose administered was 555 MBq IV. A control quality imaging was done immediately afterthe injection using a gamma-camera equipment. Static imaging at 30 min., 1 h, 2 h, 3 h and hourly during theday, if necessary, was performed until the visualization of the presence of radioactivity in the abdominal region.A last imaging was done after 24 h of injection. Two independent nuclear medicine physicians did the qualitativeimaging evaluation. The exam was considered positive of enteric protein loss when radioactivity was seen in anyabdominal imaging with subsequent migration at later images. Results: All the patients presented protein loss inthe 15 exams carried out near the diagnosis and during their follow up. Persistence of protein loss was observedin three patients 3, 5 and 7 years after treatment. Comments: These data show the sensibility of this scintigraphicevaluation and reveal a long period of protein loss in some cases, explaining the delay in their recovery.1-18Biliary ostruction due to acute paracoccidioidomycosis: Analisys of two casesA. S. G. Kono 1 , M. Yoshida. 1 , I. Giarolla 1 , J. Jukemura 2 , G. Benard 1,3 , M. A. Shikanai-Yasuda. 1,41Systemic Mycoses Outpatient Clinic, Division of Infectious Diseases, Hospital das Clínicas da Faculdade de Medicina da USP (HCFMUSP), Brazil. 2 Division of Clinical Surgery, HCFMUSP, Brazil. 3 Division of Clinical Dermatology, HC FMUSP. Brazil. 4 Department ofInfectious and ParasiticDiseases, FMUSP, Brazil.e-mail: masyasuda@yahoo.com.brParacoccidoidomycosis has been classified in chronic or acute/sub-acute form. The acute/sub-acute form ischaracterized by a rapid course and reticuloendothelial system involvement. In the absence of specific therapy,mortality is high. We describe two cases of acute/sub-acute PCM with abdominal lymphadenopathy and biliaryobstruction.Case 1: A 32 years-old male was hospitalized in 2002 due to fever, cough and weigh loss. He also complainedof abdominal pain. He performed an abdominal CT scan that revealed generalized adenomegaly and hepatomegalyof right lobe. A lymph node biopsy revealed chronic granulomatous inflammation with fungal elements compatiblewith P. brasiliensis. The immunodiffusion test was positive and counterimmunoelectrophoresis titer was 1:512. Hereceived sulfadiazine 6 g/day with improvement. However, he evolved with icterus and increased hepatic functiontests and a progressive hepatomegaly, despite the gradual fall in serology titers during the six years follow-up. Amagnetic resonance performed during this follow-up revealed important dilatation of intra and extra-hepatic biliarytract and hepatomegaly. Nowadays he is on waiting list for surgical intervention.Case 2: A 34 years-old male was hospitalized in 2006 complaining of fever, cervical adenomegaly, abdominalpain and ictericus. He was submitted to a lymph node biopsy that revealed paracoccidioidomycosis. He performedcervical, thoracic and abdominal CT scans that revealed a prominent and generalized adenomegaly. His serologyfor P. brasiliensis was reagent (immunodiffusion) with a counterimmunoelectrophoresis titer of 1:128. Hereceived trimetropim-sulphametoxazole. Patient evolved with important jaundice and increased hepatic functiontests. Another abdominal CT scan revealed biliary compression and common bile duct dilatation. Trimetropimsulphametoxazolewas replaced by amphotericin but the patient presented azotemia and no improvement of thejaundice. The sulfa treatment was re-started and the patient underwent a surgical procedure to decompress thebiliary tract. He evolved with complete total recovery. CT scan performed 6 months later didn’t show adenomegalyor visceromegaly, biliary dilatation. Serology titer fell to 1:8.Conclusions/Discussion: Although clinical treatment is the rule in PCM, clinicians must be aware of thepossibility of compression of the biliary tract. In these situations the surgical procedure can be crucial for therecovery of the patient.149

1-19Serious hepatic damage in paracoccidioidomycosis and its treatmentR Lazo 1 – 2, J Barrezueta 1 – 2, J Lazo 1 – 31.Centro de Investigaciones de Enfermedades Parasitarias y por Hongos. CIDRALAS – Ecuador, 2. Cátedra de Micología, Escuela deMedicina, Facultad de Ciencia Médicas, Universidad de Guayaquil. – Ecuador, 3. Disciplinas de Biología Celular / Curso Post Graduaciónen Patología de la Universidad Federal do Triangulo Mineiro – Uberaba, MG – Brasil.e-mail: rlazo@cidralas.med.ecWith previous experiences on Sistemic Mycosis and Amphotericine B, we use this drug on patients with severehepatic compromised, that is hepatotoxic and nephrotoxic being a contraindicated treatment. We considered thatis an opportunity to report the use of Amphoticin B on seriously ill patients with cirrhotic syndrome. We receivethe request for to examine a male patient with 26 year, tractor driver on sugar cane plantations. After 25 daysof treatment for pulmonary tuberculosis in Pulmonary Diseases Hospital presented small pustules on the back.It was made the direct microscopy examination of material obtained from the lesion and sputum, finding multiplegemation yeasts , which features corresponding to Paracoccidioides brasiliensis. The Inmunodifusion test waspositive to Paracoccidioidomycosis. It was recommended the suppression of Isoniazina, and it was not accepted.Few days after it presented marked abdominal distention. The paracentesis did not improved the symptoms. Afterthat, he was derived to our service and completely evaluated. The results of hepatic biopsy reported cirrhosis andyeast with multiple sporulation stained by Grocott method. The same was reported in the ganglionic biopsy. By thisway, it was confirmed the linfatic ganglionic visceral dissemination and seriously ill. We administered AmphotericinB, follow this Sulfametoxasole and then Sulfamotoxipiridazine. The clinical respond to the therapeutic was quick.At the First Symposium of Paracoccidioidomycosis, Medellín 1971, we commented the first findings and weproposed to study and to determine the relationship with Paracoccidioidomycosis cirrhotic syndrome. At 1982, inthe book Paracoccidioidomicose (Gildo del Negro et al.) is quoted by a new propose for the clinical diagnosis ofParacoccidioidomycosis. At 1994, in the book Paracoccidioidomycosis (Marcelo Franco et al.) is quoted the samepropose. At 2006, in Thesis for Public Health Master grade, Lazo R. F. indicated the degree of therapeutic respondto Amphotericine B, after review of 48 patients with systemic mycosis.1-20Acute respiratory failure in AIDS patients: Role of open lung biopsy to the diagnosis ofparacoccidioidomycosis (PCM)C.S.O. Bredt 1,3 ; G.L.Bredt Jr 1 ; M. Campos 1 ; D. Pavan 1 ; P. A.D. Duarte 1; F. Pedott 2 ; T. Pereira, 2 ; L. de Campos 2 ; ;D.M.Moraes 3; ; M.A.Richetti 31Associated professor. Universidade Estadual do Oeste do Paraná, Cascavel-PR- Brazil. 2 Medicine Student’s Universidade Estadual doOeste do Paraná, Cascavel-PR- Brazil. 3. Hospital Acquired Infections Practitioners. 3 e-mail: csakuma21@yahoo.com.brBackground: PCM is a systemic mycosis from Latin America. Rural regions in Paraná State (in SouthernBrazil) are notoriously endemic to this entity (Brazilian Society of Infectious Diseases). Methods: Case reports andliterature review of 3 cases of patients with co-infection of AIDS and PCM with Acute Respiratory Failure (ARF) in aRural University Hospital diagnosed by open lung biopsy. Results: 1 st case: Male, 40 y old, heterosexual, from ruralregion. He was admitted with a history of consumptive syndrome, fever, cough and and dispnea. He had previousdiagnosis of AIDS since 06 years ago without specific treatment. Chest X-Ray showed bilateral micronodularinterstitial infiltrate; pancitopenia; hypoxemia in the arterial gasometry at room air, CD4=103 mm 3 and viral load:59.400 cop/ml. It was started empiric treatment for pneumocystosis and tuberculosis. Lung biopsy revealed P.brasiliensis. PCM serology was 1:8 (IDD). Treatment was done with Amphotericin B. 2 nd case: Male, 32 y old,homosexual, admitted with a neurological disease with a diagnosis of neurocryptococosis, HIV antibodies werepositive. His chest X-ray had bilateral reticulous-nodular infiltrate. Lung biopsy showed association of pulmonarycryptococosis and PCM. 3 rd case: Female, 36 y, AIDS diagnosis since 05 years ago, without specific treatment.She was admitted at Emergency Dept with ARF. Chest X-ray with bilateral interstitial-alveolar infiltrates. Lungbiopsy revealed PCM. The three cases showed distinct radiological patterns, and in despite of all they receivedtreatment with Amphotericin B (the 1 st choice drug for severe cases), all of them evaluated to death. Conclusion:In despite of the great benefit of HAART (high active antiretroviral therapy) significantly reducing the incidence ofopportunistic infection (OI) in AIDS, there are still a lot of inpatients with non adherent patients or new diagnosiswith OI. The authors concluded that lung biopsy is very important to confirm etiology of pulmonary infections inAIDS patients, especially in areas well known to be endemic to PCM.150

1-21Paracoccidioidomycosis in a renal transplant recipient:Case report and review of the literatureAdriana S. G. 1 , M. M. Galvão 2 , M. Yoshida 1 , I. Giarolla. 1 , F. O. S, França 1 , G. Benard 1,3 , L. E. Ianhez 2 , M. A., Shikanai-Yasuda. 1,41Systemic Mycoses Outpatient Clinic, Division of Infectious Diseases, Hospital das Clínicas da Faculdade de Medicina da USP(HCFMUSP),Brazil. 2 Division of Urology, HCFMUSP, Brazil. 3 Division of Clinical Dermatology, HCFMUSP, Brazil. 4 Department of Infectious andParasiticDiseases, FMUSP, Brazil.e-mail: masyasuda@yahoo.com.brBackground and aims: Paraccoccidioidomycosis (PCM) is the most prevalent endemic mycosis in LatinAmerica but the disease is rare in immunocompromised patients, especially in transplant recipients. So far, thereare only seven cases reported in literature; all of them in renal transplant recipients. The aim of this report was todescribe another case of PCM.Case report: A 59 years old male patient who received a cadaveric renal transplantation 13 years before washospitalized due to a recent history of cough and pulmonary nodules. The initial hypotheses were lung canceror lymphoma. During his stay in hospital, he was submitted to a trans-bronchial biopsy that revealed a chronicgranulomatous inflammation with fungal elements compatible with P. brasiliensis. He was in use of prednisone 20mg/day, cyclosporine 300 mg/day. He received trimethoprim-sulphamethoxazole 160/800 mg twice a day and wasdischarged with resolution of the cough and afebrile. After 18 months of follow-up he remains asymptomatic andwith complete resolution of the pulmonary lesions.Conclusions/Discussion: This is the eighth case of PCM in renal transplant recipient reported to date. Theuse of primary prophylaxis for P. jirovecii with trimethoprim-sulphamethoxazole may explain the scarcity of casesof PCM in immunocompromised patients such as organ transplant recipients and aids patients. However, cliniciansmust be aware of this possibility when evaluating patients from endemic areas.1-22Pericardic paracoccidioidomycosis (PCM) associated with pulmonary neoplasia:Case reportC.S.O. Bredt 1 ; G.L.Bredt Jr 1 ; P.A.D.Duarte 1 ; A.A.Luiz 1; F. Pedott 2 ; T. Pereira 2 ; L. De Campos 2 . Myiamoto 2 ;A.A.Ribeiro 2 ;E.Soliva Jr 2 ; L.C.Volpolini 21 Medicine Associated Professor. Universidade Estadual do Oeste do Paraná-Cascavel-PR-Brazil. 2 Medicine Students- UniversidadeEstadual do Oeste do Paraná- Paraná -Cascavel - Brazil.e-mail: csakuma21@yahoo.com.brBackground: PCM is the most common systemic mycosis of the South America. The chronic form hasmanifestations pulmonary and / or extra pulmonary , that can be varied and hamper or delay the diagnosis.Case report: Male, 57 years old, resident of the rural area, longtime smoker, there are 30 days showsprogressive dyspnea to the efforts, chest pain, high intensity with radiation to the back and left shoulder andworsening in dorsal position and deep inspiration, 4-kg weight lost in this period. The examination showed uphypotensive, tachycardia, cardiac auscultation with sound rhythmic and muffled without further changes. The ECGshowed diffuse repolarization disorder. The echocardiogram showed paradoxical movement of interventricularseptum, impairment of systolic and diastolic myocardial functions and pericardial effusion causing major cardiactamponade. The patient was submitted to a pericardiocentesis with biopsy and drainage of 1800 ml. The studyshowed a pathological process chronic granulomatous compatible with paracoccidioidomycosis. The patientwas treated with anfotericina B. Twenty days after, the patient evaluated to dyspnea again and pulmonaryauscultation were abolished in both lung bases and lymph nodes supraclavicular palpable. The patient was thensubjected to thoracentesis whose cytological analysis showed consistent with adenocarcinoma cells. The biopsyof supraclavicular lymph node also showed adenocarcinoma little differentiated tubular suggesting lung origin.Conclusion: It is trying to do with the possibility of atypical presentations of PCM, especially in endemic areassuch as the western region of the state of Paraná in Brazil. Early diagnosis and appropriate treatment has a majorimpact on quality of life.151

1-23Disseminated Herpes simplex infection in a patient with acute/subacute paracoccidioidomycosisB.S. Souza 1 , I.X. Duarte 2 , M.P.T. Moraes 2 , K.Y.R. Coelho 2 , B.L Griva 3 , R.P. Mendes 1 .1Tropical Diseases Área; 2 Department of Pathology, 3 Nuclear Medicine Service – Botucatu Medical School – São Paulo State University.e-mail: tietemendes@terra.com.br (presenting Author)Introduction: Paracoccidioidic infection and paracoccidioidomycosis are highly prevalent in Botucatu Region(São Paulo State, Brazil), where severe cases and rare coinfections, such as this study, have been observed.Case report: A 21-year-old white housewife was hospitalized in October 2000, with a 2-month history ofincreasingly epigastric pain, cervical and inguinal lymph node enlargement, a 7.0kg-weight loss and, in the last7 days, fever of 38-39 o C, jaundice, nausea and vomiting. Physical examination revealed bad general condition,weight of 43.5 kg, blood pressure of 100/60 mmHg, pulse rate of 102/min, respiratory rate of 31/min, temperatureof 37.6 o C, conjunctival jaundice, cervical adenomegaly of the tumoral type, and hepatosplenomegaly. Abdominalcomputed tomography showed mild dilatation of the intrahepatic biliary tree and enlarged lymph nodes - hepatichilum, peripancreas and retroperitoneum. Product of fine needle aspiration of a superficial lymph node showedtypical yeast P. brasiliensis forms. As her condition improved with amphotericin B she was followed up as outpatient,with frequent readmissions due to lack of compliance to antifungal treatment and for nutritional support. An intestinalprotein loss was confirmed in October 2003. At her last admission she presented vomiting, pain in the righthypochondrium, bad general condition, weight of 36.6 kg, malnourishment, dehydration, blood pressure of 100/70mmHg, pulse rate of 120/min, and respiratory rate of 28/min. Active paracoccidioidomycosis, intestinal occlusionand pneumonia were diagnosed and treated. However, she died on March 24, 2006, despite the introductionof intravenous cotrimoxazole, appropriate diet, antibiotics and general measures of support. Autopsy showedactive paracoccidioidomycosis in lungs, epiglottis, larynx, liver, spleen, bone marrow, peritoneum, lymph nodesand intestinal mucous membrane; intestinal perforation and purulent peritonitis; laryngitis and intense necrotizingbronchopneumonia by Herpes simplex, positive for peroxidase antiperoxidase stain. Discussion: This case showsthe intense immunosupression induced by the acute/subacute paracoccidioidomycosis with abdominal lymphnodes and bowels involvement, associated with protein malabsorption syndrome. Immunosupression triggeredthe development of severe herpetic lesions in lungs and epiglottis, since she was infected by Herpes simplex, asmost of the population.1-24Lung paracoccidioidomycosis: Analysis of clinical, tomographic and functional impairmentafter adequate treatmentCosta AN 1 , Fernandes CJCS 1 , Suesada M 1 , Salge JM 1 , Kairalla R 1 and Carvalho CRR 1 .1.University of São Paulo School of Medicine. Pulmonology Service. Interstitial Lung Diseases Group. São Paulo – Brasil. e-mail: nathan.andre@gmail.com (presenting Author)Paracoccidioidomycosis is the most commonly found systemic mycotic disease in South America, in whichlungs are the main organs affected. Eighty percent of world cases are found in Brazil and are responsible forover 200 deaths per year. Nevertheless the extension of disability due to treated lung paracoccidioidomycosisis not known. We report a case series of tomographic, quality of life, functional and ergo-spirometric evaluationof adequately treated patients with lung paracoccidioidomycosis. In our service we have diagnosed in the periodof 1999 to 2005 eleven cases of lung paracoccidioidomycosis. Eight patients have completed the treatment andperformed the proposed protocol (CT scan, spirometric tests, diffusion capacity, 6MWT, cardiopulmonary exercisetest and Saint-George Respiratory questionnaire). The patients mean age was 63 ± 9 years-old and the periodbetween the end of the treatment and the protocol was 4 ± 2,4 years. Seven patients were former smokers. Thespirometric values found were FVC 3,98 ± 0,75 L (105 ± 14 % of predicted); FEV1 2,67 ± 0,47 L (89,2 ± 11 % ofpredicted); TLC 6,24 ± 1,03 L (101 ± 8 % of predicted), RV 2 ± 0,67 L (90 ± 26 % of predicted) and diffusion capacityof 84 ± 22 % of predicted . The mean distance performed in 6MWT was 500 ± 63 mts. The mean peak VO2 foundin the cardiopulmonary exercise test was 24,8 ± 5,1 ml/kg (90 ± 3 % of predicted). The relative SGRQ was 8,5 ±7,26. The CT scan findings were micronodules (4 patients, one with calcified nodules), air trapping (4 patients),apical retraction and fibrosis (3 patients) ground glass infiltrates, calcified hilar and mediastinal lymphonodes,paraseptal and centrolobolar emphysema (2 patients), bronchial thickening, septal and peribronchovascularinterstitium thickening (1 patient).Therefore in this case series adequately treated lung paracoccidioidomycosis,despite the radiologic sequelae, did not cause late disability in patients. Only a minor obstructive pattern wasfound in lung functional tests without influence in the 6MWT, cardiopulmonary exercise test and in the quality oflife evaluated by the SGRQ.152

Poster Session2Diagnosis

2-01Diagnosis of paracoccidioidomycosis in patients attended in routine services of a universityhospitalT.C. Moreto 1 , M.E.A. Marques 2 , M.L.S.C. Oliveira 2 , D.V. Moris 1 , L.R. Carvalho 3 , R.P. Mendes 1 .1 Tropical Diseases Área, 2 Department of Pathology – Botucatu Medical School – São Paulo State University.3 Biostatistic Department– Biosciences Institute – São Paulo State University. e-mail: tamoreto@yahoo.com.br (presenting Author)Introduction: Identification of appropriate laboratory measurements for confirmation of a clinical impressionis important in a routine paracoccidioidomycosis patients’ medical care and constituted the objective of this study.Methods: Clinical records and laboratory cards of 401 paracoccidioidomycosis patients with acute/subacute orchronic form attended in the Tropical Diseases Area – Botucatu Medical School (São Paulo State, Brazil) during theperiod 1974-2008 were reviewed. Their laboratory records from Department of Pathology and Tropical DiseasesResearch Laboratory were also reviewed. Direct mycological examination, cell block preparation stained by Gomori-Grocott, histopathological examination of tissues stained by hematoxylin-eosin and Gomori-Grocott, and specificantibodies serum levels carried out by double agar gel immunodiffusion test using P. brasiliensis culture filtrate asantigen were evaluated before treatment. McNemar, Tukey and chi-square tests were used in the statistical analysis;significance was set up at p0.05); 3) immunoblotting sensitivity was higher than DIDr and DID 1tests, with gp43 or gp70 (respectively, 45.8%versus 100%; 45.8% versus 96.6%; 47.5% versus 96.6%; 47.5% versus 96.6%; p0.05), but a lower sensitivity (0.0% versus 78.1%) with gp70 (p=0.02). These findings suggest that ourresults with DIDr test are limited by the sensitivity of the method itself and are not related to the antigen. In addition,negative serum on DID tests should be evaluated by immunoblotting with gp43, due to its higher sensitivity.155

2-03Combined use of Paracoccidioides brasiliensis recombinant 27 and 40-kilodalton antigens in anenzyme-linked immunosorbent assay for immunodiagnosis of paracoccidioidomycosisFernandes. V.C. 1 , Coitinho, J. B. 1 , Fonseca, J.H. 1 , Veloso, J.M.R. 2 , Araújo, S.A. 2 , Pedroso, E.P. 2 , and Goes. A.M. 1 .1 Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, UFMG, Belo Horizonte, MG - Brasil. 2 Faculdade deMedicina, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG - Brasil. e-mail: cfernandes.viviane@gmail.comParacoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it’s usuallydiagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by avariety of immunological methods, such as complement fixation and immunodifusion. Although these approachesare useful, historically their sensitivity and specificity have often been compromised by the use of complex mixturesof undefined antigens. The use of combinations of purified, well-characterized antigens appears preferable andmay yield good results. Accordingly, an indirect enzyme linked immunosorbent assay (ELISA) was design bycombination of two P. brasiliensis recombinant antigens, 27kDa and 40-kDa-molecular-mass for diagnosis andfollow-up PCM patients. The cDNA of 40-kDa protein was cloned in pET-21a vector and was expressed in aprokaryotic system as a protein with 378 amino acids. The cDNA of pb27 was cloned in pET-DEST 42 vector andwas also expressed in a prokaryotic system. A total of 109 PCM sera, 62 sera from patients with other diseasesand 22 sera from healthy individuals were studied. Detection of anti-pb27 and anti-pb40 antibodies in sera ofpatients with PCM by ELISA using a combination of the two purified proteins showed a sensitivity of 96% with aspecificity of 100% in relation to control non-infected human sera and 93,5% to sera from patients with diverseinfections. These results demonstrated an increase in sensitivity and specificity compared to results when theantigens were used separately. Thus, the use of combinations of well-defined antigens appears to offer a lot offadvantages over the use of single antigens for diagnosis of PCM. Patients undergoing treatment for more than 1year showed a reduced antibody response against pb27. These results suggest that the presence of anti-pb27antibodies might be an indicator of active disease. Financial support: FAPEMIG and CNPq2-04Laboratory measurements for therapeutic monitoring of paracoccidioidomycosis patientswith different clinical formsR. Cavalcante 1 , D.V. Moris 1 , L.R. Carvalho 2 , R.P. Mendes 1 .1Tropical Diseases Area – Botucatu Medical School – São Paulo State University; 2Biostatistic Department – Biosciences Institute – SãoPaulo State University. e-mail: mip.ricardo@gmail.com (presenting Author)Introduction: Treatment control and length of paracoccidioidomycosis are an evolving field. As clinical cure isearly reached and latent fungal cells persist after successful therapy, other criteria of cure have to be evaluated.Thus, the study of clinical and laboratory parameters in the follow up of paracoccidioidomycosis patients with differentclinical forms is our objective. Methods: Clinical records of 88 patients with confirmed paracoccidioidomycosisattended in Tropical Diseases Area – Botucatu Medical School and their laboratory cards were reviewed.Classification of clinical forms was performed according to Mendes (1994). Specific antibodies serum levels werecarried out by double agar gel immunodiffusion test using culture filtrate as antigen. Erythrocyte sedimentation rate(ESR), gamma-globulin (GG), α 1-acid glycoprotein (α 1GP), C-reactive protein (CRP) and mucoprotein (MCP) wereevaluated according to its sensitivity and time, in months, for regression to normal values. Data were presented asmedians. Kruskal_Wallis, chi-square and McNemar tests were used in the statistical analysis; significance was setup at pMCP>CRP=GG=α 1GP. ESR showed higher sensitivity [G1>(G2=G3); p

2-05Espirometric evaluation in patients with paracoccidioidomycosis (PCM)C.S.O. Bredt 1 ; G.L.Bredt Jr 1 ; P. A.D. Duarte 1; F. Pedott 2 ; T. Pereira, 2 ; L. de Campos 2 ; F. Suzin 31Associated professor. Universidade Estadual do Oeste do Paraná, Cascavel-PR- Brazil. 2 Medicine Student’s Universidade Estadual doOeste do Paraná, Cascavel-PR- Brazil. 3. Fisioterapeuta. e-mail: csakuma21@yahoo.com.brBackground: PCM is an endemic disease that causes a mortality rate of 1, 45 cases per million inhabitantsBrazilians. The chronic form represents the vast majority of cases, reaching mostly adults, knowing that the lungis one of the body most affected the identification of pulmonary sequel by spirometry, helps to characterize theseriousness of the framework and guidance therapy.Methods: 22 patients treated with itraconazole by recommendations of The Brazilian Consensus ofParacoccidioidomycosis (edited by The Brazilian Infectious Diseases Society – 2006 ) were included in this study.The patients were submitted to a spirometry evaluation and was utilized the portable equipment. The parametersevaluated were forced vital capacity (FVC), forced expiratory volume in one minute (FEV1), and the relationshipFVC/FEV1. The data were interpreted in accordance with the recommendations of the Brazilian guidelines forpulmonary function tests, 2000. The assessment was made after 15 minutes of the use of salbutamol 400 µg.Results: 22 patients were evaluated, 17 (77.27%) were male. There was standard obstructive in 16 patients(72.72%) and restrictive pattern in 2 (12.5%). Only 2 patients had no disorder. The answer occurred after theuse of bronchodilators was significant only in 6 patients (27.27%), and not significant in 16 patients (72.72%).Conclusion: The study concluded that most patients have pulmonary sequel of PCM spirometry and that is veryimportant not only to quantify the damage but also to direct the doctors the best clinical management of thesepatients. The bronchodilators may be useful to minimize the obstruction pulmonary and consequently improve thequality of life of these patients but most patients do not beneficial with the single use of this medication and needsan alternative or adjunctive therapy.157

Poster Session3Epidemiology / Ecology

3-01Determination of Paracoccidioides brasiliensis reservarea mapping the migratory story of128 patientsT.C. Moreto 1 , Barrozo L.V. 2 , Bueno R.A. 1 , Moris D.V. 1 , Mendes R.P. 11Tropical Diseases Department, Botucatu Medical School – São Paulo State University, Botucatu, Brazil. 2 Department of Geography,School of Philosophy, Literature and Human Sciences – University of São Paulo, São Paulo, Brazil. e-mail: tamoreto@yahoo.com.brIntroduction and Objectives: The long latency period observed between infection and clinical manifestationsof disease makes it difficult to determine the fungal reservarea, i.e., area where the patients were infected. The aimof this paper was to study the migratory profile of 128 paracoccidioidomycosis (PCM) patients assisted at BotucatuUniversity Hospital, to determine the fungal reservarea.Methods: Interviews were carried out with 128 patients about their place of birth, municipality of residenceat the disease onset, first, second and third municipalities in the rural area of the longest periods of residenceduring their entire lives. Using geoprocessing techniques, several maps were made: place of birth, place of thelast residence, place of birth matching the last residence and, place of birth matching the longest and the lastmunicipality of residence.Results: Out of 128 patients, 101 (78.9%) were born in 58 municipalities in São Paulo State. All 128 patientslived in a municipality in this State at the time of disease onset, corresponding to 56 municipalities. Sixty-fivepatients (50.8%) had the same place of birth and last residence, and 87 (68.0%) had spent more time at the sameplace of their last residence. Finally, 62 (48.4%) patients were born, had the longest and the last residence in 31municipalities in São Paulo State.Discussion: The migratory profile of our patients suggested as reservarea a geographic arrangementpredominantly in the SW-NE direction, confirming previous studies based only on their municipality of residenceat the disease onset.3-02Space-time cluster of acute/subacute paracoccidioidomycosis bearing relationship withthe 1982-83 El Niño episode in a hyperendemic area in BrazilL.V. Barrozo 1 , Mendes, R.P. 2 , Marques, S.A. 2 , Benard, G. 3 , Silva, M.E.S. 1 , Bagagli, E. 41 Faculdade de Filosofia, Letras e Ciências Humanas, USP, São Paulo – Brasil. 2 Faculdade de Medicina de Botucatu, UNESP, São Paulo- Brasil. 3 Faculdade de Medicina, USP, São Paulo – Brasil. 4 Instituto de Biociências, UNESP, São Paulo -Brasil. e-mail: lija@usp.brIntroduction and Objectives: The aim of this work was to analyze the space-time pattern of the series ofacute/subacute cases, in a highly endemic area in Botucatu neighboring, São Paulo State, Brazil, from 1966-99,through the application of an inference test for the detection of clusters. The possible associations between thecluster and climate factors were also investigated.Methods: PCM data (n = 91) corresponded to cases seen in the Infectious Diseases and Dermatology Sectorsof the UH-UNESP. A spatial scan statistic was applied. Cases were assumed to be Poisson distributed, adjustedfor age and gender, with constant risk over space and time under the null hypothesis. The analysis was set toinclude up to 50% of the total population at risk and up to three years of temporal window. Maps and exploratoryanalyses were made between incidence and soil water storage.Results: A significant excess risk was found during 1983-85 (P = 0.0077). There was a non-significant cluster in1998. The distribution of the total precipitation along time shows an important peak above two standard deviationsin the years 1982/83, preceding the high-risk period. Maps depict the spatial pattern of the soil water storage from1981-86 and were overlaid by cluster localization, suggesting association between excess soil water storage inthe preceding years and the cluster.Discussion: Our study area is in the region of ENSO (El Niño-Southern Oscillation) influence and experiencesgeneral increase in precipitation during El Niño years, especially during intense events. The space-time clusterseems to be related to the most intense El Niño event occurred during the past 50 years, in 1982/83. The secondmost intense in 1997/98, could be also related with the non-significant cluster of 1998. Our model for acute/subacute PCM incidence (r 2 =0.49, P

3-03Spatial distribution of chronic paracoccidioidomycosis in a hyperendemic area in BrazilL.V. Barrozo 1 , Gonzalez, C.R. 1 , Santana, M.S. 1 , Mendes, R.P. 2 , Marques, S.A. 2 , Bagagli, E. 31 Faculdade de Filosofia, Letras e Ciências Humanas, USP, São Paulo – Brasil. 2 Faculdade de Medicina de Botucatu, UNESP, São Paulo– Brasil. 3 Instituto de Biociências, UNESP, São Paulo – Brasil.e-mail: lija@usp.brIntroduction and Objectives: The aim of this work is to analyze the spatial pattern of the series of chroniccases admitted to the University Hospital of the São Paulo State University (UH-UNESP), in a highly endemicarea in Botucatu neighboring, São Paulo State, Brazil, from 1966 to 2006, through the application of an inferencetest for the detection of clusters.Methods: Human chronic PCM data corresponded to cases seen in the Infectious Diseases and DermatologySectors of the UH-UNESP. Only the cases with residence in the study area (n = 382) were selected. A spatialscan statistic was applied. Cases were assumed to be Poisson distributed, adjusted according to age andgender, with constant risk over space under the null hypothesis. The analysis was set to include up to 50% of thetotal population at risk and to find clusters of excess and low risks. Results were mapped using geoprocessingtechniques.Results: A significant excess risk was found in the following municipalities: Botucatu, São Manuel, Pratania,Pardinho, Areiópolis, Anhembi, Igaraçu do Tietê, Bofete and Itatinga (P = 0,0001). This cluster presented a meanincidence of 2.7 annual cases/100,000 inhabitants. A secondary cluster of excess risk occurred in CerqueiraCésar (P = 0,001), with mean incidence of 4.5 annual cases/100,000 inhabitants. Important low risk cluster wasfound in Cabrália Paulista, Lucianópolis, Espírito Santo do Turvo, Piratininga, Agudos and Santa Cruz do RioPardo (P = 0,0001) with mean incidence of 0.4 annual cases/100,000 inhabitants.Discussion: This approach of looking chronic cases separately can be very interesting because acute/subacute cases may be associated with temporal variability deriving from land use changes or climate anomalies,which do not represent the mean conditions of the entire period. In Brazil, estimates based on both clinical formsindicate an annual incidence rate of 1-3 per 100,000 inhabitants. Comparing the incidences for chronic casesfound here with that, Cerqueira César could be considered highly endemic while the low risk cluster is below theannual incidence rate in Brazil. Results indicate that this study area shows two opposite situations, which allowscomparing their conditions. Supported by FAPESP, PRP/IC-USP and CNPq.3-04Liquid urease test in human and animal isolates of Paracoccidioides brasiliensisS. A. G. Macoris 1 , Bosco, S. M. G. 2 , Theodoro, R. C. 2 , Richini-Pereira, V. 2 and Bagagli, E. 21 Instituto Adolfo Lutz, São Paulo - Brasil. 2 Depto. de Microbiologia e Imunologia, IBB- Unesp, Botucatu, São Paulo - Brasil.e-mail: massis@ibb.unesp.brP. brasiliensis is a thermo-dimorphic fungus and causes Paracoccidioidomycosys, the most prevalenthuman systemic mycosis in South America. Some aspects of its natural and parasitic life cycle are stillunknown. One important aspect of this interaction with the human and animal hosts is the virulence, whichwas evaluated by some experimental models. The metabolism of urea is associated with other fungalpathogens, such as Cryptococcus neoformans and Coccidioides immitis, with degrees of infection, leadingto tissue damage or compromised functionality of the affected tissue.To determine the role of urease activity of P. brasiliensis, if this enzymatic system may have someimportance for its pathogenicity, we have evaluated this activity in different isolates of P. brasiliensisobtained from human and animals hosts, belonging to the S1, PS2, PS3 and Pb01-like, genetic groups. Itwas employed the Cristenssen Urea Test and liquid urease test, described by Maslen, in order to evaluatethe urease activity of these isolates. It was observed that the liquid urease test showed to be better ratherthan Cristenssen test concerning to time and confiability of the results to discriminate the organism thathydrolyze the urea substrate. The liquid urease test was also able to discriminate some isolates whichpresented different levels of urease production. The association with genetic groups, virulence and sourceof the isolates are discussed.162

Poster Session4Genomics,Proteomics,cellular Biology andmolecular Biology

4-01Towards a molecular genetic system for the pathogenic fungusParacoccidioides brasiliensisAlmeida, A.J. 1 , Carmona, J.A. 1 , Cunha, C. 1 , Carvalho, A. 1 , Rappleye, C.A. 2† , Goldman, W.E. 2 , Hooykaas, P.J. 3 , Leão, C. 1 ,Ludovico, P. 1 , Rodrigues, F. 11. Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal. 2. Departmentof Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA. 3. Institute of Biology, Leiden University,Clusius laboratorium, Leiden, The Netherlands.e-mail (presenting author): ajalmeida@ecsaude.uminho.ptWe herein report the development of a molecular toolbox for the dimorphic fungus Paracoccidioides brasiliensis,specifically a more efficient transformation and a gene expression system. We evaluated several parametersthat influence Agrobacterium tumefaciens-mediated transformation (ATMT), such as co-cultivation conditions andhost cell susceptibility. Our results show that cellular recovery and air drying of A. tumefaciens:P. brasiliensismixtures are essential for ATMT. Overall, our data indicate a transformation efficiency of 78±9 transformants/cocultivation(5±1 transformants/10 6 target cells). P. brasiliensis GFP-expressing isolates were also constructed byinsertion of the GFP gene under the control of several fungal promoters. RT-PCR, epifluorescence microscopyand flow cytometry analysis revealed Gfp visualization for all studied promoters but without significant differencesin fluorescence and gene expression levels. Moreover, we present evidence for the occurrence of random singlegene copy integration per haploid nuclei and the generation of homokaryon progeny, relevant for the future use intargeted mutagenesis and linking mutations to phenotypes.Acknowledgments: Almeida, A.J. and Carvalho, A. and the majority of the work were financially supported bya fellowship (SFRH/BD/8655/2002 and SFRH/BD/11837/2003) and a grant (POCTI/ESP/45327/2002) from FCT,Portugal.4-02RNA interference: A potential tool to study gene function in Paracoccidioides brasiliensis- strategies to construct silencing cassettesL. Fernandes 1 , Paes H.C. 1 , Teixeira M.M. 1 , Rodrigues A.C.J. 1 , Viana D.F. 1 , Torres F.A 1 and Felipe M.S.S. 11Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília - UnB, Brasília, DF, Brazil.email: larissaf@unb.brGenetic manipulation is an important tool in general biology, evolution and the study of pathogenicity, aswell as to the identification of new therapeutic targets in fungal pathogens. Transformation systems have beendeveloped to a variety of fungi, including Paracoccidioides brasiliensis. Due to its high number of nuclei per cell,slow growth and low efficient transformation ratios, classic experiments of gene knock-out seem unfeasible in thisfungus. The RNA interference mechanism appears a good strategy to circumvent these limitations. Through an insilico search on P. brasiliensis genome database (Broad Institute) we identified all components of RNAi machinerycommon to other fungi: Dicers (dcl1 and dcl2), RISC components ago1/qde2, ago2/sms2, the RNA-dependentRNA polymerase, RdRP (qde1) and the DNA helicase (qde3). Sequence alignments revealed high degree ofconservation, indicating that the RNAi phenomenon should be present in this pathogen. Based on previous usageof RNAi to study gene function in fungi, we decided to explore the construction of cassettes to silence geneexpression in P. brasiliensis. We cloned all cassettes on the pBluescript KS ® between the cpc-1 promoter fromNeurospora crassa and the trpC terminator from Aspergillus nidulans, both of which have already been tested forP. brasiliensis. Furthermore, the cassettes were amplified using specific primers, digested with BclII and clonedinto the pAD1625 vector, which is suited to Agrobacterium-mediated transformation and contains the dominantselectable marker hph (Hygromycin B resistance). Goldoni (2005) demonstrated that the presence of an intron onthe loop increases the silencing efficiency in N. crassa by favouring hairpin formation in vivo after intron splicing.We developed three different strategies to construct the silencing cassettes: (1) one contains a loop of 100 bp andan intron of 163-bp; (2) another harbours just the loop and the last (3), just the intron. Another important feature ofthese cassettes is that they constitute a chimerical vector, since they contain our gene of interest and also invertedrepeated fragments of ura3 gene to use 5- fluororotic acid counter-selection to select for recombinants. The use ofRNAi in P. brasiliensis will certainly contribute to a better understanding of this pathogen.Financial support: CNPq.165

4-03Development of vector-based RNA interference (RNAI) for ade2 silencing on the pathogenicfungus Paracoccidioides brasiliensisPolez, V.P.P. 1 ; Fernandes, L. 2 ; Torres, F.A.G 2 & Felipe, M.S.S. 21Embrapa Recursos Genéticos e Biotecnologia, Brasília, Brazil. 2 Instituto de Ciências Biológicas, Universidade de Brasília, UnB, Brasília,DF, Brazil. e-mail: larissaf@unb.brRNA interference (RNAi) has revealed a powerful tool for transcriptional gene silencing mediated bysequence specific mRNA depletion. The multinucleated and multi-budded nature of the pathogenic fungusParacoccidioides brasiliensis have been pointed out as the reasons for the limited application of the geneknock out. To implement the potential technique RNAi, we construct a vector carrying the inverted repeatedsequences of ade2 to silence this gene in P. brasiliensis, since the loss of function of ade2 gene, whichcodifies a phosphoribosylamino-imidazole-carboxylase, promotes a red pigmentation of the colonies on limitedadenine medium, easily detected for visual screening. The silencing cassette for ade2 was amplified from thepCR79 – originally used in Histoplasma capsulatum. It presents the CBP1 (calcium binding protein) promoterfrom H. capsulatum and the ade2 inverted repeated from Aspergillus oryzae. The ade2 silencing cassettewas cloned into the pAD1625, a vector designed for Agrobacterium mediated transformation system, whichcontains the Hygromicin B resistance marker, and was called pAD1627. The pAD1627 vector was transformedinto the EHA105 and LBA4404 Agrobacterium strains by electroporation. Further, those Agrobacteriumstrains containing the silencing cassettes were used to genetic transform P. brasiliensis (Pb113) yeast cells.On the BHI rich medium, we identified Hygromycin B resistant colonies which were also confirmed by PCRamplification of the hph gene. The red pigmentation phenotype characteristic of the ade2 loss of function isbeing investigated by replica-plating the Hygromycin B resistant colonies on DYP, which is a limited adeninemedium. The appearance of red colonies certainly will validate the RNA interference phenomenon in the multibuddingyeast P. brasiliensis as a potential tool to the studies of gene function in this pathogen. Financialsupport: CNPq, MCT, FUB.4-04Protocol for establishment of Paracoccidioides brasiliensis RNA recovery from humanpneumocytes cell cultureA. Oliveira 1 , Silva, S.S. 1 , Souza C.R. 1 and Felipe M.S. 11Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazile-mail: HJaol@gmail.com (presenting Author).Paracoccidioides brasiliensis is the etiological agent of the most common systemic mycosis in Latin America,paracoccidioidomycosis. The yeast form of P. brasiliensis acts as a facultative intracellular pathogen, beingable to survive and replicate within macrophages and epithelial cells, such as pneumocytes, as a probablemechanism to evade the immune system. Recently, our group used cDNA microarray technology to find out theearly transcriptional response of P. brasiliensis to the inner environment of peritoneal murine macrophages. Thisapproach revealed that in response to the harsh macrophage microenvironment P. brasiliensis up-regulates genesrelated to the detoxification of oxidative radicals and amino acid biosynthesis. Also, genes encoding enzymes of theglycolytic pathway was down-regulated, suggesting the ability of the fungus to survive in the hostile macrophagemicroenvironment. To investigate the pathogen response to the non phagocytic cell we will analyze the interactionbetween the fungus and A549 epithelial cells during infection by cDNA microarrays. In this context, the objective ofthis study was to establish a protocol for recovery the P. brasiliensis RNA from human pneumocytes. P. brasiliensisPb01 (ATCC-MYA- 826) was co-cultivated with A549 cells in RPMI1640 medium during 6, 12, 24, 48, 72 and 96hto determine the time in which more fungi could be recovered. Followed each time, the extracellular fungi wereremoved by exhausting washing with PBS prewarmed at 37 o C. The percentage of fungi cells adhered and/orinternalized by pnemocytes was determined by CFU counting in BHI supplemented. The 48, 72 and 96h werethe most efficient times to recovery fungal cells. The A549 cells were lysed with a guanidine thiocyanate-basedsolution and intact fungi were harvested by centrifugation to extract the fungal total RNA by the TRIzol ® assay. Wehave thus succeeded in recovering RNA from the fungus during pneumocyte infection, thus setting up the protocolfor future experiments.Financial support: FAP-DF/CNPq and MCT/CNPq.166

4-05Evidence for positive selection in putative virulence factors within the Paracoccidioidesbrasilensis species complexDaniel R. Matute 1 , Lina M. Quesada-Ocampo 2 , Jason T. Rauscher 3 and Juan G. McEwen 41Department of Ecology and Evolution, University of Chicago, 1101 East 57th Street, Chicago, Illinois 60637 2 Michigan State University.Department of Plant Pathology 3 University of Puerto Rico, Rio Piedras. Department of Biology 4 Corporacion para InvestigacionesBiologicas (CIB), and University of Antioquia, Medellin, CiolombiaParacoccidioides brasilensis, a dimorphic fungus, is the causative agent of paracoccidiodomycosis, one of themost importnat systemic mycosis in Latin America. Recently, the existence of three different genetically isolatedgroups in P. Brasilensis was demonstrated, thus enabling comparative studies of molecular evolution among P.Brasilensis lineages. Thirty two gene sequences coding for putative virulence factors were analyzed to determinewheter or not they were under positive selection. Our maximum likelihood-based approach yielded evidence forselection in 12 genes all involved in different cellular processes. This proportion is significantly higher than thenumber of housekeeping genes under positive selection. An in-depth analysis of four of these genes showedthem to be either antigenic or involved in pathogenesis. In this paper, we present evidence inidicating that severalreplacement mutations in the gp43 gene are under positive balancing selection. The other three genes (fks, cdc42and p27) exhibit very little variation among the P. Brasilensis lineages and appear to be under positive directionalselection. Our results are consistent with the more general observations indicating that selective constrains arevariable across the genome, and that even in the genes under positive selection, only a few sites are altered.We present our results within an evolutionary framework that may be applicable to studies on adaptation andpathogenesis in P. Brasilensis and other pathogenic fungi.4-06Phylogenetic analysis of PRP8 intein and mycological features in Paracoccidioidesbrasiliensis species complexR. C. Theodoro 1 , Bagagli E. 1 and Trinca L. A. 21 Depto. de Microbiologia e Imunologia, IBB- Unesp, Botucatu, São Paulo - Brasil. 2 Depto. de Bioestatística, IBB-Unesp, Botucatu, SãoPaulo - Brasil. e-mail: raquel@ibb.unesp.brA recent species status investigation in Paracoccidioides brasiliensis described three cryptic species (S1,PS2 and PS3). This study aimed to evaluate the potential of the PRP8 Intein, a parasitic genetic element, aswell as some mycological characteristics for the recognition of these species in P. brasiliensis. The PRP8 inteinsequence from a total of 22 P. brasiliensis isolates, 20 from the three genetic groups and 2 unidentified isolates,were determined for phylogenetic analysis by Maximum-Parsimony, Maximum Likelihood and Bayesian Analysis.Additionally, mycological analysis, such as conidia production (in Potato Dextrose Agar and Soil Extract Agarmedia), mycelia-yeast transition and yeast morphometry were performed. All the isolates presented a full-lengthintein, although the Endonuclease domain seems to be inactive due to substitutions in the second essentialaspartic acid residue. The phylogenetic analysis clearly separated the isolates from the three species and revealeda significant difference between the isolate Pb01 and the remaining ones. The Pb01 isolate does not belong to anyof the three genetic groups, suggesting the occurrence of a new species. While most of the isolates from S1 groupproduced high number of conidia (5-50/field), the isolates from PS2 and PS3 groups did not produce conidia.Pb01 isolate produced few conidia per field (0-5), which were peculiar due to their longer shape when comparedto S1 group. Concerning the yeast morphology, the isolate Pb01 presented a large number of giant cells (>30µmof diameter) and some isolates from PS2 group presented pseudo-hyphae at 36°C. The M-Y transition experimentsuggested that some elongated yeast isolates from PS2 take more time, in days, to start the transition. In conclusionthis study presented a reliable molecular marker for species recognition, including the new species Pb01-like andpointed some potential morphological markers for species differentiation. If the groups are genetically separated,they are supposed to accumulate some differences, reflecting in the occupation of new ecological niches or indifferent strategies for survival in saprobic or host environment. Thus, the monitoring of the genotypes that arecausing PCM disease is extremely important for the comprehension of the different clinical aspects and treatmentresponses.Financial support: Fapesp (Grant Numbers: 06/03597-4 and 07/01306-5)167

4-07A high degree of polymorphism and recombination of the gp43 gene among phylogeneticspecies of Paracoccidioides genusTeixeira, M. M. 1 , and Felipe, M.S.S. 11 Instituto de Biologia, UnB, Brasília - Brasil.e-mail: marcus.teixeira@gmail.comBackground and aims: Paracoccidioidomycosis (PCM) diagnosis is mainly performed by using serological testagainst the immunodominant antigen GP43, a molecule that is secreted under both in vitro and in vivo conditions.Many studies demonstrate that the gp43 gene is highly variable among isolates and the genealogy of this locusrepresents the real evolutionary scenario of the genus Paracoccidioides. Our aim is to investigate the polymorphismand the possibility of recombination of this gene among the phylogenetic species S1, PS2, PS3 and the crypticisolated group “Pb01-like”. Methods: We amplified and sequenced fragments of gp43 gene (promoter+exon1and exon2) from 17 isolates (“Pb01-like”) and compared to the 65 sequences previously deposited for the threephylogenetic species of P. brasiliensis (S1, PS2 and PS3). The sequences were aligned using the ClustalWalgorithm and phylogenetic analyses were performed by parsimony and Bayesian methods for this locus. Also, wehave analysed and compared the deduced entire protein sequences of GP43 from isolates Pb18, Pb3, Pb eslavaand Pb01, each one representing the four phylogenetic groups. Recombination analysis was performed using thesplit decomposition method that allowed the identification of recombination nets. Results: The DNA sequencesobtained from the four phylogenetic groups showed a high degree of polymorphism in the gp43 gene (π=0.03978).In a total of 880 nucleotides analyzed 159 were polymorphic (18%). Phylogenetic analyses showed that geneticallyisolated groups could be recognized by gp43 genealogy, suggesting that this molecular marker could be useful totrack phylogenetic species in the Paracoccidioides genus. The amino acid sequences of GP43 from isolates of thefour phylogenetic groups showed exclusive amino acid substitutions and indicates high genetic variability betweenPb01-like group and the other three phylogenetic species (S1, PS2 and PS3). Recombination analysis in the exon2 of this gene revealed that recombination events occurs inter-species (“Pb01-like”- S1 - PS2), which is showedby the nets connecting these phylogenetic species. Conclusions: Here we concluded that the gp43 gene mayhave been a recombination hotspot during speciation in Paracoccidioides. This gene could be a potential markerto distinguish between phylogenetic species in this genus. Financial support: CNPq, FAP-DF, FUB.4-08Virtual screening of Paracoccidioides brasiliensis genome – a new drug design approachA.K.R. Abadio 1 , Carvalho M.J.A. 1 , Martins N.F. 2 , and Felipe M.S.S. 11 Instituto de Ciências Biológicas, Universidade de Brasília, Brasília – Brasil.2 Embrapa Recursos Genéticos e Biotecnologia, Brasília – Brasil.e-mail: anakarina.abadio@gmail.comIntroduction and aims: Paracoccidioides brasiliensis is the dimorphic fungus responsible forparacoccidioidomycosis (PCM), one of the most important systemic mycosis in Latin America. Among the areas ofPCM incidence there are Brazil (80 % of the cases), Venezuela, Colombia and endemic regions that extend fromMexico to Argentina. The mycosis current treatment with conventional drugs normally causes serious side effects,such as nephrotoxicity. We focused in protein sequences of Aspergillus fumigatus (54 genes) and Candidaalbicans (26 genes), previously described as essential genes, representing broad biological functions such ascellular metabolism, cell wall organization and biogenesis, ergosterol biosynthesis, ribosome biogenesis, proteinmodification. The objective of this work was to identify possible essential genes of P. brasiliensis and also in otherhuman pathogenic fungi as C. albicans, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitisand A. fumigatus that are also absent in the human genome, by comparative genomic analysis.Methods: These protein sequences were analysed through multiple sequence alignments against severaldatabases using a comparative approach. Next, a conservative domain screening was made by multiple alignmentsusing Clustal and the structural domains were identified using InterProtScan. Furthermore, phylogenetic studieswere performed.Results and conclusions: The virtual screening allowed the selection of 8 candidate genes such as: NOB1that encode an essential protein for processing the 20S pre-rRNA to the mature 18S rRNA; NOC3 that encodea nuclear complex-associated protein required for ribosome maturation and transport. This approach stronglysuggests the potential use of those candidate genes to new antifungal drug development.168

4-09Formamidase of Paracoccidioides brasiliensis: Cytolocalization, purification and analysisof the native proteinC.L Borges 1 , Barbosa, M.S.. 1 , Parente, J.A. 1 , Feitosa, L.S. 5 , Santana, J.M. 3 , Báo, S.N 4 ., Sousa, M.V. 4 , Richart, C.A.O. 4 ,Soares, C.M.A. 1 .1Instituto de Ciências Biológicas, UFG, Goiânia - Brasil. 2 Instituto de Ciências Biológicas, UnB, Brasília – Brasil. 3 Faculdade de Medicina,UnB, Brasília – Brasil. 4 Instituto de Biologia, UnB, Brasília – Brasil. 5 Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP,São Paulo, Brasil. e-mail: clayton@icb.ufg.brParacoccidioides brasiliensis is a thermally dimorphic fungus causing paracoccidioidomycosis, an endemicdisease widespread in Latin America. The dimorphic transition from the mycelial (22 ºC) to the yeast (37 ºC)form is induced by a shift from the environmental temperature to that of the mammalian host. The formamidasegene of P. brasiliensis was described as highly expressed in mycelia of P. brasiliensis, isolate Pb01. Formamideaminohydrolase (formamidase, EC 3.5.1.49) catalyzes the highly specific hydrolysis of formamide to produceammonia and formate. In a previous work we identified the formamidase of P. brasiliensis, which reacts withantibodies present in sera of P. brasiliensis infected patients. In this work we continue the characterization ofthis highly expressed protein of P. brasiliensis. In this vein, the recombinant formamidase was used to producepolyclonal antibody in mice, which showed high specificity in Western blot assays with total protein extract of P.brasiliensis yeast cells. We sought to determine the cellular localization of the native protein in fungal yeast cells byconfocal and transmission electron microscopy. The P. brasiliensis formamidase was found in cytoplasm and cellwall. Due to descriptions of the protein in other systems as a tetrameric molecule we also performed purificationof the native protein in two steps of column chromatography, DEAE sepharose and phenyl sepharose columns.Fractions with formamidase activity were selected and analysed by PAGE, which revealed a protein with molecularmass of 200 kDa. The purified protein was submitted to trypic digestion and confirmed by MALDI-TOF massspectrometry as a formamidase. Additionally, SDS-PAGE assay with boiled purified protein revealed a proteinwith molecular mass of 50 kDa. Those results suggest that P. brasiliensis formamidase is a tetrameric protein.The function of P. brasiliensis formamidase remains unclear. In order to elucidate the role of this molecule, thecDNA encoding formamidase was used to screen a library constructed with P. brasiliensis yeast cDNAs using aSaccharomyces cerevisiae two hybrid system. Proteins related with protein folding, processing and destinationwere found, which can be related with the cell wall localization of formamidase of P. brasiliensis. A model forformamidase interactions is provided. Supported by: FINEP/CNPq.4-10Two subunits of β1,3-glucan synthase complex: Gene expression (RHO1 and FKS1) andfunctionality (RHO1) in Paracoccidioides brasiliensisF. Sorais, Niño-Vega, G., and San-Blas, G.Instituto Venezolano de Investigaciones Científicas, Centro de Microbiología y Biología Celular, Apartado 20632, Caracas 1020A, Venezuela.e-mail: fsorais@ivic.veA multigene family of six known RHO members (RHO1-RHO5, and CDC42) has been identified in yeasts.Several functions have been ascribed to these GTPases, among which regulation of cell wall glucan synthesis,organization of the actin cytoskeleton, regulation of secretion and control over polarity are highly relevant. Rho1 isrequired for β-1,3-glucan synthase (Fks1) activity in yeast and Candida albicans. In Paracoccidioides brasiliensis,little is known about this synthase complex, apart from the sequencing of its FKS1 gene (Pereira et al., 2000).Therefore, we searched the gene coding for the second subunit of this enzymic complex, RHO1, and compare theexpression of both genes. Also, functionality of the P. brasiliensis RHO1 gene was demonstrated by complementinga temperature-sensitive Saccharomyces cerevisiae rho1 null mutant with an expression vector containing the P.brasiliensis RHO1 gene. P. brasiliensis RHO1 is 950 bp long, harbours four introns, and codes for a 191 aminoacid-long protein (accession number: AY392528). Gene expression comparison of both putative subunits of the β-1,3-glucan synthase complex in P. brasiliensis, Fks1 and Rho1, were assessed by northern blot at intervals duringthe mycelial (M) to yeast (Y) transition and in the extreme (M and Y) stages. Opposite patterns of expressionwere seen in both genes, a result that may be linked to the fact that Rho1 plays roles in several events within thecells, not only in controlling Fks1. To test whether P. brasiliensis RHO1 could complement a S. cerevisiae rho1mutation, a temperature-sensitive mutant rho1-104 (S. cerevisiae strain HNY21) was transformed with the plasmidconstruct pYES2-RHO1. As negative control, the mutant strain was transformed with pYES2. P. brasiliensis RHO1successfully restored growth of S. cerevisiae rho1 mutant under restrictive temperature conditions.Whether P. brasiliensis Rho1p regulates β-1,3-glucan biosynthesis, or any other function related to fungalRho1p orthologs, remains for further studies.169

4-11β1,3-glucan synthase: Recombinant protein, cytolocalization, activity and studies understress conditionP. K. Tomazett 1 , Félix, C. R 2 ., Barbosa, M. S 1 ., Santana, J. M 3 ., Faria, F. P 1 ., Báo, S. N 4 ., Soares, C. M. A 1 ., Pereira, M 1 .1 Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia-GO-Brazil. 2 Laboratóriode Enzimologia, Instituto de Ciências Biológicas, Universidade de Brasília, Goiânia-GO-Brazil. 3 Faculdade de Medicina, Universidade deBrasília, Goiânia-GO-Brazil. 4 Laboratório de Microscopia Eletrônica, Instituto de Ciências Biológicas, Universidade de Brasília, Goiânia-GO-Brazil. e-mail: patriciakt@hotmail.comThe cell wall of fungi is an essential and antigenic structure. The β-1,3-glucan polymer possibly givesshape to the cell and its synthesis, which occurs at the plasma membrane, may be the result of the activityof β-1,3-glucan synthase. In P. brasiliensis just one gene homologue of β-1,3-glucan synthase (PbFKS1) hasbeen characterized. Here, the catalytic subunit of glucan synthase (PbFKS1c) was used to obtain a recombinantprotein in Escherichia coli. The pGEX-4T-3 vector was used to produce the recombinant protein in fusion withGlutathione S-transferase (GST) in its active form. The enzymatic assay of the recombinant and native glucansynthase was performed using a radioisotopic method where the substrate UDP-glucose is radioactively labeled.The purified recombinant PbFKS1c was used to generate specific rabbit polyclonal serum. This antibody was usedto perform an immunocitolocalization of glucan synthase in P. brasiliensis yeast cells. Glucan synthase activity iscurrently assayed by the radioisotopic method. However, this method is expensive and requires special handlingand disposal methods for radioactive wastes. Based in a method described to soybean seeds galactinol synthasewe have adapted a new colorimetric method to determine the glucan synthase activity. The colorimetric methodhas been standardized with the native glucan synthase. The reaction time, protein concentration and substrateconcentration have already been determined. The colorimetric assay is still being tested to the recombinant proteinPbFKS1c. The growth of P. brasiliensis was evaluated under stress conditions to determine the concentration inwhich the fungus is sensible to the stressors agents calcofluor white, congo red, SDS, NaCl, KCl and sorbitol,including cell wall damage and osmotic stress. By using the concentration in which the fungus is visually sensibleto those agents, the transcript level glucan synthase was evaluated by real time RT-PCR, and the enzymaticactivity was performed by using colorimetric method.4-12The α-amylase (amy1) gene in Paracoccidioides brasiliensis, identification andsemiquantitative expressionE. Camacho, Niño-Vega, G., and San-Blas, G.Instituto Venezolano de Investigaciones Científicas, Centro de Microbiología y Biología Celular, Apartado 20632, Caracas 1020A,Venezuela. e-mail: ecamacho@ivic.veIn Paracoccidioides brasiliensis, an α-1,3-glucan is present as the outer layer of the cell wall in the yeastlikephase, a polysacharide that is absent in the mycelial phase. α1,3-Glucan has been proposed as a virulence factornot only in P. brasiliensis but also in Blastomyces dermatitidis and Histoplasma capsulatum. An α1,4-amylase isessential for the synthesis of cell wall α1,3-glucan and expression of the virulence in H. capsulatum. With theaim of exploring the possibility of similar functions in P. brasiliensis, we identified, isolated and characterized itsα-amylase (amy) gene.P. brasiliensis IVIC Pb73 (ATCC 32071) was grown in PYG medium, at 23ºC (mycelial phase, M) or 37ºC(yeastlike phase, Y). Genomic DNA was extracted from freeze-dried M cultures. Total RNA was extracted withTRIzol from frozen Y cells. To amplify the gene, degenerate and gene-specific oligonucleotides were sequentiallyused, the latter by means of a RACE reaction. Semiquantitative RT-PCR was applied for gene expression, using 3µg total RNA as template, in 10-30 cycles, to determine the exponential phase in the expression of P. brasiliensisamy1 and 18S genes.A gene with high identity with H. capsulatum α-1,4-amylase, expressed preferentially in the pathogenic Yphase, was identified. In silico amino acid analysis of the deduced protein led to the identification of all fourconserved regions of the αamylase family, the critical moieties for biological activity and amino acids associatedwith the specificity to glucosidic α-1,4-linkages. Currently, the participation of such gene in the remodeling of thecell wall architecture in P. brasiliensis, pathogenic Y phase is under study.Acknowledgements: To FONACIT (Caracas, Venezuela) and ICGEB (Trieste, Italy) for partial financial support.170

4-13Mating type genes MAT1-1 and MAT1-2 and sexual reproduction in Paracoccidioides brasiliensisTorres I1,5, García AM1,4, McEwen JG1,2,3, Gonzalez A4, Restrepo A1, Arango M1,2.1. Unidad de Biología Celular e Inmunogenética-Corporación para Investigaciones Biológicas-CIB. Medellín, Colombia. 2. Departamento deMicrobiología y Parasitología-Universidad de Antioquia. Medellín, Colombia. 3. Departamento de Fisiología y Bioquímica -Universidad de Antioquia.Medellín, Colombia. 4. Universidad Pontificia Bolivariana-Facultad de Medicina. Medellín, Colombia. 5. Estudiante de doctorado en Biología-Facultadde Ciencias Exactas y Naturales- Universidad de Antioquia. Medellín, Colombia. 4. Escuela de Microbiología U de A. e-mail: isaurap10@gmail.com,myrthaarango@yahoo.com.Introduction: Paracoccidioides brasiliensis (Pb) taxonomic classification has not yet been defined.Nevertheless, some morphological, molecular and phylogenetic studies have placed the fungus as a member ofthe phylum Ascomycota even if its sexual cycle and mating type system remain unknown. The aim of this work wasto demonstrate the presence of both MAT genes in Pb and perform some crosses between potentially compatiblepartners by plating in different culture media. Methods: 76 Pb isolates from a variety of sources (geographic,clinical, environmental and phylogenetics species) were analyzed in an attempt to find mating type genes.Specific primers were designed from Pb ESTs libraries and the MAT genes sequences derived from Histoplasmacapsulatum. Samples corresponding to the above isolates were assessed by PCR by means of these specificprimers and the mating genotype was assigned according to the amplicons’ size thus obtained. Some ampliconswere sequenced and analyzed by means of the BLAST and Expasy tools. Some isolates were selected accordingto certain characteristics such as mating type, phylogenetic species, country of origin and were then crossed onsynthetic media, Results: The PCR assays were successful as the presence of both mating type genes wasmade evident in all Pb isolates tested with an amplicon of 400pb corresponding to the MAT1-1 gen (heterothallic)and fragments of 300pb and/or 1000pb corresponding to the MAT1-2 gen (heterothallic). Interestingly, there wasa group that showed both the MAT1-1 and the MAT1-2 amplicons (“homothallic”). Homology analysis from Pbsequences performed with BLAST tools, showed a high grade of identity and importat e-values when comparedwith MAT genes from others Ascomycetes. However, the same analysis in “homothallic” isolates showed that one ofthese genes was a truncated copy, not being therefore true homothallism. Protein analysis (Expasy) revealed thatPb MAT1-1 and MAT1-2 had the characteristic alpha and HMG boxes, respectively. When the crosses performedon synthetic media were analyzed some structures resembling fruiting bodies were observed, but their content didnot correspond to the expected reproductive structures; there were neither asci nor ascospores. Conclusions:This study allowed to develop a PCR assay that used specific primers and permited identification of MAT genes inPb isolates. In the future, this finding may allow to improve the taxonomic classification of this fungus The presenceof both genes suggest that sexual reproduction may occur in Pb and that, at the moment, additional crosses withdifferent partners, with emphasis in crossing the phylogenetic species, are under way, and may point towards amore precise definition of the concept of biological species in the Pb population.4-14Molecular evidences of sexual reproduction in P. brasiliensisTeixeira, M. M., Paes, H. C, Fernandes, L., Silva, S. S. and Felipe, M.S.S.Instituto de Biologia, UnB, Brasilia - Brasil.e-mail: marcus.teixeira@gmail.comBackground and aims: The thermodimorphic fungus P. brasiliensis is the aetiological agent ofParacoccidioidomycosis (PCM). The teleomorph form of this fungus has never been observed either in vitro or invivo. Recently, through phylogenetic analysis by concordance and non-discordance methods and by recombinationanalysis, it was suggested that P. brasiliensis has a sexual stage in its life cycle (Matute et al., 2006). Here weshow that this pathogen has sex-suggestive features and contains homologues to all genes that comprise themating machinery in other fungal species. Methods: Based on the transcriptome of the isolate Pb01 and thestructural genome of the isolate Pb3 we designed two pairs of primers to amplify the transcriptional factors α-boxand HMG that correlate to both idiomorphs (MAT1.1 and MAT 1.2 respectively). We amplified the cognate regionsfrom 29 isolates that represent the three phylogenetic species of P. brasiliensis (S1, PS2 and PS3) and isolatesfrom “Pb01-like”. We also performed in silico analysis of the structural genome of Pb3 to search for potentialgenes involved in mating as described in Histoplasma capsulatum, Aspergillus fumigatus and Saccharomycescerevisiae. We performed a direct blastX of these genes against the structural genome of P. brasiliensis isolatePb3. Results: For the 29 isolates we were able to amplify three populations: i) 15 isolates that present only theα-box domain (MAT1.1); ii) 8 isolates that present only the HMG (MAT1.2); and iii) 6 isolates that present boththe α-box and HMG (MAT1.1 and MAT1.2). The in silico analysis showed that P. brasiliensis isolate Pb3 containsthe core genes involved in mating with high similarity to H. capsulatum, such as pheromone-processing enzymes(ste14, ste24, ste6, ste13, ram1, ram2, kex1 and kex2), components of a pheromone-response pathway (GBA1,ste4, ste18, ste20, ste11, ste7, fus3) and pheromone receptors (ste2 and ste3). Conclusion: These resultssuggest that P. brasiliensis may undergo mating and then meiosis during its life cycle. This work opens newavenues of investigation into the possibility of in vivo or in vitro mating in this pathogen.171

4-15α 1,3-glucanase is important in the remodelling of Paracoccidioides brasiliensis cell wallH. Villalobos, Niño-Vega, G., and San-Blas, G.Instituto Venezolano de Investigaciones Científicas, Centro de Microbiología y Biología Celular, Apartado 20632, Caracas 1020A,Venezuela. e-mail: hvillalo@ivic.veAlthough not a frequent component of the fungal cell wall, α-1,3-glucan is present in Schizosaccharomycespombe, some Aspergilli and the yeastlike forms (not the mycelial ones) of Histoplasma capsulatum, Blastomycesdermatitidis, and Paracoccidioides brasiliensis, among a few other species, where it plays decisive roles indimorphism and virulence. To degrade this polysaccharide and allow separation of the daughter cells, α-1,3-glucanases are required. A strict control on such activity is important in order to prevent any possibility of autolysis.Therefore, the study of this enzyme, its encoding gene and regulation are important for the understanding ofmorphogenetic processes in these fungi.This work aims to decode the full sequence of the agn1 gene in P. brasiliensis, verify the presence of introns,analyze the deduced amino acid sequence, and quantify its expression by RT-PCR. In view of the observed boostin cell wall α1,3-glucan when P. brasiliensis is grown in medium supplemented with horse serum (HS), agn1expression is also measured in genomic material extracted under such experimental conditions.The agn1 5’ region was obtained by means of oligonucleotides designed on a partial sequence from a HindIIIlibrary (SMART RACE cDNA Amplification Kit). New oligonucleotides designed on agn1 end regions allowed theamplification of the genomic and codifying sequences, and detection of introns.The genomic sequence is 1494 bp long, interrupted by two introns, for an ORF 1370 bp long. Its expression iscurrently under study. The deduced protein sequence is composed of 456 amino acids, with high similarity to otherfungal glucanases. The presence of a signal peptide and putative sequences for post-translational modificationsare detected in the deduced protein.Acknowledgements: To FONACIT (Caracas, Venezuela) and ICGEB (Trieste, Italy) for partial financialsupport.4-16Characterization of Paracoccidioides brasiliensis Chs3 by overexpression in SaccharomycescerevisiaeL. Barreto, Sorais, F., Niño-Vega, G., and San-Blas, G.Instituto Venezolano de Investigaciones Científicas, Centro de Microbiología y Biología Celular, Apartado 20632, Caracas 1020A,Venezuela. e-mail: lbarreto@ivic.veCytoplasmic membrane-associated chitin synthases are responsible for the biosynthesis of cell wall chitin infungi. Six chitin synthases (Chs1-Chs6) have been detected in Paracoccidioides brasiliensis. Northern analysesshow that, contrary to other CHS genes, CHS3 is only expressed in the pathogenic yeast phase and at theend of the mycelium-to-yeast transition in P. brasiliensis, suggesting a possible involvement in morphology andvirulence. Heterologous expression of the CHS3 cDNA under the regulation of the inducible GAL1 promoter inSaccharomyces cerevisiae has been successfully performed, to be followed by determination of Chs3 enzymaticactivity.The CHS3 gene (accession number: AF107623) contains a 3817 bp-long open reading frame, interrupted bytwo introns (79 bp and 86 bp), and encodes for a protein 1220 amino acid-long, highly homologous to Class IVchitin synthases. The first cDNA strand was synthesized by RT-PCR, using gene-specific primers designed on thebasis of the genomic sequence; it included an engineered NotI restriction site around the start codon and a secondengineered XbaI restriction site around the stop codon.The CHS3 cDNA was cloned into NotI / XbaI sites of the yeast expression vector pYES2, and the plasmidconstruct was used to transform a S. cerevisiae chs3 null mutant (ATCC 4003160), using the EasyComp TMTransformation Kit (Invitrogen).Transformants were grown in galactose-raffinose medium, where expression of the cloned cDNA was inducedunder the GAL1 promoter. As controls, yeast cells transformed with the empty vector were used. Determination ofchitin synthase activity, according to Orlean (1987), is currently under progress in our laboratory.172

4-17Autophagy in the human pathogenic fungus Paracoccidioides brasiliensis duringdimorphismSarah M. Pedroso, Cláudia L. B. Campos, Francisco G. Nóbrega, Flavia V. Morais.Universidade do Vale do Paraíba (UNIVAP), São José dos Campos, Brazil.e-mail: cbcampos@univap.brThe degradation of macromolecules and organelles in eukaryotic cells occurs by a highly conservedprocess, named macro-autophagy (autophagy), through the action of the lysosomal/vacuole compartments.This process is crucial for survival of several microorganisms, mainly in stress conditions, being involved inmany biological events such as development, differentiation and adaptation.The human pathogenic fungus Paracoccidioides brasiliensis is the etiological agent of a deep mycosis,paracoccidioidomycosis, which has to undergo mycelium to yeast dimorphism inside the host prior to theestablishment of the disease. There are many genes involved in autophagy in fungi, as previously reportedin Aspergillus species and Saccharomyces cerevisiae. In Paracoccidioides brasiliensis, some of them wereidentified in studies of gene expression during mycelium to yeast transition, suggesting that autophagy may befunctional and may contribute to the differentiation of the fungus. In this work, we investigated the involvementof autophagy in dimorphism of Paracoccidioides brasiliensis induced by temperature rise from 25 o C to 36 o C.We show that autophagy is mostly absent in mycelia.However, the amount of autophagic vacuoles, visualized by mono-dansylcadaverine labeling, progressivelyincreases during mycelium to yeast transition, but is low by the end of this process. Both in mycelia andyeast cells at stationary phase, the level of autophagic vacuoles are much higher than in the actively growingfungi, which suggest a role for autophagy during nutrient starvation. We also investigated the effect of twoautophagy inhibitors, N-ethylmaleimide (NEM) and 3-methyladenine (3-MA), on mycelium to yeast dimorphism.NEM potently induced death during dimorphism, while 3-MA reduced autophagy and inhibited P. brasiliensisdimorphism. Our results suggest a potential role for autophagy during P. brasiliensis dimorphism and nutrientstarvation. Key words: Paracoccidioides brasiliensis, autophagy, dimorphism.4-18Niche specific regulation of Paracoccidioides brasiliensis genes in the infectiousprocessA.M. Bailão, Borges C.L., Chagas R.F., Salem-Izaac S.M., Pereira M. and Soares C.M.A.Laboratório de Biologia Molecular, UFG, Goiania - Brasil.e-mail: alexandre.bailao@gmail.comThe fungal pathogen Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis(PCM). The disease may present a large spectrum of clinical manifestations with the involvement of differentorgans and tissues. We have been studying genes that could be relevant to the fungal parasitic style and to theinfectious process. Our hypothesis is that during the parasitic phase it would occur genes whose expressionwill perform a control role in the well-succeeded parasitic life style. Additionally, the fungus invades differentniches in the host, a factor that may induce expression of different set of genes.Our results, obtained by transcriptome analyses and by confirmation experiments strongly suggest thatP. brasiliensis presents a metabolic program in which: (i) the nitrogen metabolism, the glicolytic pathway andthe lipid synthesis predominate during liver infection; (ii) a change to a lypolitic style and increased nitrogenmetabolism , as well as, cell wall remodeling are predominant during fungal dissemination through thebloodstream; (iii) the transport of copper and iron are exacerbated during fungal infection in liver and duringdissemination through bloodstream. Due to the relevance to the fungal pathogenesis of understanding thegene expression in host niches, and also considering the potential of this knowledge for improving the diseasetherapy, studies had been conducted adopting genomic and proteomic strategies in order to extend the initialstudies and to evaluate the fungal global adaptation to host different micro environments. A model for fungaladaptation to the host niches is presented.Supported by: CNPq e FINEP173

4-19Differential gene expression in virulent and avirulent isolates of ParacoccidioidesbrasiliensisC. Arruda 1 , Beatriz C. A. Alves 1,2 , Vera L. G. Calich 1 and C. A. Moreira-Filho 1, 21Department of Immunology, Biomedical Sciences Institute, University of São Paulo, São Paulo, SP, Brazil; 2 Albert Einstein Research andEducation Institute, Hospital Israelita Albert Einstein, São Paulo, SP, Brazil.e-mail: celinaarruda@ol.com.br (presenting Author)P. brasiliensis is a dimorphic fungus which causes paracoccidioidomycosis (PMC), a systemic granulomatousdisease. Different clinical manifestations are due to host related factors or to intrinsic characteristics of the fungus,like virulence. Some virulence factors especially that involved in dimorphism have been proposed. This workwas focused on the identification of differentially expressed sequences between yeast forms of virulent and lessvirulent Paracoccidioides brasiliensis (Pb) isolates by means of mRNA Differential Display.A genetic murine model of paracoccidioidomycosis (PCM) characterized B10.A and A/J mice as susceptibleand resistant strains to Paracoccidioides brasiliensis (Pb) infection, respectively. The disease developed by thesemice strains mimics the severe and benign forms of human PCM. In the present work, two isolates of P. brasiliensisPb 18 (Pb 18V and Pb 18AV) were used to infect intratracheally (i.t.) B10, susceptible mice. The inoculation ofPb 18V, a highly pathogenic sample, promotes a severe and disseminated infection in susceptible mice. Onthe other hand, Pb18AV, obtained after several years of in vitro subculture of Pb 18, has low pathogenicity andcauses a restrained pulmonary infection in the same mice. Total RNA were obtained from lungs at weeks 4 and8 post-infection and from in vitro cultures. The cDNA were synthesized using oligo-deoxythymidine-anchoredprimers, and amplified by PCR with oligo dT plus random primers. The products were submitted to polyacrylamidegel electrophoresis analysis. One band of approximately 250 bp expressed only by Pb 18V inoculated mice wasobtained with the random primer OPA 05. This fragment was cloned and sequenced. Its nucleotide sequenceshowed no similarity with any fungus sequence described so far. RT-PCR experiments are being conducted inorder to quantify this differential gene expression.This work was supported by research grants from FAPESP and CNPq.4-20Gp43 isoforms of Paracoccidioides brasiliensis as ligands of laminin, fibronectin andcollagen type ISilva, J.F 1 ; Benard, G 2 .; Mendes-Giannini, M.J.S 1 .1Faculdade de Ciências Farmacêuticas de Araraquara – UNESP – Brasil and 2 Faculdade de Medicina de São Paulo – USP – Brasil.e-mail: silvajf@fcfar.unesp.The capacity of the Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis (PCM), toprovoke human illness, and to cause mycosis with great variety of clinical manifestations, from localized formsuntil spread disease which may cause death, depends probably on factors such as: fungal virulence, its ability ininteracting with the host superficial structures, invading them and on the host immunological response. Studiesof the correlation among some of the components (gp43) and their role in the pathogenicity are subjects of greatimportance in paracoccidiodomycosis. It has also been shown that different fungal strains produce gp43 with atleast four isoform profiles with pIs ranging from 5.8 to 8.5. The 43 kDa glycoprotein, ligand of laminin, and thepeptide 1 (NLGRDAKRHL), corresponding to residues 76-85, competed with gp 43 and significantly inhibitedthe adhesion of P. brasiliensis to the Vero cells by ~ 60 %. Aiming to understand the mechanisms that regulatethe interaction of this fungus with host epithelial cells, this study investigated the protein expression profile offour isolates of P. brasiliensis and the capability of these proteins, in special gp43 isoforms, binding to differentcompounds of extracellular matrix proteins (ECM). Depending on the isolate, proteins were expressed differentially.The gp43 isoforms of pIs 6.0 and 6.3 (presented in Pb339 and Pb265 isolates) and pI 5.7 (presented only in Pb339isolate) binding to laminin, while the gp43 isoforms of pI 6.7, 6.6 and 5.5 (presented only Pb339 isolate) binding tofibronectin and collagen type I, confirming previous data and increasing our knowledge about the different isoformsof the gp43 and their behaviour as adhesin and as ligand to ECM compounds. Then, the several gp43 isoformscan binding to different ECM compounds, depending on the isolate and maybe this fact is very important frombetter understanding of their role in the pathogenicity of P. brasiliensis.Suppoted by: FAPESP; PADC-FCF; CNPq and CAPES174

4-21Transcription regulation of the PbGP43 gene with nitrogen in the human pathogenParacoccidioides brasiliensisA. A. Rocha, and Puccia R.Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP, São Paulo, Brasil. e-mail: rpuccia@unifesp.brThe present work shows the involvement of NIT2-like binding motifs in transcription modulation of thePbGP43 gene, which encodes an important antigen from the human pathogen Paracoccidioides brasiliensis.This investigation has been motivated by the finding of a high number of NIT2-like motifs within the first 2,047nucleotides of the PbGP43 5’ intergenic region from Pb339 isolate, recently cloned and sequenced by our group.This fragment contains 23 NIT2-like sites, which form 4 putative clusters, two of them identical. Our analysis wasbased on electrophoretic mobility shift assay (EMSA) with selected probes and real time reverse transcription(RT)-PCR of PbGP43 from P. brasiliensis cultures exposed to, or depleted of, ammonium sulfate and glutamine.The results suggested that at least some NIT2-like motifs are functional and that PbGP43 is modulated by nitrogenprimary sources. We have mapped 4 oligonucleotides containing TATC motifs that formed DNA-protein complexespossibly involving a NIT2-like factor. This suggestion is based on the following observations: i) they formed moreintense EMSA bands with extracts from ammonium sulfate-depleted cells; ii) a point mutation in the TATC core(to GATC) decreased shifted band intensity; iii) all the complexes migrated similarly. Both ammonium sulfate andglutamine provoked rapid decrease in PbGP43 mRNA accumulation, but this effect could be reversed upon saltdepletion. This kind of modulation was observed not only in Pb339, but also in Pb3 and Pb18. The oligonucleotidesthat prime amplification of a 2,047-bp PbGP43 intergenic fragment using Pb339 DNA elongated a fragment ofsimilar size with template from Pb18 and six other isolates, while for Pb2, Pb3, Pb4 and Pb5 the amplicon was~1,500 bp and for Pb9 and Pb17 it was ~3,000 bp. The Pb18 genome sequence of this amplicon is 98% identicalto that of Pb339. In Pb3, the number of NIT2-like motifs and clusters is lower. This is the first report about PbGP43transcription modulation with primary nitrogen sources. That will help understand the antigen biological functionand its modulation during infection.Financial support: Fapesp and CNPq4-22Identification of Paracoccidioides brasiliensis adhesins through representational differenceanalysisS.V. Nogueira 1 ; Bailão, A.M. 1 ; Borges; C.L. 1 ; Pereira, M. 1 ; Mendes-Giannini, M.J. 2 ; Winters, M. 3; Soares, C.M.A. 11 Laboratório de Biologia Molecular,UFG,Goiás-Brasil. 2 Faculdade de Ciências Farmacêuticas, UNESP-Brazil. 3 Division of InfectiousDiseases, UC, College of Medicine, Ohio - USA. e-mail: alexandre.bailao@gmail.comParacoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM), the most prevalentsystemic mycosis in Latin America. As a dimorphic pathogenic fungus it grows as saprobic mycelium, resultingin the formation of propagules, which initiates the infection in humans when inhaled into the respiratory tract.Subsequently, in the lung, the mycelia propagules develop into yeast cells. Adhesion of microorganisms to hostcells and tissues represents a critical step in the process of infection. The propagules that lodge in the alveoliadhere and invade the alveolar cells and/or the basal lamina. Alveolar basal lamina is composed of a specializedextracellular matrix (ECM), in which laminin, types IV and V collagen, entactin, and fibronectin can be found. Innormal tissues, most ECMs are covered by epithelial or endothelial cells and hence are not available for binding.However, any type of trauma that damages host tissues may expose the ECM and enable microbial colonizationand infection. The aims of the present study are to identify and select genes induced in P. brasiliensis adheredto collagen, to promote heterologous expression of recombinant proteins and to characterize the predictableadhesins. Representational difference analysis (RDA) was applied to two cDNA populations of P. brasiliensis: fromcontrol yeast cells and from yeast cells in vitro adhered to collagen type I. Our analysis identified 47 transcriptsdifferentially expressed. Among them we found enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11), anenzyme that catalyzes the dehydration of 2-phospho-D-glycerate (PGA) to phosphoenolpyruvate (PEP) in thecatabolic direction in the second half of the glycolytic pathway. Despite the absence of a signal sequence andtypical motifs required for membrane anchoring, the attachment of enolase to cell surfaces of some microorganismsis well established. The cDNA was cloned into the pGEX-4T-3 vector and expressed in Escherichia coli pLYS cellsas a glutathione S-transferase fusion protein. The purified recombinant enolase was used to generate specificmouse polyclonal and both were used in affinity ligand and binding assays to characterize enolase as an adhesin.Also, biochemical characterization of the P. brasiliensis enolase was performed.Supported by: Capes, CNPq, FINEP175

4-23Adhesion profiles and extracellular matrix ligands of Paracoccidioides brasiliensis isolatesobtained from armadillos (Dasypus novemcinctus)Silva, R.P. 1 , Bagagli, E. 2 ; Mendes-Giannini,M.J.S. 11Faculdade de Ciências Farmacêuticas de Araraquara-UNESP-Brasil and 2 Instituto de Biociências de Botucatu-UNESP-Brasil.e-mail: rbtps@hotmail.comParacoccidioidomycosis (PCM) is human systemic mycoses caused by the thermodimorphic fungus P.brasiliensis, with a wide distribution in Latin America, mainly in Brazil. The clinical manifestations includecutaneous and systemic forms, and can attack various tissues, especially the lung. Recently, P. brasiliensisstrains with typical morphology have been isolated from Dasypus novemcinctus, confirming as the primarynatural reservoir of this fungus. Its geographic distribution is similar to that of human PCM. The isolates fromarmadillos were virulent in the animal model, with dissemination to many organs. The isolates were classifiedinto virulence categories according to number of cfu per gram of tissue. The ability of the pathogen to interactwith the host superficial structures is essential to its virulence. P. brasiliensis expresses proteins that interactin various ways with the extracellular environment and generally involves ligands produced by the pathogenand is capable to adhere to extracellular matrix proteins (ECM). This approach has not been studied with thearmadillo’s isolates. Then, we studied the capacity of different P. brasiliensis armadillos isolates to adhereto pulmonary epithelial cells (A549), as well as the protein profile of these isolates and ECM ligands. Ourdata confirmed previous studies that more virulent isolates have greater adhesion capacity. The comparativeanalysis of the “cell-free” isolates extracts showed that T10B1 (more virulent) presented remarkable difference.The isolates also showed distinct patterns for the ECM ligands. T10B1 presented an elevated number of ligandsto fibronectin, I and IV collagen and similar to laminin. Therefore, one of the armadillo isolate has additionalligands to three ECM proteins. This pattern may be related to microniche of the fungus in the host as well as,the difference that may occur in the first contact with the human tissues.FAPESP, PADC-FCF and CNPq4-24Use of in vivo-induced antigen technology in the identification of Paracoccidioidesbrasiliensis proteins potentially expressed during infectionT.C.V. Rezende 1 , Dantas. S.F.I.M. 1 , Bailão. A.M. 1 , Castro. N.S. 1 , Tomazett. M.V. 1 , Deepe Jr. G.S. 2 , Pereira. M. 1 andSoares. C.M.A. 1* .1 Laboratório de Biologia Molecular, UFG, Goiânia - Brazil. 2 Division of Infectious Diseases, UC, College of Medicine, Ohio - USA.e-mail: celia@icb.ufg.brIn vivo induced antigen technology (IVIAT) is a technique that has been used in organisms for identificationof immunogenic proteins that may play a role in virulence or pathogenesis. The objective of this work was toscreen immunogenic proteins of Paracoccidioides brasiliensis potentially expressed during human infection.Sera were collected from patients with paracoccidioidomycosis. Equal volumes of sera were pooled andreacted with whole yeast cells and yeast cell lysates of the fungal isolate Pb01. We constructed a cDNAexpression library with RNAs of P. brasiliensis yeast cells recovered from livers of infected mice. Seraadsorbed with P. brasiliensis yeast cells were used to screen the library. We identified 35 clones whichcDNAs encoded predicted proteins related to cell metabolism, transport, energy, transcription, protein fate,signal transduction, biogenesis of cellular components. Of all positive clones identified by IVIAT we selectedtwo encoding the sera reactive aromatic - L- amino acid decarboxylase (DDC) and lumazine synthase (LS)of P. brasiliensis for further studies. We characterized the complete cDNA of Pbddc and Pbls that wereoverexpressed in an Escherichia coli host to produce high leves of recombinant fusion protein with GST. Therecombinant proteins DDC and LS were recognized by sera of patients with confirmed paracoccidioiomycosisand not by sera of healthy individuals. Real time RT-PCR was used to analyze the expresion of the Pbddcand Pbls in the two forms of P. brasiliensis and in yeast cells infecting macrophages. The accumulation ofboth transcripts was higher in the yeast form and in yeast cells infecting murine macrophages. The resultssuggest that PbDDC and PbLS can be considered immunogenic proteins up-regulated during the infectiveprocess.176

4-25Changes in the transcription levels of Paracoccidioides brasiliensis CHS5 and CHS4 genes,mycelial phase, respond to external osmolarityG. Niño-Vega, Sorais, F., and San-Blas, G.Instituto Venezolano de Investigaciones Científicas, Centro de Microbiología y Biología Celular, Apartado 20632, Caracas 1020A,Venezuela. e-mail: fsorais@ivic.veSix chitin synthase (CHS) genes have been identified in the genome of Paracoccidioides brasiliensis.From the deduced amino acid sequences of their encoded products, they belong in six out of seven (I-VII)proposed Chs classes: PbrCHS1 (I); PbrCHS2 (II); PbrCHS3 (IV); PbrCHS4 (VII); PbrCHS5 (V) and PbrCHS6(VI). Herein we report the arrangement of PbrCHS5 in a head-to-head configuration with PbrCHS4, bothgenes sharing a common 5’UTR. Also, changes in transcription levels for both genes, following alteration ofexternal osmolarity, are studied.The 5’UTR sequence comprised 3.8 kb between their respective translational start codons. In silicoanalysis of the shared promoter region also yielded sequences that matched three potential sites for Rlm1p.This fact made us wonder whether the transcription of PbrCHS4 and PbrCHS5 might be stimulated by hypoosmoticstress, as reported for their respective orthologous A. nidulans genes, csmB and csmA. To evaluatechanges in transcription levels in response to alterations of the external osmolarity, the fungus was grownin its mycelial form in 0.3 M KCl-supplemented YPD medium. Lower transcription levels were found for bothPbrCHS4 and PbrCHS5 when the fungus was grown under hyper-osmotic conditions. Although a commonregulatory control for both genes, mediated by a Rlm1p-like transcription factor acting in a shared 5’UTR,might be at work during osmotic stress in P. brasiliensis, the chromosomal head-to-head arrangement ofboth genes does not necessarily imply a similar response to every external challenge. In fact, while bothhave high expression in the mycelial form of the fungus, PbrCHS5 but not PbrCHS4 was expressed in theyeast form, as previously reported (Niño-Vega et al., 2004).Research into gene regulation of P. brasiliensis could help us to understand the relationship amonggenes involved in cell wall synthesis, and their role in cell wall maintenance.4-26A Paracoccidioides brasiliensis aconitase is induced by ethanol and acetate and repressedby glucose as carbon sourcesW.A. Brito. 1,2,3 , Castro. N.S. 3 , Pereira. M. 3 and Soares. C.M.A. 31 Curso de Farmácia, UnUCET, UEG, Anápolis – GO – Brasil. 2 Curso de Farmácia, UniEVANGÉLICA, Anápolis – GO – Brasil. 3Laboratório de Biologia Molecular, ICBII, UFG, Goiânia – GO – Brasil. e-mail: wesleyfarmacia@uol.com.brParacoccidioides brasiliensis is a thermal-dimorphic fungus, the causative agent of Paracoccidioidomycosis(PCM), an important endemic mycosis in Latin America. In this work a protein species preferentially expressedin yeast cells with a molecular mass of 82kDa and isoeletric point (pI) of 6.1 was isolated from the proteomeof P. brasiliensis and characterized as an aconitase (E.C. 4.2.1.3). Aconitase is an enzyme that catalyzes theisomerization of citrate to isocitrate in both the tricarboxylic acid (TCA) cycle and the glyoxalate cycle (GC).We report the cloning and characterization of the cDNA encoding the aconitase of P. brasiliensis (PbACO1).The cDNA showed a 2400 bp open reading frame (ORF) and encoded a predicted protein with 779 aminoacids. The expression analysis of the PbACO1 gene was performed through quantitative real time RT-PCRand results demonstrated an increasing expression during differentiation from mycelium to yeast cells. Realtime RT-PCR assays showed that PbACO1 transcript is over expressed when the fungus grows on mediawith acetate and ethanol as carbon sources and is repressed by glucose. Those results point that PbACO1may be required when glucose is unavailable and other precursors of acetyl-CoA such as ethanol or acetateare available. These adaptations to nutrient-limited growth may involve a differential compartmentalization ofsome enzymes. Since mitochondrial, peroxisomal and cytosolic aconitases has been described in fungi, weobtained a polyclonal antibody against the purified recombinant PbACO1 in order to analyze the subcellularlocalization of the molecule in P. brasiliensis yeast cells. The protein localization in yeast cells compartmentsis under progress.Supported by FINEP, CNPq.177

4-27Comparative analysis of gene expression during macrophages infection by pathogenicfungi (Paracoccidioides brasiliensis and Histoplasma capsulatum)Simoneide, S.S. 1 and Felipe M.S.S. 11Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil. e-mail: simoneide.silva@gmail.com (presenting Author)Paracoccidioides brasiliensis and Histoplasma capsulatum are the human pathogenic thermo-dimorphicfungi that cause the paracoccidioidomycosis and histoplasmosis, respectively. The establishment of thesemycoses occurs by propagules inhalation in where they will be in contact with the lung epithelial cells andthe fungi are phagocytosed by resident macrophages. Once inside the phagocytic cells, the pathogensmay be eliminated or create a favorable microenvironment in where they could multiplicate and establishthe disease. In the present work we investigated by microarray technology and compared the kineticprofile of ex vivo peritoneal macrophage infected with P. brasiliensis and H. capsulatum. Total RNA frominfected and non-infected macrophages by these fungi at different times were extracted with the TRIzol ®reagent (Invitrogen, USA) and hybridized onto arrays glass. After normalization, the Significance Analysisof Microarrays software (SAM - http://www.stat.standford.edu) was used to assess the significant variationsin gene expression between infected and non-infected macrophages. Significantly modulated genes wereidentified by passing both a statistical tests q value

4-29The high affinity copper transporter of Paracoccidioides brasiliensis is up-regulated duringmacrophage infectionR.S. Santos 1 , Bailão. A.M. 1 , Borges. C.L. 1 , Salem-Izacc. S.M. 1 , Dantas. S.F.I.M. 1 , Deepe Jr. G.S.2 and Soares. C.M.A. 1* .1- Laboratory of Molecular Biology, Department of Biochemistry and Molecular Biology, Universidade Federal de Goiás, Goiânia - Brazil.2 – Division of Infectious Diseases, College of Medicine, University of Cincinnati, Ohio - USA. e-mail: celia@icb.ufg.brParacoccidioidomycosis (PCM) is a granulomatous mycosis caused by the dimorphic fungus Paracoccidioidesbrasiliensis. The fungus occurs as mycelium at 26ºC and as yeast at 36ºC. The fungus invades the human bodythrough inhalation of propagules, which can establish infection once they undergo phase transition to the yeastcells in the pulmonary alveolar epithelium. The success of host tissues colonization is intimately associated topathogen capacity on uptake essential nutrients from the host milieu. Copper is one of those nutrients and theability in acquiring this metal is considered a virulence factor. Copper is an important cofactor for a wide rangeof enzymes involved in many biological processes such as respiration, cell growth, iron uptake and oxidativestress. Transcriptional studies have shown that ctr3 (high affinity copper transporter) of P. brasiliensis is inducedin yeast cells recovered from liver of infected mice. In the present study we isolated and characterized the cDNAand gene sequences coding to CTR3 of P. brasiliensis. The cDNA showed a 582 bp open reading frame (ORF)encoding a predicted protein with 193 amino acids, predicted molecular mass of 21.5 kDa and of pI 8.6. Thegenomic sequence presented four exons interrupted by three introns. The transcriptional behavior of the ctr3 genewas analyzed during exposure of P. brasiliensis yeast cells to conditions of depletion of iron, copper and zinc bysemi-quantitative RT-PCR. It was shown a significant increase in the transcription level of the ctr3 gene in thecombined absence of the three metals, in absence of copper as well as in absence of iron. Real time RT-PCR wasused to analyze the expression of ctr3 in the two fungal morphological forms of P. brasiliensis and in yeast cellsinfecting macrophages. The ctr3 expression was upregulated in the yeast phase and in yeast cells residing intomacrophage phagosomes. Taken all together those data suggest the importance of ctr3 and of the copper/ironuptake system during the infective process. In order to better understand the biological function of this molecule,proteic interactions of CTR3 are under progress using a Saccharomyces cerevisiae two hybrid system.Financial support: MCT/CNPq, CAPES, FINEP and FUNAPE-UFG.4-30Alternative oxidase is transcriptionally stimulated at 36°C and is required for mycelium toyeast transition and viability of Paracoccidioides brasiliensisNatália M. Junqueira, Guilherme N. D. Souza, Hilsamara Y. Alves, Mariana P. Santos, João Paulo T. Di Benedette,Claudia B. L. CamposIP&D, Universidade do Vale do Paraiba, São José dos Campos, SP, Brazil. e-mail: cbcampos@univap.brAlternative oxidase (AOX) is an enzyme present in the mitochondrial respiratory chain of plants, fungi, manytypes of yeasts and some protistis, but absent in animal cells. Likewise cytochrome oxidase, AOX reduces themolecular oxygen to H 2O at the final step of the respiration process. AOx activation has been associated witha fine tuning of ATP production and control of cellular redox balance under environmental stress conditions.Recently, AOX has also been associated with virulence in C. neoformans. In this work, we studied the role of AOXin temperature-induced mycelium to yeast (M-Y) transition of P. brasiliensis using biochemical, molecular andcellular biology approaches. The AOX inhibitor salicyl hydroxamic acid (SHAM) at 0.5 mM reduced proliferationand viability of P. brasiliensis during M-Y transition without effecting dimorphism. At 2 mM, SHAM impaireddimorphism and killed the fungus. In yeast cells, SHAM also delayed proliferation, but fungi remained viable. Inoxygen consumption experiments performed with antimycin A, a respiratory complex III inhibitor, the total capacityof the AOX pathway was slightly increased in mycelia at 36°C compared to 25°C. At 36°C, the rate of oxygenconsumption and generation of H 2O 2by mitochondria was increased in mycelia, but respiration was coupled,which suggest that AOX might be controling energy production while prevents excessive ROS generation.Quantitative real time PCR showed that AOX transcripts highly increased 15 min after temperature shift to 36°C.The profile of AOX expression during M-Y transition followed a bimodal curve, increasing early after temperatureshift, decreasing to the basal level at 24 hours and rising again from 48 hours until day 7. In yeast cells, AOXtranscripts were about 30x the basal mycelial level. Our data show that alternative oxidase is biochemically andtranscriptionally stimulated in P. brasiliensis in response to temperature shift to 36°C, contribute to survival duringM-Y transition and to proliferation of yeast cells. We propose that AOX is required to orchestrate metabolic demandwhile avoid oxidative stress. Considering that AOX is absent in mammals, this enzyme might be a promizing targetfor therapeutical proposals.This work was supported by Fapesp. Key words: Paracoccidioides brasiliensis, alternative oxidase,mitochondria, dimorphism.179

4-31Transcriptional profile of Paracoccidioides brasiliensis induced by oenothein B, anantifungal agent from Brazilian Savannah plant Eugenia unifloraP. F. Zambuzzi 1 , Rezende, R. V. 1 , Borges, C. L. 1 ,Ferri, P. H. 2 , Santos, S. C. 2 , Soares, C. M. A. 1 and Pereira, M 11 Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiania-GO-Brazil. 2 Laboratório deBioatividade Molecular, Instituto de Química, Universidade Federal de Goiás, Goiania-GO-Brazil. e-mail: mani@icb.ufg.brParacoccidioides brasiliensis is a soil-borne fungus and the causative agent of paracoccidioidomycosis, asystemic mycosis that cause chronic and progressive infection. The long-time treatment, the high toxicity of thedrugs, and the appearance of resistant or multi-resistant strains have imposed the need for a permanent searchand development of new therapeutic approaches. A large variety of compounds extracted from plants has beenused to the discovery of new antimicrobial agents. The active compound oenothein B, purified from leaves ofEugenia uniflora, a Brazilian Savannah plant, has been evaluated on the growth, viability and gene expressionof P. brasiliensis. That compound interferes with yeast cell morphology and inhibits glucana synthase transcriptsaccumulate; suggesting that oenothein B can be a good candidate to antifungal agent. Aiming elucidate themechanism of action of oenothein B on P. brasiliensis, was realized Representational Difference Analysis (RDA).P. brasiliensis yeast cells were grown on MMcM (Mc Veigh & Morton) medium in the presence (tester) and in theabsence (driver) of oenothein B for 90 min, at 37 o C. After extraction of total RNA, the cDNA was obtained andused to RDA experiments according with modified protocol previously described by Pastorian et al. (2000). RDAexperiment originated 280 ESTs that were successfully sequenced and originated 28 contig and 24 singlets. Usingthe BLATX program, was possible classified the ESTs in agreement with the functions. The analyses indicated thepresence of transcripts with functions related with cell wall and membrane, elongation and transcription factors andhypothetic proteins. The elucidation of the action mechanism of oenothein B will facilitate the design and synthesisof related compounds with enhanced pharmacological profiles.4-32Transcriptional profile of murine macrophages infected with pathogenic fungus HistoplasmacapsulatumSimoneide.S.S 1 , Passos-Silva. D. G. 2 ., Teixeira. S.M.R 2 , Junta, C. 3,4 , Medeiros. A. I. 6 , Silva. C. L. 5 , Faccioli. L. H. 6 , Passos.G.A.S. 3,4 , Felipe. M.S.S. 11Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazil. 2 Departamento de Bioquímica e Imunologia, UFMG,Belo Horizonte, MG, Brazil. 3 Departamento de Genética, USP, Ribeirão Preto, SP, Brazil. 4 Faculdade de Odontologia, USP, RibeirãoPreto, SP, Brazil. 5 Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto 14040-900, SP, Brazil. 6 Faculdade de CiênciasFarmacêuticas, USP, Ribeirão Preto, SP, Brazile-mail: simoneide.silva@gmail.comHistoplasma capsulatum is the thermo-dimorphic fungus that causes histoplasmosis. The yeast form of P.brasiliensis acts as a facultative intracellular pathogen, being able to survive and replicate within macrophages asa probable mechanism to evade the immune system. In the present work the kinetic profile of murine macrophagesinfected with H. capsulatum was investigated. A clinical isolate of the fungus was used for macrophage infection.Total RNA from infected and non-infected macrophages at 6, 24 and 48 hours was extracted with the TRIzol ®reagent (Invitrogen, USA) and hybridized against glass arrays. After normalization, the Significance Analysis ofMicroarrays software (SAM- http://www-stat.standord.edu/) was used to assess the significant variations in geneexpression between experimental and control conditions. Genes were considered to be modulated if they crossedtwo statistical thresholds (q value

4-33Analysis and identification of three P. brasiliensis genes possibly implicated in virulenceO. Hernandez 1 , AM Garcia 1 A Restrepo 1 and JG McEwen 1,2 .1 Corporación para Investigaciones Biológicas, CIB, Medellín – Colombia. 2 Facultad de Medicina Universidad de Antioquia, Medellín– Colômbia. e-mail: orvillehr@hotmail.comIn general, fungal dimorphism is considered an essential trait for successful adaptation of pathogenic fungito the human host, and this ability that has been studied among different dimorphic fungi and characterizedby several physiological and structural modifications. P. brasiliensis conidia are consider to be the infectiousparticles which once inhaled into the lungs begin their morphological transition to the yeast phase. This processhas been previously studied, both morphologically and at the transcriptome level either during batch culturegrowth or in the infection process itself. Some of the major alterations described in the transcriptome profileduring the morphological switch include differential expression of genes involved in the rearrangement of cell wallcomposition, stress response and diverse metabolic pathways. Nonetheless, conidia-to-yeast transition remainsgreatly uncharacterized and, by this reason, it is important to analyze the relevance of specific proteins during suchprocess. Recently, the complete genome and the corresponding annotation of P. brasiliensis strain Pb 03 hasbeen published by the Broad Institute with 90% of all possibly proteins expressed by the fungus being incorporatedinto this genome. The aims of this work was to analyze some important gene sequences considered as virulencefactors in other fungi, such as: Glucan synthase, involved in the generation of beta-glucan, important in the cellwall -and an alternative oxidase, involved in the electron transport chain. The latter provides an alternative routefor electrons passing through the electron transport chain to reduce oxygen and glyceraldehide 3 phosphatedeshidrogenase (GAPDH), catalyzes the sixth step of glycolysis and serves thus to break down glucose for energyand carbon molecules. Based in the Pb 03 genome “in silico” analyses were made using the partial sequencesof the genes mentioned above; additionally, PCR primers were designed to amplify these genes in strain ATCC60855, and the PCR products were sequenced and compared with the database published by the Broad Institutein order to confirm their presence. Results: All the sequences corresponding to virulent genes searched for werefound with similar levels above 90% and e-value of 0.0 for all of them. The probability of these results being atrandom is near zero, confirming the existence of these genes in P brasiliensis. ClustalW analysis showed highsimilarity between the sequence of these genes in 60855 and Pb03 strains. These results made of these genessuitable targets for knockout–knockdown studies aimed at confirming their role in pathogenicity.4-34Internalization of magnetic nanoparticles by Paracoccidioides brasiliensis yeast cellsA.C. Amaral 1, 3 , Braun S. 1 , Nunes E.S. 4 , Bocca A.L. 1 , Lima E.C.D. 4 , Báo S.N. 1 , Azevedo R.B. 1 , de Morais P.C. 2 and Felipe M.S.S. 11Instituto de Ciências Biológicas e 2 Física, Universidade de Brasília, UnB, Brasília, Brazil. 3 Ciências Genômicas e Biotecnologia,Universidade Católica de Brasília, UCB, Brasília, Brazil. 4 Instituto de Química, Universidade Federal de Goiás, UFG, Goiânia, Brazil.e-mail: amaralandre@yahoo.com.brIntroduction and aim: Functionalized magnetic nanoparticles can be used for several applications in biology,such as cell labeling, isolation and purification of specific molecules or cells from a culture (J. Biosci. Bioeng.,100(1):1-11, 2005). When properly functionalized these nanoparticles, with average size between 3 and 20nm caneasily penetrate the cells. Once inside the cell, this magnetic nanoparticle can separate or drive the cell using anexternal magnetic field. Functionalization is possible by linear linker molecules with reactive organic groups at bothends. One group is attached to the nanoparticle surface and the other is used to link biocompatible molecules,antibodies, fluorophores, etc (J. Nanob., 2:3, 2004). Here we report the internalization of magnetic nanoparticlesby P. brasiliensis Pb18 yeast cells. Methods: To investigate the internalization of nanoparticles, the yeast cells(3 × 10 4 cells/mL) were incubated during 1, 4 and 8 hours in YPD medium containing maghemite nanoparticlesfunctionalized with dimercaptosuccinic acid (10 14 particles/mL), followed by routine techniques for transmissionelectron microscopy (TEM). To evaluate the toxicity, after the exposure times to magnetic particles 3 × 10 2 cells wereplated in BHI supplemented with 4% horse serum, 5% P. brasiliensis 192 culture filtrate and 40mg/L Gentamicin.The plates were incubated at 36 ºC and CFU was counted at 21 st day post-plating to asses the cell viability. Resultsand discussion: The TEM has revelead the uptake of magnetic nanoparticles by the P. brasiliensis Pb18 after4 and 8 hours of incubation. The majority of nanoparticles were found between the cell wall and the plasmaticmembrane, and a very small number were observed within the cytoplasm. These magnetic nanoparticle uptakesdid not interfere with the cell viability, once no differences on CFU between the treated groups and controls werenoted. These findings could be useful, for example, to accompaning the establishment of the paracoccidioidomycosisin animal model. Since the magnetic nanoparticles have the capability to be used as contrast agents in magneticresonance imaging (J. Biosci. Bioeng., 100(1):1-11, 2005), animals can be infected with P. brasiliensis containing thenanoparticles to investigate the kinectic of the fungal infection. Financial support: CNPq.181

4-35Molecular typing and evolution of the Paracoccidioides genusTeixeira, M. M. 1 , Paes, H. C. 1 , Fernandes L. 1 , San-Blas, G. 2 and Felipe, M.S.S. 11 Instituto de Biologia, UnB, Brasilia - Brasil. 2 Instituto Venezoelano de Investigaciones Cientificas, IVIC – Venezuela.e-mail: marcus.teixeira@gmail.comBackground and aims: Paracoccidioidomycosis is a systemic illness with an area of occurrence coveringmost of Latin America, affecting mainly young male individuals living in the countryside. Studies on differentisolates of the causative agent, Paracoccidoides brasiliensis, have revealed a great degree of genetic variabilityin this pathogen. The Pb01 isolate, in particular, differs from the three phylogenetic species (S1, PS2 and PS3)previously described by the genealogical concordance method (GCPSR). This work aimed at looking into theexistence of a systematically significant group of “Pb01-like” isolates through GCPSR. Methods: 88 isolates wereanalyzed by phylogenetic methods of Maximum Parsimony and Bayesian analysis in thirteen (13) polymorphicregions, including individual and concatenated loci. The polymorphisms were evaluated and the divergence timeof speciation was measured between the phylogenetic groups. We also assessed the possibility of recombinationboth within each group and between them by split decomposition analysis. Results: Phylogenetic data enabledthe grouping of “Pb01-like” isolates in a clade distinct from S1, PS2 and PS3. In keeping with GCPSR, the “Pb01-like” clade can be considered a new phylogenetic species, since the clade corresponding to “Pb01-like” isolatesis strongly supported by all phylogenetic trees – separately and in a concatenated analysis – generated frompolymorphism data of the thirteen loci, with highly significant values of posterior probability (1.0) and bootstrapagreement (100%). A high number of fixed polymorphisms were identified between the “Pb01-like” clusterand that composed of the other three species, which suggests a blockage of genetic flow between them. Thespeciation event that defined the new phylogenetic group is sympatric relative to S1 and PS2. The “Pb01-like”cluster and the group that includes S1, PS2 and PS3 have been genetically isolated for about 19 million years.Recombination analysis revealed that recombination events occur independently inside the two groups, whichsuggests reproductive isolation. Conclusion: Added to the morphological exclusive data for this genetic isolatedgroup (Bagagli, E., personal communication) we suggest that the phylogenetic species encompassing the “Pb01-like” clade should be named Paracoccidioides lutzii, as a tribute to medical mycologist Adolpho Lutz, who firstdescribed human pathogen P. brasiliensis one hundred years ago. Financial support: CNPq, FAP-DF, FUB.4-36Adhesion and expression of ligands of extracellular matrix (ECM) by differentParacoccidioides brasiliensis isolatesJ.F Silva 1 , G. Benard 2 , M.J.S. Mendes-Giannini 1 .1Faculdade de Ciências Farmacêuticas de Araraquara – UNESP – Brasil and 2 Faculdade de Medicina de São Paulo – USP – Brasil.e-mail: silvajf@fcfar.unesp.Paracoccidioides brasiliensis is a dimorphic fungus and the causative agent of paracoccidioidomycosis, a mycosisusually characterized by multiple organ involvement and a chronic evolution. P. brasiliensis virulence can be attenuatedor lost after subsequent cycles of subculturing for prolonged periods and reestablished after passage in animals or inepithelial cells cultures. The success of colonization is a complex event, generally involving a ligand encoded by thepathogen (adhesins) and a cell receptor. An important aspect in the interaction between P.brasiliensis and its host isthe ability to adhere to matrix extracelular components (ECM). The understanding of this process and identificationof these molecules may eventually provide new targets for more efficient treatment strategies. The aim of this studywas to investigate the protein expression profile of P. brasiliensis isolates during infection of pulmonary epithelial cells(A549), and more specifically, the pattern of proteins expressed by four different isolates (Pb01, Pb113, Pb339 andPb265) with ability to ligate to ECM components, before and after passage in cell cultures. All P. brasiliensis isolatespresented different capacity to adhere to epithelial cells, but specially before cell cultures passage. The profiles of fourP. brasiliensis “cell-free” extracts from repeatedly subcultured strains and from reisolated strains were analyzed by twodimensionalelectrophoresis. Differentially expressed proteins were identified. Furthermore, a large series of ligandsto ECM components was observed: 62 ligands to collagen type I, 23 to fibronectin, and 18 to laminin, of a total of 420proteins. The isolates Pb01 and Pb339 presented fibronectin ligands only after reisolation from epithelial cells, apparentlyshowing that these ligands may have a role in pathogenicity. The same occurred with Pb01 in relation to collagen.Pb113, even after reisolation, did not present laminin ligands, whereas Pb265 did not present fibronectin ligands. Thus,this study suggested that different patterns of the ECM ligands can occur depending on the isolate and that multipleproteins may function as adhesins, depending on the challenged posed by the microenvironment. Expressive number ofproteins, with characteristics of ECM ligands, was observed mainly after passage in epithelial cultures. These differentialprofile ligands could be associated with the ability of this fungus to express different proteins in fungal-host interaction.Suppoted by: FAPESP; PADC-FCF; CNPq and CAPES182

4-37The heath shock factor of Paracoccidioides brasiliensis and its complementation ofSaccharomyces cerevisiaePaes HC, Matos LF, Teixeira MM, Torres FAG,Felipe MSS.Laboratorio de Biologia Molecular, Instituto de Biologia Celular, Universidade de Brasilia, Distrito Federal, Brasil.e-mail: sorumbatico@gmail.com (presenting author)The heath shock factor, a ubiquitous transcription factor of eukaryotes, is responsible for the regulation ofseveral stress response genes in baker´s yeast and metazoans, but has not been thoroughly studied in filamentousor dimorphic fungi so far. It might be of importance in the control of morphogenetic programmes in the lattergroup, in view of its evolutionary specialization to respond to changes in temperature. Therefore, we set outto characterize its homologue in P. brasiliensis, and have identified, cloned and sequenced it. The hsf gene ispresent in a sole copy in this fungus, and the corresponding sequence is the largest heat shock factor found so far.The ORF codes for protein 840 residues long, wich contains easily identifiable DNA binding and oligomerizationdomains. However, is presents other regions that are conserved among other Eurotiomycetidae as Coccidioides,Histoplasma and aspergilli, but that bear no resemblence to gous expression of hsf in S. cerevisiae was abble torescue the loss of the native gene, wich would normally be lethal to the yeast. The recombinant strain presented agrowth defect that we have beeb unable to account so far. The functional evidence of trans – complementation hasprompted us to device gel shift experiments to further assess the behavior of this protein, and these are currently inprogress. We intend to examine the binding of this new hsf to canonical and non-canonical heat shock elements(HSEs) from promoters of several genes of P. brasiliensis and to the classic HSE described in S. cerevisiae. It maybe that genes other than heat shock proteins respond to this factor in P. brasiliensis , including differential genesfor dimorphic transition and determinants of virulence – a possibility we also envision to investigate.183

Poster Session5Immunology:Patients / Experimental Animals

5-01Phenotypic and functional characterization of human dendritic cells induced by low andhigh-virulence strains of Paracoccidioides brasiliensis2Fornazim MC, 1Mamoni RL, 2Spago MC, 1Oliveira RTD, 2Gabetta CS, 2Nowill AE, 1Blotta MHSL.1Laboratório de Imunologia Celular e Molecular - Departamento de Patologia Clínica – FCM - UNICAMP e 2Laboratório de ImunologiaCelular - CIPOI –FCM - UNICAMP Campinas – São Paulo – Brasil.e-mail: fornazim@unicamp.brParacoccidioidomycosis (PCM) is the most important deep mycosis in Latin America. Dendritic cells (DCs)play an essential role in the initiation and modulation of the immune response, since cells they are the onlyprofessional antigen-presenting cells able to stimulate naive T lymphocytes. To determine whether DCs becomephenotypically and functionally competent in the presence of P. brasiliensis, peripheral blood monocytes obtainedfrom healthy individuals were differentiated in the presence of GM-CSF and IL-4 for 7 days and pulsed in vitrowith yeast cells (Pb18, high virulence and Pb265, low virulence) for 24 hours. After that, DCs were stainedwith monoclonal antibodies against surface antigens and analyzed by flow cytometry. Cell cultures (DCs and Tlymphocytes) supernatants were stored at -20°C for cytokines determination (TNF-a, IL-6, IL-12p40, IL-10 e IFNg)by ELISA. DCs pulsed with the both strains of the fungus present morphology and phenotype characteristic ofmature cells with increased expression of CD83, CD80, CD86 and CCR7 and decreased phagocytic capacity. Theexpression of CD83 and the production of inflammatory cytokines such as TNF-a, IL-6 e IL-12p40 were higherin DCs pulsed with low virulence yeast cells (Pb265) compared with Pb18. Moreover, DCs pulsed with Pb265were also able to induce higher levels of T lymphocytes proliferation and IFN-g production than those generatedin the presence of Pb18. Altogether the results indicate that DCs generated in the presence of the fungus exhibitphenotype of mature APC (expression of CD83, CD80, CD86 and CCR7), are able to produce pro-inflammatorycytokines as well as to stimulate adaptive immune response inducing theCD4+ T lymphocyte proliferation and IFN-g production. Furthermore, the higher response obtained with thestrain of lower virulence demonstrates the ability of DCs to sense small changes in pathogens and to appropriatelymodulate the adaptive immune response.5-02Paracoccidioidomycosis patients dendritic cells: Effect of Paracoccidioides brasiliensisantigens on surface molecules expressionP. K. Sato 1 , Oshiro. T.M. 2 , Diogo. C.L. 2 , Passos. E.C. 3 , Sadahiro. A. 4 , Shikanai-Yasuda. M.A. 2,51 Pós-graduação do Depto de Moléstias Infecciosas e Parasitárias da Faculdade de Medicina da USP(FMUSP), São Paulo, Brasil.2 Laboratório de Investigação Médica em Imunologia (LIM-48) do Hospital das Clínicas da FMUSP, São Paulo, Brasil. 3 Curso deAprimoramento Fundap do Depto Moléstias Infecciosas e Parasitárias da FMUSP, São Paulo, Brasil. 4 Instituto de Ciências Biológicas- Universidade Federal do Amazonas, Manaus, Brasil 5 Depto de Moléstias Infecciosas e Parasitárias da FMUSP, São Paulo, Brasil.e-mail: lim48imuno@yahoo.com.brIntroduction: Paracoccidoidomycosis (PCM) is an endemic infectious disease caused by the pathogenicand dimorphic fungus Paracoccidioides brasiliensis (P. brasiliensis). Previous studies have shown that Th1immune response confers protection in PCM whereas Th2 is associated with susceptibility. Recently, as antigenpresenting cells, dendritic cells (DCs) able to activate Th1 response were described in resistant mice infected withP. brasiliensis. However, their role in human cellular response for this disease is not clear.Objective: To investigate the effect of P. brasiliensis 43KDa-glicoprotein (gp43) and cell-free antigen (CFA)on DCs’ surface molecules expression.Materials and Methods: Monocyte-derived DCs were generated from peripheral blood of cured PCM patients(positive paracoccidioidin skin test and serum specific antibody titles measured by CIE≤1: 4). Then they werepulsed with CFA or gp43 and/or activated with inflammatory recombinant cytokine TNF-α, after differentiation(CD11c + , CD1a + , CD14 - ). DCs’ surface molecules expression was verified by flow cytometry.Results: Both Gp43 and CFA stimuli induced DCs maturation resulting on high expression of CD86, HLA-DRand DC-Sign molecules. Gp43 stimulus is associated with higher percentage of positive cells for these markerswhen compared with TNF-α activation or CFA + TNF-α activation.Conclusions: DCs were more efficiently activated by GP43 than CFA. CFA components may be common toother fungi resulting in non-specific and weaker maturation of DCs.Financial support: Fapesp 04/14955-3 e FFM.187

5-03Altered expression of stats 1,3,4,5 and their modulation by cytokines in mononuclear cellsfrom paracoccidioidomycosis patients1,2Romano C.C.; 2 Mendes-Giannini, M.J.S., 1 Duarte, A.J.S. 1 Benard. G.1Universidade Estadual de Santa Cruz- UESC, Ilhéus, Bahia, Brasil. 2 Laboratório de Investigação Médica 56- FMUSP, São Paulo, Brasil.3UNESP, Araraquara, São Paulo, Brasil. e-mail: ccromano@uesc.br (presenting Author)Introduction/Objective: We have previously shown a specific imbalance in the IL-12/INFγ/IL-10 regulationin PCM patients. In this regard, it has recently been shown that cytokines modulate the expression and activity ofthe STATs family of proteins. In this study we analysed the expression of inactive and active STATs 1, 3, 4 and 5proteins by intracellular cytometry.Methods/Results: The mononuclear cells of cured controls and patients with PCM was gotten through gradientof Ficoll and the cells cultured for 72h with medium plus ionomycin and PMA (basal) or stimulated with gp43. Weobserve higher levels of gp43 STAT 1 (inactive and active ) in the cured controls when compared with the patientscells (25±3,7 controls vs 13,8±3,9 patients)( active: 28.4 ± 2.8 controls vs 8.8 ± 1.3 patients).Similarly, the gp43STAT 4 expression was higher in the cured control in relation to patients for the two isoforms (26,6±3,2 vs 19,8±4,6for inactive and 33,4 ±4,5 vs 10,5±3,3 for active). However, the active STAT 3 expression was significantly biggerin patients’ group when compared with controls (7,0±2,2 vs 37,3±8,5 for basal; and 8,8±2,2 vs 35,6±8,7 for gp43).We also observed bigger expression of inactive STAT5 in the patients cells, when compared with control (8,7±3,2vs 24.1±4,5, for basal; 10.9±1,8 vs 22,5±4.6 for gp43). Upon gp43 stimulus, only controls’ cells were able toactivate this factor of transcription (17,2± 4,4 STAT 5 active for basal; 28,5±7,5 STAT 5 active for gp43). In vitroneutralization of endogenous IL-10 resulted in increase of gp43- active STAT 1 in patients to levels displayed bythe cured subjects and IL-12 addition restored STAT 4 expression in PCM patients to levels expressed by thecured subjects for the two isoforms.Conclusion: These data suggest that the PCM infection can inhibit the activation of transcription factorsas STAT1 and 4, which is important for IFN-g and IL-12 production. In parallel, the activation of STAT3 andconstitutive expression of STAT5 in cells of patients related, respectively, with the control of lymphocytes activationand protection against apoptosis.5-04Increased T cell regulatory activity in patients with paracoccidioidomycosisM.C. Ferreira, M.H.S.L Blotta, R.L. Mamoni.Department of Clinical Pathology - Faculty of Medical Science – UNICAMP – Campinas / SP – Brazil.e-mail: mcf@fcm.unicamp.br (presenting Author)Introduction and Aims: It has been shown that Paracoccidiodomycosis (PCM) patients present a suppressedcellular immune response characterized by negative DHT to P. brasiliensis antigens, elevated apoptosis ofactivated lymphocytes, high expression of CTLA-4, and production of IL-10 and TGF-β. These data point towardthe involvement of regulatory T cells (Tregs), which have emerged as a crucial T cell population regulating theimmunological response in several infectious and non-infectious diseases. The aim of this study was to verifywhether Tregs are involved in the immunosuppression observed in PCM patients, through the analysis of thenumber and the phenotype, as well as the functional activity of Treg cells (CD4 + CD25 + T cells) in peripheral bloodof patients before and after antifungical treatment.Methods and Results: Ex vivo expression of mRNA for FoxP3 in PBMC evaluated by qRT-PCR was higher inPCM patients with active disease than in the healthy controls. The number and phenotype of Treg cells (CD4 + CD25 +T cells) were determined by flow cytometry. In accordance to mRNA analysis, the results showed that before thetreatment, PCM patients present higher number of Treg cells (CD4 + CD25 + ) than patients after treatment or healthycontrols. Furthermore, we detected an increased expression of CTLA-4, LAP-1 and GITR on TCD4 + CD25 + ofpatients with active disease in relation to healthy individuals. In order to compare the functional activity of Tregs,cells obtained from patients (with active or treated disease) and healthy controls, we analyzed the suppressiveeffect of these cells in cultures of PBMC stimulated with the mitogen ConA. The results demonstrate that the Tregsfrom patients with active disease exhibited stronger regulatory activity than cells from control or treated patients ina dose dependent manner.Conclusions: The elevated expression of Foxp3 mRNA and increased number of Treg cells in peripheralblood of patients with active disease, which presented higher expression of regulatory markers and suppressiveactivity in vitro, suggest an elevated regulatory activity during the disease and indicate the potential role of Tregsin the immunosuppression observed in PCM. Supported by FAPESP and CNPq188

5-05Polymorphisms analysis of CTLA-4 gene in paracoccidioidomycosis patientsV. F. Lozano¹, Paes H.C.², Teixeira M.M.¹, Lins T.C.L.³, Vieira R.G.³, Blotta M.H.S.L. 4 , Goes A.M. 5 , Pereira R.W. 3 , BoccaA.L .¹,2 , Felipe M.S.S 1,2 .¹ Faculdade de Medicina, UNB, Brasilia – Brasil. ²Instituto de Biologia, UNB, Brasília – Brasil. 3 Pós-graduação em Ciências Genômicas eBiotecnologia, UCB, Brasília – Brasil. 4 Faculdade de Ciências Médicas, UNICAMP, Campinas – Brasil. 5 Instituto de Ciências Biológicas,UFMG, Belo Horizonte - Brasil.e-mail: vivianefurlan74@yahoo.com.br (presenting Author)The CTLA-4 protein is mainly expressed in activated T cells and plays an essential role in the immune responsethrough its regulatory effect on T cell activation. Polymorphisms of the CTLA-4 gene have been correlated toseveral autoimmune pathologies and, more recently, to neoplastic and infectious illnesses. Paracoccidioidomycosis(PCM) is a systemic mycosis caused by the dimorphic pathogen Paracoccidoides brasiliensis. Its symptoms areassociated to various factors, including altered secretion of cytokines (higher IL-10 and lower IFN-γ production),hypergammaglobulinaemia and suppression of cellular immunity. This low responsiveness is also attributed to ahigher expression of CTLA-4 in the cells of patients relative to control individuals. This work aimed at studyingpossible associations of PCM and two single nucleotide polymorphisms (SNPs) of CTLA-4, -318C/T in the promoterand +49A/G in exon 1. To this end, 74 PCM patients and 76 controls from different regions of Brazil had theirallelic and genotypic frequencies determined. The comparison of genotypic and allelic frequencies had not shownsignificant differences between patient and control groups that could link either CTLA-4 SNP to PCM. The analysisof results concerning haplotypic frequencies revealed a strong linkage disequilibrium (D’=1) between SNPs -318and +49 for both groups. Nevertheless, the analysis did not reveal significant differences of haplotypic frequenciesbetween groups, which reinforce the absence of correlation between those SNPs and PCM. Furthermore, thegenetic structure of the patient and control groups was also evaluated in order to eliminate the bias of ancestry.There was a predominance of European over Amerindian and African ancestries in both groups. From the resultsit was possible to determine that the population used in this study was genetically homogeneous, and that theabsence of an association between polymorphisms of CTLA-4 and PCM cannot be attributed to ancestral bias.This work shows that there is no association of SNPs -318 and +49 of CTLA-4 and resistance and/or susceptibilityto paracoccidioidomycosis.Financial Suport: CNPq5-06Lymphoproliferation, levels of antibodies and cytokines to ab2-β anti-idiotipic antibodiesin patients with paracoccidioidomycosis (PCM)C.R. Furuchó 1 , Oliveira 1 C.C.U., Bertossi 1 , D.R.T., Fonseca 1 , C.A., Shikanai-Yasuda, M.A 1,2 .1 Laboratório de Investigação Médica em Imunologia (LIM-48), Hospital das Clínicas da Faculdade Medicina da USP(FMUSP), São Paulo,Brasil. 2 Departamento Moléstias Infecciosas e Parasitárias, FMUSP, São Paulo, Brasil.e-mail: lim48imuno@yahoo.com.brThe development of specific, sensitive, and easily accessible methods for monitoring of antibody levels andanalysis of cellular immune response represent important strategy for diagnosis and host-parasite studies inparacoccidioidomycosis. The performance of an anti-idiotipic 7.B12 (Ab2-β) antibody was compared with gp43in lymphoproliferative and serological tests in patients with PCM. Methods: 51 patients with PCM, 36 previouslytreated/cured (CT) patients and 14 with active disease (AD). The criteria for inclusion were: positive mycologicaland histopathological tests and/or high levels of serum antibodies (CIE>1/32) to P. brasiliensis and negative CIEfor 25 healthy individuals (CO). Ab2-β (1 and 4µg/ml) and gp43 (1µg/ml) were employed in lymphoproliferativeassay, PHA (5µg/ml) was used as mitogen. IgG, IgG 1, IgG 2, IgG 3, IgG 4ELISA anti- gp43 (5µg/ml) and anti-Ab2-β(15µg/ml) were analysed. Results: Ab2-β induced lymphoproliferation in patients with PCM. The specificity wasbetter using 1µg/ml of Ab2-β, however, the sensitivity was higher using 4µg/ml. Analyze of lymphoproliferationusing gp43 showed that CT and CO groups are different. There was no cross reactivity in CO group using Ab2-β(1 and 4 µg/ml) and gp43; there was no difference between levels of IL-10 secreted by cells from CT, AD or COgroups. Levels of IFN-γ from CT and AD groups were related to the presence of lymphoproliferation and low levelsof IL-10; specificity for gp43 or Ab2-β was higher than 95%. Higher Ig G and IgG 2anti-gp43 and IgG anti-Ab2-βwere registered in CT and AD than in the CO group; IgG 4levels of anti-gp43 were significantly higher in the CTthan CO group, and the highest levels was observed in the AD group. Conclusions: The Ab2-β antibody showeda good specificity but lower sensitivity than gp43 in the analysis of lymphoproliferation and humoral immuneresponse. Financial support: CNPq 472809/04189

5-07Paracoccidioidomycosis: Study of citotoxic immune response in cutaneous and mucosal lesionsC. Pagliari 1,2 , N. V. Pereira 1,2 , M. I. S. Duarte 1 and M.N. Sotto 2 .Laboratório da Disciplina de Patologia de Moléstias Transmissíveis 1 Departamento de Patologia e 2 Departamento de Dermatologia. Faculdade deMedicina da USP, São Paulo – Brasil. e-mail: cpagliari@usp.brSome studies have related the role of CD8+T cells and Natural-killer (NK) cells in immune response in PCM,both capable to produce immune mediators. Experimentally, NK cells are associated to a depressed immuneresponse. Studies focusing CD8+T cells in PCM suggested their role in the control of fungi due to perforin andgranzyme production. Our objective was to contribute to the study of in situ PCM immune response addressing theparticipation of NK cells, CD8+T cells and granzyme B expression. Sixty biopsies of PCM skin and mucosa wereclassified according to tissue reaction: presence of compact granulomas (G1), poorly-organized granulomas (G2)and both kinds of granuloma in the same lesion (G3). CD8+T cells, NK cells and granzyme B were demonstratedby immunohistochemistry. The cells were quantified and the results compared by Mann-Whitney test. There wasa statistically significant increase in the number of CD8+T cells over NK cells in cutaneous G1 and G2 lesions.There was no statistical difference regarding such cells in G3 lesions, although CD8+ T cells were abundant insuch lesions. In mucosal lesions, CD8+T cells were increased in number over NK cells in all groups (p

5-09Alterations in pulmonary mechanics during experimental infection by ParacoccidioidesbrasiliensisJ.F. Fracon 1 , Bocca A.L. 2 , Siqueira I. M. 2 Zin W.A. 3 , Faffe D.S. 3 , Jerônimo M.S. 2 , Soares M.A.S. 4 , Russo R.C. 5 , Figueiredo F. 11- University Católica of Brasília – UCB - Brazil; 2 –University of Brasília – UnB - Brazil, 3- Federal University of Rio de Janeiro – UFRJ- Brazil; 4- Sabin Laboratory – DF - Brazil; 5-Federal University of Minas Gerais – UFMG - Brazil.Paracoccidioidomycosis, a systemic mycoses restricted to Latin America, is caused by dimorphic fungusParacoccidioides brasiliensis. When the patients are treated with specific therapy there are improvements butlesions usually remain as sequelae. The remission is often accompanied by significant pulmonary fibrosis. Inthe present work, mice were infected intravenously with variable yeast cells of P. brasiliensis and were analyzedthe morphology and pulmonary mechanics in the course of the experimental infection caused by the fungus,at different days pos infection. The ventilated, static (Est), dynamic (Edyn), elastances, their differences (ℵE),resistive (ℵP1), viscoelastic/inhomogeneous (ℵP2) pressures and the total variation of pressures (ℵPtot) of thelungs, were obtained by end-inflation occlusion method. Arterial blood samples were collected for arterial bloodgas analysis of the parameters: hidrogenionic potential (pH), partial pressures of oxygen (PaO2), carbon dioxide(PaCO2), oxygen saturation (SaO2), base excess (BE) and bicarbonate (HCO3). After the collecting data theanimals were sacrificed and the lungs were removed and were analyzed for morphology and for the concentrationof chemokines. Histopathology revealed progressive inflammatory response, leading to granuloma formation, withincrease in the fungus counts and formation of fibrosis, gradual to the development of infection, compromisingthe pulmonary tissue. The dosage of chemokines detected the presence of: MIP-1 α, RANTES, MIG and KC,demonstrating significant increase directly proportional to the time of infection. The presence of mist acidosisfollowed by metabolic acidosis, indicating alterations more pronounced in the acute phase, was observed in thearterial blood. In the evaluation of pulmonary mechanics showed increases in static and dynamic elastancesand increases in viscoelastic/inhomogeneous pressure and in the total variation of pressures of the lungs. Thisresults indicate that the lung injury is progressive, with the establishment of tissue fibrosis, probably determining arestrictive functional pattern as a result of lower distensibility of lung tissue.Financial Suport: CNPq.5-10TLR-2, TLR-4 & DECTIN-1 expression in human macrophages and neutrophils stimulatedby Paracoccidioides brasiliensisBonfim, CV; Mamoni, RL; Blotta, MHSL.Laboratório de Imunologia Molecular e Celular. Departamento de Patologia Clínica – Faculdade de Ciências Médicas – UNICAMPe-mail: camilinha@gmail.comThe pattern of the immune responses to P. brasiliensis determines the disease progression and clinicaloutcome. Innate immune response is mediated by phagocytic cells, such as macrophage and neutrophils, whichingest and kill invading pathogens and then trigger the adaptive immune system through the secretion of cytokinesand chemokines. C-type like lectin receptors (CLR) and Toll-like receptors (TLRs) are the two main PRRs involvedin fungus recognition. Therefore, the purpose of the present study was to evaluate the expression of TLR-2, TLR-4 and dectin-1 (CLR) in macrophage and neutrophils from healthy individuals by flow cytometry analysis, afterstimulation with Pb18 (high virulence) and Pb265 (low virulence) yeasts of P. brasiliensis. As positive controlswe used specific ligands to TLR-4 (LPS), TLR-2 and dectin-1 (zymosan). Our results demonstrated a decreasedTLR-2 expression on macrophages and neutrophils as soon as 30 minutes after yeast cells stimulation. Thisdecrease was similar to the one caused by zymosan stimulation. A marked decrease of TLR4 expression wasdetected only after 60 minutes of stimulation and in this case, it was possible to verify that Pb265 was able toinduce a higher decrease of TLR-4 expression than Pb18. The low-virulence isolate (Pb265) also provided ahigher decrease on dectin-1 (a β-glucan receptor) expression in both cells, but mainly in macrophages, showing anelevated potential to activate the host’s defense response. The highest decrease of the dectin-1 expression wasobserved on macrophages after 30 minutes of stimulation with yeast cells. Altogether, our data suggest that thedecrease of TLR-2, TLR-4 and dectin-1 on phagocytes surface can be related to the recognition and internalizationof the fungus, resulting in activation of the immune response.Supported by FAPESP and CNPq191

5-11Absence of TLR2 results in less severe paracoccidioidomycosis but increased inflammatoryresponse caused by PMN & TH17 cellsLoures, FV & Calich, VLGInstituto de Ciências Biomédicas, USP, São Paulo, Brasil.Email: loures@icb.usp.brMacrophages are the first host cells that interact with Paraccocidioides brasiliensis. Toll-like receptors (TLRs)present in macrophages recognize molecular patterns of microorganisms and influence innate and adaptativeimmunity. However, the role of TLR2 in paracoccidoidomycosis was never investigated. The aim of our work wasto characterize the involvement of TLR2 in murine paracoccidioidomycosis using TLR2 -/- and C57BL/6 control(WT) mice. In vitro cultivated macrophages, primed or not by IFN-γ, were challenged with yeasts and fungicidalactivity, cytokines and nitric oxide (NO) production assessed 48h later.Mice were in vivo infected by the intratracheal route with 1x10 6 yeasts. After 48h, 2 and 11 weeks, the micewere sacrificed and severity of infection was evaluated by CFU counts, presence of NO and cytokines in lunghomogenates besides flow cytometric analysis of lung infiltrating leucocytes. We verified that macrophages fromTLR2 - / - mice secreted lower levels of NO, MCP-1 and IL-10 and have a decreased ability to interact with fungusthan those from WT mice resulting in decreased number of viable yeasts recovered 48h postinfection. In vivo,diminished fungal burdens and NO levels were detected in the lungs of toll-deficient mice at all postinfectionperiods assayed. Augmented levels of IL-17 were detected in the lungs of TLR2 - / - mice by 48h postinfection.At week 2, higher amounts of IL-17 and IL-23 associated with decreased levels of MCP-1 were found. Atweek 11, increased IL-23 was associated with decreased concentrations of MCP-1, IL-10 and IL-12. At bothpostinfection periods, increased numbers of polymorphonuclear (PMN) cells were observed in the lungs of TLR2 −/− .Furthermore, by week 2 decreased number of macrophages associated with augmented numbers of lymphocyteswere recovered from TLR2 - / - mice relative to WT mice.Lungs of TLR2 - / - mice presented decreased number of activated CD4 + and CD8 + T cells at weeks 2 and11 although lower numbers of regulatory CD4 + CD25 + FoxP3 + T cells were detected in this strain by week 11.Equivalent patterns of mortality and lung lesions were observed in both mouse strains. Conclusion: absence ofTLR2 results in less severe infection but increased inflammatory response and lung pathology caused by PMNand TH17 cells.5-12MYD88 is a dispensable factor in resistance to Paracoccidioides brasiliensis in a murinemodel of blood-borne disseminated infectionAngel González 1, 2 , Alberto Yañez 3 , Daniel Gozalbo 3 and Maria Luisa Gil 3 .1Microbiology School, Universidad de Antioquia, Medellín-Colombia. 2 Medical and Experimental Mycology Group, Corporación paraInvestigaciones Biológicas (CIB), Medellín-Colombia. 3 Departamento de Microbiología y Ecología, Universitat de Valencia, València,Spain. e-mail: agonzalezm@cib.org.coWe aimed at determining the role of MyD88, the universal toll-like receptor (TLR) adaptor protein, in murinedefenses against Paracoccidioides brasiliensis yeast infection.Wild-type (WT) and MyD88-deficient mice infected i.v. with P. brasiliensis yeast cells had an equivalent fungalburden as measured by colony forming units, as well as similar levels of pro-inflammatory cytokines (IL-1β, IL-6,IL-12p70, TNF-α and MIP-2), Th1 (IFN-γ) and of Th2 (IL-4) in tissue homogenized supernatants. In both types ofmice the in vitro production of TNF-α, IFN-γ and IL-12p70, by antigen-stimulated splenocytes from mice previouslyinfected with the fungus were also similar.Production of Th1 cytokines correlated with a similar frequency of IFN-γ producing CD4+ T cells. Recruitmentof neutrophils to the peritoneal cavity of i.p. infected mice was not affected in TLR2, TLR4 and WT mice, butsignificantly decreased in MyD88-deficient mice. Additionally, in response to P. brasiliensis yeasts the in vitroproduction of TNF-α by peritoneal macrophages from MyD88-, TLR2- and TLR4-deficient mice, was undiminished,as compared with TNF-α production by macrophages from WT mice, even in the presence of laminarin.Taken together, these data suggest that recognition of P. brasiliensis yeast occurs independently of the adaptormolecule MyD88, as well as of other molecules such as TLR2, TLR4 and dectin-1.192

5-13The influence of ß2 integrin in the fungal burden in Paracoccidioides brasiliensis infected miceJ. Nunes 2 , Catão. E. 1 , Fraga C. L. F. 1 Amaral.A 1 , Figueiredo. F. 2 , Felipe M. S.S. 1 Faccioli. L. H. 3 , Bocca A. L. 1,21Instituto de Ciências Biológicas - Dept. de Biologia Celular, Brasília, Brazil, 2 Área de Patologia - Faculdade de Medicina, Brasília, Brazil,3Dept. Análises Clínicas, Toxicológicas e Bromatológicas - Faculdade de Ciências Farmacêuticas de Ribeirão Preto, São Paulo, Brazil.e-mail: albocca@unb.brParacoccidioides brasiliensis is a facultative intracellular fungus that causes aracoccidioidomycosis (PCM),a chronic and granulomatous disease. As in other systemic mycoses, the host’s principal defense mechanismagainst PCM is the cellular immunity mediated mainly by IFN-gamma activation of macrophages. Macrophagesappear to play a fundamental role in this infection acting as the first line of defense for organism. The first interactionbetween macrophages-fungi is very important in this mycosis progression and some molecules are involved inthis interaction, like ß2 integrin. To investigate the importance of ß2 integrin to progression of the disease weused gene knockout mice (KO) of ß2 integrins and C57Bl/6 (WT) both intravenous infected with the virulent P.brasiliensis strain (Pb18). We analyzed the points 15, 30, 45, 60 days after infection. After 15 days of infection, inß2KO mice, no fungal cells were detected in the liver of mice, althougth we observed more fungus in the lung of KOmice than in the control mice. After 30 and 60 days of infection, The KO mice there were less fungal burden whencompared with the control (WT mice). The spleen cell proliferation stimulated by concanavalin-A is increased in allpoints of analyzes when compared with the WT. We observed also that macrophages infected with P. brasiliensisin vitro had decreased in internalized yeast after 24h of co-culture. Recently was demonstrated that P. brasiliensisregulates up-genes related to inflammation and phagocytosis, and this genes can be involved in fungi survive inmacrophages. Our data suggest that ß2 integrins that are probably involved with the fungi internalization contributewith the survival of the fungi inside macrophages and this phagocytosis serve as a protected environment for theP. brasiliensis. Financial Suport: CNPq.5-14Surface antigens increase susceptibility to Paracoccidioides brasiliensis infection viainterleukin-4 productionOliveira L.L. 1 , Cavassani K.A. 1 , Rocha F.A. 1 ; Vancim J.O. 1 ; Moreira A.P. 1 ; Campanelli A.P. 1,4 ; Milanezi C.M. 1 , Martinez R. 2 ;Rossi M 3 , Oliveira E.B. 1 ; and Silva J.S. 11 Department of Biochemistry and Immunology, 2 Internal Medicine and 3 Department of Pathology, School of Medicine of Ribeirão Preto-USP, Ribeirão Preto, SP; 4 Department of Biological Sciences, Bauru Dental School, University of São Paulo, Bauru, SP; Brazil.e-mail: licursi@usp.brIntroduction and Objectives: The paracoccidioidomycosis (PCM) is characterized by a chronic inflammatorygranulomatous reaction. The Th1/Th2 paradigm of acquired immunity to fungi is essential for a better understandingof the immunoregulation which occurs during fungal infections. Classical studies on the immune responsesdeveloped by patients with polar forms of paracoccidioidomycosis (PCM) have demonstrated a Th1-biasedimmune response in the asymptomatic and mild forms of PCM, whereas a Th2 pattern has been associated withthe severe disease. In this work, we evaluated the ability to induce specific suppression in a murine model by theinoculation of surface/secreted P. brasiliensis antigens (sPbAg). These results can provide new information thatmay be important in the understanding of immunoregulatory perturbations observed in paracoccidioidomycosis.Methods and Results: C57BL/6 and IL-4KO mice were immunized three times by subcutaneous route (threeweeks apart) with sPbAg. Yeast cells (Pb18) were shook vigorously (in PBS) for 30s in vortex and the supernatant(sPbAg) was used in the assays. Control mice were injected with PBS. Two weeks after the last boost, the animalswere challenged (iv) with viable yeast forms of P. brasiliensis. The results showed the inoculation of sPbAg priorto infection resulted in a severe granulomatous lesions and fungal dissemination to the all organs analyzed.Clearly, increased fungal growth after sPbAg inoculation was dependent of IL-4, since high level of this cytokinewas found in the homogenates of lungs, spleen and liver of mice injected with sPbAg, but not in the control group.Moreover, the injection of sPbAg did not affect the course of infection in mice deficient of IL-4, suggesting that theexacerbation of PCM after inoculation of sPbAg is mediated by IL-4. Conclusion: Based on these data, our resultsdemonstrated that sPbAg is able to increased secretion of IL-4 and this fact impaired Th1 responses associatedwith the cure of PCM. So is suggested that sPbAg play important role in the modulation mechanisms for theinstallation of primary infection in hosts. Financial support: CNPq, FAPESP.193

5-15FBP plays a role in the imunosuppression on the course of Paracoccidioides brasiliensisinfectionMariano, V.S. 1 , Oliveira, L.L. 1 ; Coltri, K.C. 1 ; Vendruscolo, P.E. 1 , Ferraz, D.B. 1 , Roque-Barreira, M. C. 1 and Panunto-Castelo, A. 21 Faculty of Medicine of Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil. 2 School of Nursing of Ribeirão Preto, USP, Ribeirão Preto, SP,Brazil.e-mail: vaniasmariano@usp.brParacoccidioidomycosis (PCM) is a chronic, granulomatous and progressive disease, which is associatedwith various degrees of suppressed cell-mediated immunity. In the present study we described the isolation inthe NaCl 1M-eluted fraction (FBP) fraction upon affinity chromatography on immobilized fetuin of the solubleantigen fraction from P. brasiliensis. Although these fetuin-binding proteins were divalent cation-dependent,they did not present either enzymatic or lectin activity.Electrophoresis analysis of FBP revealed a major protein of 60kDa and four minor proteins of 50, 35, 24 e14kDa. In a blotting assay, we showed that 60kDa protein was bound by pulmonary extracellular matrix frommouse, suggesting that FBP can play important role in the paracoccidioidomycosis (PCM) and, consequently,be a therapy target. When mice were treated 21 days postinfection with FBP or saline emulsified in completeFreund adjuvant (CFA), a strong adjuvant to protective Th1 immune response against PCM, we observed thatCFA-treated mice were protected, whereas FBP+CFA-treated mice reversed the protection.Although lungs from FBP+CFA-treated mice presented organized granulomas with few compromisedtissue, there was a large number of viable fungi inside the granuloma. Consequently, the CFU number in theorgans of these mice was higher than that from CFA-treated mice. In addition, lung homogenates from FBPmice had high levels of TGF-β, a known immunosuppressor cytokine. In vitro analysis, FBP had a proliferativeeffect under B cells and induced IL-10 production.On the basis of these results, we hypothesized that FBP suppressed the protector immune responsetriggered by CFA, leading to persistent viable fungi infection inside the granulomas. Moreover, high levelsof TGF-β were observed in the pulmonary epithelium from FBP+CFA-treated mice. Altogether, these resultsindicate that FBP plays an important role in the imunosuppression produced on course of P. brasiliensisinfection, and open perspectives of intervention in this suppressive process, leading to a beneficial effect onthe infection severity. Support: CAPES, CNPq, FAPESP.5-16Paracoccidioides brasiliensis inhibits human neutrophils apoptosisM.J. Acorci 1 , Dias-Melicio L.A. 1 , Golim M.A 2 , Bordon-Graciani A.P. 1 , Peraçoli M.T.S. 1 , and Soares A.M.V.C. 11Department of Microbiology and Immunology, Biosciences Institute, São Paulo State University, Botucatu, São Paulo, Brazil. 2 BotucatuBlood Center, School of Medicine, São Paulo State University, Botucatu, São Paulo, Brazil.e-mail: ladiasmelicio@ibb.unesp.brParacoccidiodomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presentsa wide spectrum of clinical manifestations. Because of the great number of neutrophils (PMNs) found in theP. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the developmentof the infection. This fungus is found intracellularly in PMNs and monocytes/macrophages, suggesting that itis capable of evading damage and surviving inside these cells.Thus, the goal of this study was evaluate if P. brasiliensis induce PMNs apoptosis suppression, andif this process was related to IL-8 production, a chemokine involved in apoptosis delay. PMNs apoptosisand intracellular levels of IL-8 were analyzed by flow cytometry, and culture supernatants IL-8 levels wereevaluated by ELISA. We showed that Paracoccidioides brasiliensis inhibited human neutrophils apoptosis.We also demonstrated that PMNs challenged with the fungus produced high levels of IL-8.These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis toescape killing, P. brasiliensis can extend the life span of PMNs by inducing autocrine IL-8 production, probablymaking these cells suitable for survival and multiplication194

5-17Prostaglandin inhibits Paracoccidioides brasiliensis killing by human monocytes: Reversalby activation with IFN-γ, TNF-α & GM-CSF due to increased H 2O 2productionA.P. Bordon-Graciani, Dias-Melicio L.A., Acorci M.J., Peraçoli M.T.S., and Soares A.M.V.C.Department of Microbiology and Immunology, Biosciences Institute, São Paulo State University, Botucatu, São Paulo, Brazil.e-mail: ladiasmelicio@ibb.unesp.brParacoccidioides brasiliensis (Pb), the etiological agent of paracoccidioidomycosis, is a dimorphic fungusthat survives within nonactivated human monocytes/macrophages. Previous studies have demonstrated thatthe lack of fungicidal activity by nonactivated human monocytes is associated to fungus capacity of inducingprostaglandins release, since a significative fungicidal activity was detected after monocytes treatment withindomethacin (INDO), a cyclooxigenase inhibitor. Thus the purpose of this work was to assess if the inhibitoryeffect of prostaglandins could be reversed by cells activation with IFN-γ, TNF-α and GM-CSF.Moreover, we asked if this process could be associated with alterations on the levels of killing effectormolecules such as NO and H 2O 2and/or cytokines such as TNF-α, IL-6 and IL-10. Peripheral blood monocytesobtained from 18 healthy donors were treated only with INDO or activated with IFN-γ, TNF-α or GM-CSF inpresence or absence of INDO for 18h, and further challenged with high (Pb18) or low (Pb265) virulent strain ofPb for 4h. Then, cultures were evaluated for fungicidal activity and for H 2O 2and NO release, and expression ofinducible nitric oxide synthase (iNOS) mRNA by real-time RT-PCR. The concentrations of TNF-α, IL-6 and IL-10on supernatants of cocultures were evaluated by ELISA.As expected, nonactivated cells lacked fungicidal activity against both strains. Killing activity against twostrains was significantly increased after cells incubation with INDO and/or cytokines. However, Pb265 killingwas always higher than that detected for Pb18. A clear association between killing and increased H 2O 2and TNFαlevels was observed. The iNOS mRNA expression showed interesting results, indicating a modulatory effectof prostaglandin upon this expression via TNF-α production, what could indicate the role of NO in this process.Nevertheless, both strains were capable to inhibit the NO production.Taken together, these results strongly suggest that human monocytes challenged with Pb releaseprostaglandins that inhibit the capacity of these cells to produce adequate levels of H 2O 2and TNF-α. This effectmay be compensated by cells incubation with cytokines that activates them to release higher levels of thesemediator with consequent Pb killing.5-18Production of leukotriene B 4By different strains of Paracoccidioides brasiliensisL.A. Dias-Melicio, Biondo G.A., Bordon-Graciani A.P., Acorci M.J., Peraçoli M.T.S., and Soares, A.M.V.C.Department of Microbiology and Immunology, Biosciences Institute, São Paulo State University, Botucatu, São Paulo, Brazil.e-mail: ladiasmelicio@ibb.unesp.brLeukotrienes (LT) are eicosanoids derived from arachidonic acid (AA) metabolism and produced vialipoxygenase (LOX) followed by conversion into leukotriene B 4(LTB 4). Host cells are one source of eicosanoidsduring fungal infection; however, another potential source of eicosanoids is the fungal pathogen itself.Independently of the source, the leukotrienes production is critical for the fungal survival and/or growth and forthe modulation of host immune response.Thus, the purpose of this study was to investigate if different strains of P. brasiliensis (Pb18, Pb265, Bt79,Bt192) have the capacity to produce LTB 4in vitro, and if this production is related to the fungal survival and/orgrowth. Additionally, we evaluated if these strains could utilize exogenous source of AA to this production. Thefour strains were cultured during 4 and 8 hours, and after the LTB 4levels were measured in supernatants cultureby ELISA.The results demonstrated that all strains produced high levels of LTB 4at 4 hours of culture, however, after8 hours of culture, almost all strains showed significant decrease in LTB 4levels. The addition of AA in thefungal cultures significantly increased the LTB 4production in all cultures. The treatment of fungal cultures withMK 886, an inhibitor of leukotrienes synthesis, significantly inhibited the synthesis of this mediator, as well asdecreased the fungal viability. Then, our results show that P. brasiliensis produce LTB 4utilizing endogenous andexogenous sources of AA and this production is involved in the fungus survival.195

5-19Comparative analysis of the infection evolution between isolates Pb 18 and Pb 01 of theParacoccidioides brasiliensisC. L. F. Fraga 1 , Siqueira I. M. 2 , Ribeiro, A.M. 2 , Nunes J. 1 , Figueiredo F. 1,3 , Felipe M. S.S. 1,2 , Bocca A.L. 1,21Pós-graduação em Patologia Molecular, Faculdade de Medicina, UNB, Brasilia – Brasil. 2 Departamento de Biologia Celular, Instituto deBiologia, UNB, Brasília – Brasil. 3 Área de Medicina, Universidade Católica de Brasilia, Brasilia – Brasil. e-mail: albocca@unb.brThere is a remarkable genetic diversity between different isolates of Paracoccidioides brasiliensis. After thefinding of genetic variability within the species, it was concluded that the Pb 01 isolate occupied a special positionin the group, differing from the Pb 18 isolate. However, all the knowledge about the immune response during thedevelopment of the Paracoccidioidomycoses in experimental models is based on protocols that use either thevirulent strain Pb 18 or the non-virulent strain Pb 265, obtained from the state of São Paulo, Brazil. Little is knownabout the Pb01 strain, specially regarding its virulence and the induced immune response in experimental animalmodels. The aim of this study was to verify the immune response in mice infected with Pb 01 compered to the Pb18strain which is known the pathogenicity in vivo. For that purpose, mice C57Bl/6 was infected with 1x10 7 cells/ml e.v.and the evolution of the disease was analyzed, by histopathological studies, counting colony-forming units (CFUs)as well as by spleen cells proliferation on days 15 th , 30 th , 45 th , 60 th , 75 th and 100 th post-infection. It was observedthat Pb18 and Pb01 strain have different pathogenicity in the Paracoccidioidomycosis infection. Compared withPb01 strain, Pb18 caused more inflammatory tissue reaction such as a decrease lymphoproliferative activity. Thelung from mice infected with Pb01 revealed a lower progressive inflammatory response, leading to few granulomaformations after 45 days of infection and a significant reduction in pulmonary fungal burden when compared withmice infected with Pb18. In additional, the analysis of the immune response is in progress, measuring the levels ofIL2, IL4, IL10, IL12, IFNγ, TNFα, IgGs and NO, in the supernatants of cell cultures.Financial Support: CNPq.5-20Paracoccidioides brasiliensis infection determines dendritic cells to differentiate to theplasmocytoid subpopulation which induces a more severe pulmonary infection whentransferred to resistant miceA. Pina, and Calich, V.L.G.Instituto de Ciências Biomédicas, Universidade de São Paulo, SP, Brazile-mail: adripina@usp.brB10.A and A/J mice were previously characterizes as susceptible and resistant strains to pulmonaryParacoccidioides brasiliensis (Pb) infection. Resident alveolar macrophages (AM) and dendritic cells (DC), thefirst cells that interact with Pb in lungs play a fundamental role in the immunity that further develops. Differentlyfrom expected, resident AM from susceptible mice present an increased ability to kill fungal cells and to secretepro-inflammatory cytokines. Thus, the aim of this work was to analyze the role of another antigen presenting cell,the dendritic cell, in the genetic resistance to Pb.Bone-marrow derived DC from B10.A and A/J mice were in vitro challenged with Pb yeasts (1:25 fungus:DCratio) or soluble Pb antigen (100 µg/ml) and cytokines and oxide nitric (NO) were determined 48h later in culturesupernatants. Costimulatory molecules were analyzed by flow cytometry and some DC preparations were usedto study their stimulatory activity using homologous and heterologous naïve lymphocytes previously stained withCSFE. Pb-activated DC of B10.A mice produced higher levels of IL-12, TNF-α and NO when compared with DC ofA/J mice. Viable yeast cells were more efficient activators than the soluble Pb antigen. DC from A/J mice presentedaugmented co-expression of CD11c and B220 whereas Pb yeasts were able to induce high expression of CD11c +CD11b + in B10.A DCs. This indicates that A/J cells preferentially maturate to a plasmocytoid subpopulation butthe myeloid subset was observed with B10.A cells. Differently from B10.A cells, A/J DCs induced IL-2 secretionand a high lymphoproliferation of homologous lymphocytes. Previous treatment of resistant but not susceptiblemice with A/J DCs led to a more severe infection as determined by organ fungal loads, secretion of cytokinesand activation of regulatory T cells. B10.A DCs, however, did not substantially alter the pattern of infection andadaptative immunity of both mouse strains. Thus, differently from expected, DCs from resistant mice are able totransfer a pattern of immunity which results in more severe infection.FAPESP196

5-21IL-10 deficiency determines a better fungicidal ability associated with overproduction ofIFN-γ and nitric oxide by Paracoccidioides brasiliensis infected macrophagesCosta, T.A. 1 and Calich, V.L.G. 11 Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil. e-mail: tacosta@usp.brIn systemic mycosis, the interaction between tissue macrophages and fungal cells play a fundamental rolein the protective mechanisms of innate immunity and influences adaptative immunity that further develops.These phagocytes exert their function by ingesting and inactivating invading organisms, producing cytokines andinteracting with other cells of the adaptive immune system. Several clinical observations suggest that there is aninverse relationship between interferon (IFN)-γ and IL-10 production in patients with fungal infections. Studies ina pulmonary model of paracoccidioidomycosis demonstrated that the resistance in mice is linked to a preferentialsecretion of IFN-γ whereas susceptibility is associated with very low levels of this cytokine. Both susceptible andresistant mice produced IL-10, a macrophage deactivating cytokine, throughout the course of the disease. Tobetter understand the role of IL-10 in murine PCM, macrophages from IL-10-deficient mice (IL-10 KO) and theirnormal (WT) C57BL/6 counterparts were comparatively studied after P.brasiliensis infection. IFN-γ (10,000 pg/mL) primed and unprimed peritoneal macrophages (1x10 6 cells/well) of both mouse strains were co-cultivated invitro with 4x10 3 /mL P.brasiliensis yeasts (1:50 fungus : macrophage ratio). 48h after infection fungicidal activitywas assessed by CFU counts, and supernatants used to measure the levels of nitric oxide (Griess reagent) andcytokines (ELISA). It was verified that macrophages from IL-10 KO mice produced higher levels of NO than thosefrom WT (WT, 1.35±9.35 µM; IL-10 KO 60.31±1.47 µM, p

5-23Nitric oxide but not treg cells plays a major immunoregulatory role in a pulmonary modelof fungal infectionS. Bernardino and Calich V.L.G.Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazile-mail: sibern@icb.usp.brParacoccidioidomycosis (PCM), caused by the inhalation of Paracoccidioides brasiliensis spores, is a majorpulmonary fungal disease in South America. The protection in PCM is mainly mediated by cellular immunity.Inducible nitric oxide synthase (iNOS) is a nitric oxide (NO) generating enzyme which plays an important role inclearance of microbial infections, but its overproduction can lead to immunessupression of cellular immunity. Weinvestigated the role of NO in the acute and chronic phases of the disease using C57BL/6 iNOS KO and their WTcounterparts infected by the intratracheal route with 1 million yeasts. Compared with WT, at week 2 post infectioniNOS KO mice presented decreased fungal loads and high levels of TNF-alpha in the lungs. These findingswere associated with increased number of infiltrating pulmonary CD4 + CD69 + , CD4 + CD25 + , CD8 + CD69 + T cells andactivated alveolar macrophages expressing CD40 + , CD80 + , CD86 + , Ia k and CD11 molecules. In contrast, at week10 post-infection KO mice presented higher fungal burdens in the lungs which, however, were still associated withelevated number of CD4 + CD69 + and CD4 + CD25 + T cells, macrophages expressing CD80 + and CD40 + moleculesbut an equivalent number of regulatory T cells TCD4 + CD25 + FoxP3 + . In agreement, iNOS KO mice developed wellorganized pulmonary granulomas which control the fungal dissemination and resulted in similar mortality ratesfor both mouse strains. Early in vivo TNF-alpha depletion was able to abrogate the CFU differences between KOand WT mice observed at week 2 of infection. Furthermore, this treatment resulted in increased and precociousmortality of KO, but not WT mice. In conclusion, despite its importance in fungal clearance, NO exerts an importantsuppressive effect in cellular immunity and TNF-alpha production which plays a fundamental role in the organizationof lesions, control of yeasts dissemination and host survival.Supported by FAPESPKey words: Paracoccidioidomycosis, nitric oxide, TNF-alpha, T cells, granuloma5-24Role of CCR4 in the experimental infection by Paracoccidioides brasiliensis: Migrationcontrol of CD4 + CD25 + T cells to lesion siteRocha F.A. 1 , Oliveira L.L. 1 , Massafera Tristão FS 1 ; Milanezi C.M. 1 and Silva J.S. 11 Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto-USP, Ribeirão Preto, SP; Brazile-mail: nandaffer@usp.brIntroduction and Objectives: Paracoccidioidomycosis (PCM) is a chronic and granulomatous diseasecaused by the inhalation of conidia forms of Paracoccidioides brasiliensis. The infection results in the formation ofgranulomas containing viable yeast cells that are the fungal sources for disease reactivation. Because CD4 + CD25 +regulatory T cells (Tregs) are in the lesions of patients with paracoccidioidomycosis, the migration of Treg cellsis dependent on the axis chemokine-chemokine receptors, and CCR4 ligands are produced in P. brasiliensisinducedlesions, we aimed to evaluate the role of this receptor during the P. brasiliensis infection in CCR4 -/- andC57BL/6 wild-type mice. Methods: Mice were infected with 1x10 6 yeast cells of a P. brasiliensis strain (Pb18)and analyzed weekly until 8 weeks. The lung, liver and spleen from mice were used for histopathology and flowcytometry analyses, and for quantification of viable fungal. We observed an increase in fungal burdens in the lungsof CCR4 -/- mice in the early infection. The cell surface expression of CD3, CD4, CD8, CD19, NK, Gr1, CD103,CTLA-4 (CD152), CD11c, CD11b and GITR, and intracellular Foxp3 were assessed by flow cytometry, for bestchraracterization of leukocytes isolated from the lungs. The histopathological section of organs were stained withH&E and analysed for Morphology and morphometry of granulomas. Conclusion: Probably, the aspects of themaintenance of chronicity during the infection with P. brasiliensis can be influenced by CD4 + CD25 + T cells expressingCCR4. The potent suppressor activity of Tregs that migrated to the granulomas, in response to chemokine ligandsproduced in the lesions, became this chemokine receptor an important target in immunotherapeutic strategies tocontrol PCM.Financial support: FAPESP, CAPES, CNPQ, FAEPA198

5-25Granulomatous imflammation and fibrosis in mice with chronic pulmonary Paracoccidioidomycosis:Immune-related associationsNaranjo TW 1 , Lopera DE 1 , Duque JJ 2 , Diaz-Granados LR 3 , Restrepo A 1 and Cano LE 1,41 Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas, CIB, Medellín – Colombia. 2 Departamento de Patología, Facultadde Medicina, Universidad de Antioquia. 3 Facultad de Medicina, Universidad Pontificia Bolivariana, Medellín-Colombia. 4 Molecular Microbiology Group, Escuelade Microbiología, Universidad de Antioquia, Medellín – Colombia. e-mail: tnaranjo@cib.org.coBackground: In our experimental model of pulmonary paracoccidioidomycosis (PCM), infection is induced bythe intranasal (i.n.) inoculation of Paracoccidioides brasiliensis conidia. This model has the advantage of reproducingseveral aspects of the human disease allowing to study pulmonary tissue response at different post-infection periodstaking into consideration aspects as diverse as cytokine production, inflammatory response, fibrosis development,treatments evaluation and others. Aim: To determine if during the chronic stages of experimental pulmonary PCM animmune-related association could be established between the granulomatous inflammation and fibrosis Materialsand methods: BALB/c male mice, 7-8 weeks old, were inoculated i.n. with 4x10 6 P.brasiliensis viable conidia, controlanimals received PBS. Ten mice were sacrificed at different times, 2h, 4, 8, 12 and 16 weeks post-inoculation. Thelungs of 5 mice/period were embedded in paraffin, sectioned and stained with H&E, Masson’s trichromic and reticulinfor histologic analysis; two independent pathologists evaluated granulomatous inflammation and fibrosis. Fibrosiswas defined as the observation of thick collagen fibers around inflammatory lesions in the presence of reticulin fibers.The lungs of the remaining 5 mice/period were homogenized in 2 ml of proteases’ inhibitors cocktail and resultantsupernatants were frozen at -70 o C for cytokine (TNF-α, IFN-γ, IL-13, IL-1β, TGF-β determination and PGE2 assaysby commercial ELISAs. Results: Sequential histopathologic analyses showed that P.brasiliensis infection induceda significant (p≤0.001) increase of the peribronchial granulomatous inflammation with involvement of 10 to 35%of the lung’s area, with values of 10±7, 22±15, 20±11 and 15±10% at 4, 8, 12, and 16 weeks, respectively. Proinflammatorycytokine levels, (TNF-α, IL-1β and PGE2 were increased in same periods where high inflammatoryresponses were observed. In addition, pro-fibrotic cytokines (IL-13, TGF-β) showed a significant increase during theperiods immediately before establishment of fibrosis (8 weeks post-infection); this fibrous process progressed until the16 weeks post-infection. IFN-γ levels did not show significant increase during the studied periods. Conclusion: Thisstudy reveals association, albeit not direct, causality between pulmonary histopathologic evidence (granulomatousinflammatory response, fibrosis) and cytokine behavior. Financial support: CIB, UdeA, Colciencias (ProjectNo.2213-04-16439).5-26Alterations of granuloma architecture, cellularity and severity of the early infection in murineparacoccidioidomycosis by IFN-γ, celecoxib or lumiracoxib treatmentR.F.S. Molina, Nishikaku. A.S, Albe. B.P, Burger. E.Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo – Brasil. e-mail: raphaelmolina@usp.brIn the present study we sought to interfere in the fibrotic process, which worsens the quality of life in patients withparacoccidioidomycosis (PCM). We infected intraperitoneally B10.A female mice, susceptible to Paracoccidioidesbrasiliensis (Pb), with the highly virulent Pb18 isolate. We treated these animals with IFN-γ (with antifibrotic andfungicidal activity) or the anti-inflammatories drug Lumiracoxib or Celecoxib (which affect neutrophil migrationand possibly inhibit components of the extracellular matrix - ECM). Control groups received no drug treatment.After 15 days of inoculation, we collected the spleen, liver, lungs and omentum. We observed that the omentumof mice treated with either anti-inflammatory drug had more than twice the size and number of lesions comparedwith the same organ of only infected animals or those treated with IFN-γ. Through analysis of histological slidesstained with HE, Giemsa, Grocott and Picrosirius we observed, in omentum of the non-treated infected mice, few,loose granulomas with thin collagen fibers. These lesions had Pb with typical morphology confined in granulomas,extensive neutrophil infiltration, numerous giant cells and few eosinophils. Mice treated with IFN-γ showed few,compact granulomas, with large quantity of thin and coarse collagen fibers circumscribing few Pbs with morphologyranging from typical to atypical, and major presence of neutrophils, giant cells and eosinophils. Mice treated witheither anti-inflammatory drug showed a large number of loose granulomas, with reduced numbers of thin collagenfibers. In this group there was a high number of fungi with typical morphology disseminated throughout the tissue,reduction of neutrophils and eosinophils, but presence of many giant cells. We recovered more viable Pb from theomentum of animals treated with either anti-inflammatory drug than from the other groups, and the other hand,mice treated with IFN-γ yielded the fewest number of Pb. Our results suggest that in susceptible animals to PCM,at this period of infection, the treatment with IFN-γ restrained the infection through segregation within compactgranulomas by ECM fibers, causing a decrease of fungal load, and the treatment with anti-inflammatory drugs didnot contain the infection but, instead, facilitated fungal dissemination in the entire tissue examined.Financial Support: FAPESP – 06/60091-6 and 07/56745-3; CNPq – 307492/2006-0.199

5-27Therapeutic activity of a DNA vaccine using the gene hsp65 from Mycobacterium lepraefor experimental paracoccidioidomycosisA. M. Ribeiro 1 , Souza A. C. O. 2 , Amaral A.C. 5 , Fraga C. L. F. 1 , Nunes J. 1 , Salles B. C. 2 , Coelho-Castelo A. A. M. 3 ,Figueiredo F. 4 , Silva C. L. 3 , Felipe M. S. S. 2 , Bocca, A. L. 11Área de Patología, Faculdade de Medicina, UNB, Brasilia – Brasil. 2 Departamento de Biologia Celular, Instituto de Biologia, UNB, Brasília– Brasil. 3 Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, USP, São Paulo – Brasil. 4 Área deMedicina, Universidade Católica de Brasilia, Brasilia – Brasil. 5 Ciências Genômicas e Biotecnología, Universidade Católica de Brasilia,Brasilia – Brasil.e-mail: ribeiroalice@yahoo.com.brHeat shock proteins are recognized as important molecules in the modulation of the immune system andare highly conserved among different organisms. The DNA-hsp65 vaccine from Mycobacterium leprae has beenshown to have prophylactic and immunotherapeutic effects against various diseases, for instance, tuberculosis,arthritis and leishmaniasis. In this work, we evaluated the effectiveness and immunomodulatory potential of theDNA-hsp65 treatment in model BALB/c mice infected with Paracoccidioides brasiliensis, the etiological agent ofParacoccidioidomycosis, the most important endemic mycosis in Latin America.The DNA-hsp65 vaccine conferred protection against this pathogen for therapeutic assays, as indicated by:1) a significant reduction in pulmonary fungal burden; 2) a decrease in pulmonary damage and in the presenceof collagen in granulomas (revealed by histological analyses of the pulmonary tissue); 3) the reestablishment ofspleen cells proliferation; increase levels of IL-12, IFN-γ, TNF-α and IgG2a and unaltered levels of IL-4, IL-10and IgG1 compared with the control mice. Together, these findings indicate that, in mice, the treatment with theDNA-hsp65 vaccine protect mice against paracoccidioidomycosis. Previously we demonstrated that DNA-hsp65immunization protects mice against paracoccidioidomycosis. Our results open new perspectives on preventionand treatment of other systemic mycoses.Financial Suport: CNPq.5-28Evaluation of the protection induced by the immunization with radioattenuated yeast cellsof Paracoccidioides brasiliensis in animal modelEstefânia M.N. Martins 1,2 , Bernardo S. Reis 2 , Alfredo M. Goes 2 and Antero S. R. Andrade 11Laboratório de Radiobiologia, CDTN / CNEN, Belo Horizonte – Brasil. 2 Departamento de Bioquímica e Imunologia, UFMG, Belo Horizonte– Brasil.e-mail: estefaniabio@yahoo.com.br (presenting Author)Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in LatinAmerica. Currently there is no effective vaccine. In our laboratory yeast cells of P. brasiliensis were attenuated bygamma irradiation, which lose the reproductive ability, while retaining the morphology, the synthesis and secretionof proteins, the oxidative metabolism and the expression of the antigens present in the native yeast. The aim ofpresent work was evaluate the protection elicited by the immunization of BALB/c mice with radioattenuated yeastcells of P. brasiliensis.BALB/c mice were divided in two groups that were immunized one or two times, respectively. For each groupthe mice were divided in three sub-groups that were challenged 30, 45 and 60 day after immunization. Recovery ofCFUs, histopathological analysis and cytokines determination (IFN - γ, TNF - α, IL - 10 and IL – 5) were performedone and three months post challenge. The sera were collected weekly to evaluate the IgG antibody titers and theIgG1 and IgG2a pattern in the course of infection. To evaluate the type of elicited immune response the cytokineswere determined by real time PCR. The mice immunized once presented a significant reduction in the CFUsrecovery when examined 30 days after the challenge.However, 90 days post challenge the number of CFUs recovered from all organs increased. The use of twoimmunizations increased the protector effect and a remarkable protection was verified 90 days post challenge,showing that a long lasting protection able to eliminate the fungi cells of the tissues was elicited. At the same timethe levels of IgG2a and IFN - γ, were high while a very low production of IL-10 and IL-5 was verified, suggestingthat a Th1 pattern was dominant. The current study showed the efficacy of radioattenuated yeast cells of P.brasiliensis for induction of protection in experimental PCM.200

5-29Modulation of experimental paracoccidioidomycosis by monoclonal antibodies against themajor diagnostic antigen, GP43F. A. Pinto 1 , R. Puccia, 2 C. J. da Silva 1 , J. E. Muñoz 1 , L. R. Travassos 2 , C. P. Taborda 11Instituto de Ciências Biomédicas, Departamento de Microbiologia, Universidade de São Paulo, São Paulo, Brasil. 2 Departamento deMicrobiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo, Brasil. e-mail: fel-ipe@hotmail.comBackground: Antifungal chemotherapy is required for paracoccidioidomycosis (PCM) treatment, thougheven after prolonged administration there is no assurance of complete destruction of the fungus. The period oftreatment depends on the drug used and the severity of disease. The role of antibody-mediated immunity inhost resistance to P. brasiliensis is less recognized. In several other systems, however, there is considerableevidence that administration of monoclonal antibodies (mAbs) can modify the course of disease in mice infectedwith fungi such as Cryptococcus neoformans, Candida albicans, Histoplasma capsulatum, Pneumocystis spp,Fonsecaea pedrosoi and Aspergillus spp. In the present study a panel of monoclonal antibodies against gp43 wasutilized for evaluating the effect of passive immunization in mice intravenously infected with a virulent strain of P.brasiliensis.Methods: BALB/c mice were immunized with 1mg of the monoclonal antibody (mAb) 3E, 32H or an irrelevantone by intraperitoneal route, 24 h before the intravenous infection with 3x10 5 yeast cells of P. brasiliensis (Pb18).A maintenance dose was given every week for a month. A week after the last immunization, mice were sacrificedand lung, spleen and liver were removed for measuring the fungal burden, cytokine levels (IL12, IL4, IL10 andIFN-γ) and histology.Results: Animals immunized with mAb 3E showed significant reduction of colony forming units (CFU) in lungand spleen compared to the groups that received mAb 32H or irrelevant mAb. Histological analysis confirmed theresults obtained in the CFU assay although a significant difference in the cytokine levels between the groups wasnot observed.Conclusion: The anti-gp43 mAb 3E but not mAb32H or the irrelevant mAb significantly reduced the fungalburden in BALB/c mice intravenuouly infected with P. brasiliensis, The present work also raises the possibility ofa potential therapeutic use of mAb3E.Supported by Fapesp and CNPq.5-30The influence of Fonsecaea pedrosoi cell wall in peritoneal macrophage activationY.K.M. Nóbrega 1 , Lozano V.F. 1 , Figueiredo. F. 3 , Bocca A. L. 1,21Área de Patologia - Faculdade de Medicina, Brasília, Brazil, 2 Instituto de Ciências Biológicas - Dept. de Biologia Celular, Brasília, Brazil,3University Católica of Brasília – UCB - Brazil.e-mail: albocca@unb.brChromabastomycosis is a chronic supporative granulomatous mycosis that exists all around the world. TheFonsecaea pedrosoi is the most frequent etiologic agent in the worls. The infection begins with the trauma and theimplantation of conidia or hyphae fragments in the subcutaneous tissue producing the initial lesion. Inside the host,a fungus structure differentiates into sclerotic forms allowing the establishment of the disease.Considering the importance of an efficient immune cellular response, through the interaction between thecells of immunologic system with the components of the fungus wall, the objective of the work was to analyzethe influence of the fractions from the F. pedrosoi cellular wall in the activation of murine peritoneal phagocytes.Our results demonstrated that after 4 hours after the inoculation of the solutions the migration was constitutedpredominantly by neutrophils and after 72 hours predominantly by macrophages.The F2 fraction and the inactivate fungus stimulates predominantly B lymphocytes migration. The F1 and F2fraction induced the high production of IL-12 and IL-10 in macrophages respectively. The macrophages stimulatedwith F2 fraction showed a decreased phagocytosis index and an increased production in IL-10 production. Ourresults suggest that, in chromoblastomycosis, the cell wall components presents in F1 or F2 fraction contribute toinduce a Th1 response or Th2 response.Financial Suport: CNPq.201

Poster Session6Treatment

6-01Timing of itraconazole therapy onset and its impact on the progression of fibrosisinduced by Paracoccidioides brasiliensis conidia: Results in an experimental model ofparacoccidioidomycosisDE. Lopera 1 , TW. Naranjo 1 , JJ. Duque 2 , Diaz-Granados LR 3 , RA. Jiménez 4 , A. Restrepo 1 , AF. Zuluaga 5 and LE. Cano 1,6,1Grupo de Micología Médica y Experimental, Corporación para Investigaciones Biológicas, CIB. 2 Departamento de Patología, Facultad deMedicina, Universidad de Antioquia, UdeA. 3 Facultad de Medicina ,Universidad Pontificia Bolivariana. 4 University of Nebraska, Nebraska– USA. 5 Grupo GRIPE, Facultad de Medicina, UdeA. 6 Grupo de Microbiología Molecular, Escuela de Microbiología, UdeA, Medellín –Colombia. e-mail: dlopera@cib.org.coBackground and aim: The active stages of Paracoccidioidomycosis (PCM) are usually controlled by Itraconazole(ITZ) therapy; however, patients often (~30-50%) develop pulmonary fibrosis and exhibit important functional respiratorylimitations. Our aim was to determine if in an experimental model of PCM, fibrous sequelae could be avoided or modifieddepending on the time of initiation of ITZ therapy. Materials and methods: Male BALB/c mice (n=50) were infectedintranasally with 4x10 6 Paracoccidioides brasiliensis (Pb) conidia and distributed in three experimental groups: (i) nontreatedmice (control), (ii) orally treated with ITZ (1mg/day by 8-weeks) starting at 4-weeks of infection (before fibrosisconsolidation) or (iii) orally treated with ITZ starting at 8-weeks of infection (when fibrosis is well-established). Sacrifice wasevery four weeks from 0 to 16 and lungs were processed histologically using Masson’s trichrome and reticulin stainings.Then, two pathologists registered the appearance (thin or thick) of collagen and reticulin fibers and scored their relativeincrease respect to control as 0=no changes, 1=minor, 2=moderate or 3=major. Fibrosis was defined as the observation ofthick collagen fibers around inflammatory lesions in the presence of reticulin fibers whereas incipient fibrosis was taken aspresence of only thin fibers. Results: At 4-weeks post-infection, 80% of the Pb infected mice showed incipient pulmonaryfibrosis that, later on, (8 and 12-weeks) progressed to well-established fibrous sequelae in 60% of the animals, attaining100% of them by 16-weeks. Group (ii) showed an early and significant reduction (p≤0,05) in the score assigned to thinreticulin and collagen fibers deposition along with a score=0 to thick fibers (p≤ 0,05); consequently, at 16-weeks, 80% hadincipient fibrosis and none (0%) had well-established sequelae. In Group (iii) but only at the end of treatment (16-weeks),a significant reduction in the score of thin fibers was observed, although formation of thick fibers was not completelyprevented. In fact, in sharp contrast with Group (ii), 40% of the mice had incipient fibrosis and 20% showed severe sequela.Conclusions: The progression of pulmonary fibrosis in Pb-infected mice can be controled by an early ITZ treatment.These experimental results open the possibility of avoiding fibrosis in the human patients by early therapy.Financial support: Colciencias (Proyecto N° 2213-04-16439), UdeA, CIB.6-02Posaconazole for the treatment of paracoccidioidomycosis: First clinical resultsTobón AM 1,2 , Agudelo CA 1,3,4 , Restrepo A 11 Corporación para Investigaciones Biológicas, CIB, Medellín – Colombia. 2 Hospital La María, Medellín – Colombia 3 Universidad PontificiaBolivariana, Medellín – Colombia. 4 Hospital Pablo Tobón Uribe, Medellín - Colombia. e-mail: atobon@cib.org.coThe introduction of azolic derivatives in the treatment of paracoccidioidomycosis (PCM) drastically reduced the durationof therapy, as well as the relapse rate. Posaconazole is a new antifungal azolic, liposoluble with a wide antifungal spectrum,making an interesting option for PCM treatment. The results of posaconazol therapy in 4 adult PCM patients are presented.Three of these patients had the chronic multifocal and one the subacute form. All patients were male, smokers, and hada mean age of 52 years (range 24-73), One had sequelae of lung (fibrothorax, cor pulmonale) and kidney tuberculosis,as well as adrenal insufficiency. In these chronic patients duration of their clinical course was 8.3.months; constitutionaland respiratory symptoms predominated accompanied by oral mucosae ulcerations. The X-rays revealed mixed bilateralinfiltrates and in one patient, a cavity in the upper field. As for the patient with the subacute form, an important gastrointestinalcomponent was recorded with diarrhea of two months duration, involvement of the colon, lymph nodes, liverand spleen. In the chronic patients diagnosis was accomplished by direct examination and isolation of Paracoccidioidesbrasiliensis from clinical samples (sputum, oral exudates); these patients had reactive serological tests. In the subacuteform patient, diagnosis was made by lymph node and colon biopsies; his serologic tests were non-reactive. In thesefour patients, the standard treatment with ketoconazole or itraconazole had failed, in one because concomitant anti-TBtherapy, another patient due to worsening of adrenal insufficiency and the last patient due to gynecomastia with clinical andserologic failure. The juvenile case had failed to both amphotericin and itraconazol therapies. Once the failure was verified,posaconazole at a 400 mg BID was instaurated. One of the patients with chronic disease died a month after therapyinitiation secondary to lung sequelae and adrenal insufficiency due to previous PCM and TB. Other two chronic patientsshowed rapid clinical improvement and significant decreases in serologic titres after starting posaconazole. The infiltrates inchest X ray´s dissapeared, leaving mild fibrotic sequelae. Both patients were followed up along three years, without relapseof disease. The patient with juvenile form improved of his symptoms in the first month after start the posaconazole, followedby disappearance of adenopathies; he remained without symptoms until end of therapy at six month. The colonoscopydone at fifth month of therapy showed absence of any commitment. This report shows that posaconazole is a therapeuticalternative for patients with PCM and failure of first treatment.205

6-03Variable effect of caspofungin on the mycelial and yeastlike phases of several strains ofParacoccidioides brasiliensisS. Rodríguez-Brito, Niño-Vega, G., and San-Blas. G.Instituto Venezolano de Investigaciones Científicas (IVIC), Laboratorio de Micología, Apartado 21827, Caracas 1020A, Venezuela.e-mail: srodrigu@ivic.veCaspofungin inhibits the activity of β-1,3-D-glucan synthase (GS), enzyme responsible for the synthesisof β-1,3-glucan in the fungal cell wall. This polysaccharide is an essential component of this outer fungal cellstructure. In Paracoccidioides brasiliensis, β-1,3-glucan constitutes 36% and 5% of the mycelial and yeastlikewalls, respectively. Instead, the yeastlike cells synthesize α-1,3-glucan as the main wall component (45%), apolysaccharide absent in the mycelial cell wall.To study the effect of caspofungin on both morphological phases of P. brasiliensis, isolates IVIC Pb73 (ATCC32071), IVIC Pb300 (soil), IVIC Pb377 (armadillo), IVIC Pb381 and IVIC Pb444 (patients), were grown for 4 days inRPMI 1640 (GIBCO) medium, in the presence of caspofungin (0; 0.1; 0.5 and 1.0 µg/ml). Yeast cells were incubatedat 37ºC; cell density was followed by turbidimetry in Klett units, every 24 hours. Mycelia grew at 23ºC; growth wasmeasured daily by dry weight. At 1 µg/ml, caspofungin inhibited yeast growth in different proportions dependingon the isolate: Pb73 (65%) > Pb381 (52%) > Pb300 (35%) > Pb377 (22%) > Pb444 (21%) Considering the lowamount of β-1,3-glucan in the yeast cell wall of P. brasiliensis, the performance of caspofungin against the yeastphase of P. brasiliensis was surprising. The mycelial phase, as expected, was highly susceptible to caspofungin,inhibition fluctuating between 74% (Pb381) and 81% (Pb73). Scanning electron microscopic observations indicatedstructural modifications in the cell walls of both phases as a consequence of caspofungin action.We are currently working on cell wall composition analysis for both phases of the isolates under study, inorder to determine whether differences in P. brasiliensis cell wall composition, particularly with regards to theparticipation of β-1,3-glucan and the related activity of its synthesizing enzyme (GS), could somehow explain theobserved isolate–dependent inhibition by caspofungin.Acknowledgement: Merck, Sharp & Dohme (Caracas, Venezuela), for partial support.6-04Assessment of the efficacy of voriconazole, comparativily to other antifungals, inparacoccidioidomycosis experimental of the ratsD. S. Granzoto 1 , Vitali, L. H. 1 ; Martinez, R. 11 Departament of Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo-USP, Ribeirão Preto, Brazil.emal: danielagranzoto@yahoo.com.brIn this study it was evaluated the effectiveness of voriconazole in comparison to ketoconazole, fluconazole,itraconazole and sulfamethoxazole-trimethoprim in the experimental infection of female Wistar rats byParacoccidioides brasiliensis. The parameters used to quantify the response to the drugs were counts of colonyforming units (CFU) of Paracoccidioides brasiliensis in the lung and spleen and animal survival. The antifungaltreatment was started seven days after infection. The medications were administered by gavage for 6 to 15 days,according to the experiment, the following daily doses in mg/kg weight: voriconazole-5, 7, 10 and 20; ketoconazole-10, 12 and 15; fluconazole-6; itraconazole-4; sulfamethoxazole-trimethoprim-100, 120 and 150(sulfamethoxazole).In evaluation of the effectiveness of the drug made by counting the CFU, the animals treated with voriconazole-7mg/kg/dia showed a significant reduction of infection splenic compared to the control group (averages of 36,9x10³and 68,8x10³/body). The daily dose of 10mg there was decrease of fungal infection in the lung (averages 20,1x10 6 and 96,5 x10 6 ). With a daily dose of 10mg reduced the fungal infection in the lung (averages 20,1 x10 6and 96,5 x10 6 ) and in the spleen (averages of 9,1 x10³ and 108,9 x10³) compared to untreated rats. However,itraconazole and fluconazole showed greater efficacy in reducing the fungal load in the lung and spleen thanvoriconazole. In two studies, voriconazole (10mg/kg body weight/day) prolonged discreetly the survival of animalstreated with the drug. In relation to the control group, the period of mortality of 50% of the animals treated with6 doses of voriconazole, fluconazole, itraconazole and sulfamethoxazole-trimethoprim was respectively, 16, 22,22, 39, 28 and 26 days. With 12 doses, the values corresponding to 50% of deaths were, respectively, 16, 17,14, 36, 25 and 32 days after inoculation. Voriconazole showed a dose-dependent effect, because there was aless effective not significant in the dose recommended for clinical use (5 and 7mg/kg/day) in relation to the higherdosages (10 and 20mg/kg/day). The treatment with voriconazole provided a reduction of the load fungal tissueand increased survival of rats, and may represent a new therapeutic option in human paracoccidioidomycosis.Keywords: Paracoccidioidomycosis, voriconazole, azoles, experimental infection.206

6-05Amphotericin B-PLGA-DMSA nanoparticle: A new alternative to treat ParacoccidioidomycosisA.C. Amaral 1, 3 , Bocca A.L. 1 , Ribeiro A.M. 1 , Nunes J. 1 , Falcomer C.L. 1 , Peixoto D.L.G. 1 , Simioni A.R. 4 , Pinto F.L. 4 , LacavaZ.G.M. 1 , Azevedo R.B. 1 , Titze-de-Almeida R. 1 , Tedesco A.C. 4 , de Morais P.C. 2 and Felipe M.S. 11Instituto de Ciências Biológicas e 2 Física, Universidade de Brasília, UnB, Brasília, Brazil. 3 Ciências Genômicas e Biotecnologia,Universidade Católica de Brasília, UCB, Brasília, Brazil. 4 Instituto de Química, Universidade de São Paulo, USP, Ribeirão Preto, Brazil.e-mail: amaralandre@yahoo.com.brIntroduction and aim: Biodegradable polymers used to prepare controlled release systems are attractiveby the possibility to control the release of a drug slow and gradual through pre-designing its properties, such asdegradation kinetics and incorporation of target-specific molecules. Polymers used in these nanocarriers canbe natural (e.g. chitosan and alginate) or synthetic (e.g. PLGA-poly(lacti-co-glicolic acid)) and they have beenchoosing mainly because of its biocompatibility and biodegradability. During in vivo applications biocompatiblepolymers are broken down into molecules that can take part in normal metabolic pathways to be removed fromthe body (Trends in Biotech. 24(1):39-47, 2006; J. Interventi. Cardiol. 19(6):500-506, 2006). Here we describethe antifungal efficacy of a newly developed formulation of desoxycholate amphotericin B (D-AMB) encapsulatedwithin PLGA-DMSA nanoparticles (NanoAnf) in the murine model of paracoccidioidomycosis (patent filled INPI #0700446-0, Antmicrob. Agents Chemother., submitted). Methods: Balb/C mice infected by the intravenously routewith the virulent strain Pb18 of P. brasiliensis received NanoAnf (6mg/Kg of encapsulated D-AMB, ip, interval of72hs, which is equivalent to 2.7mg/Kg of pure amphotericin B) or D-AMB (Fungizon ® 2mg/kg, ip, interval of 24hs,which is equivalent to 0.9mg/Kg of pure amphotericin B). The treatments started 30 days after the fungal infectionand run for 30 and 60 days. Results and discussion: NanoAnf treated animals presented a marked antifungalefficacy noted by the decrease on the lung fungal burden, followed by absence of renal and hepatic abnormalities,as well as genotoxic and cytotoxic effects. Also, the animals received this new preparation, presented betterparameters of healthy status, like absence of piloerection, hypotrichosis, and reduced the loss of body weightobserved in D-AMB-treated animals (12.4%) after 30 daily doses. Moreover, this preparation allows a sustained,gradual drug release and lung tropism, due to the presence of DMSA, and a favorable 72h extended dosing interval.In conclusion, our results indicate the novel D-AMB-coated PLGA-DMSA, NanoAnf, is a promising preparationuseful to treat paracoccidioidomycosis such as other fungal infections. Financial support: CNPq.6-06Fungicidal activity of amphotericin B-magnetic fluid conjugate in vitro againstParacoccidioides brasiliensisMedeiros P.B. 1 , Amaral A.C. 1, 3 , Saldanha C.A. 1 , Bocca A.L. 1 , Guilherme R.B. 4 , Silva J.R. 4 , Nunes E.S. 4 , Lima E.C.D. 4 ,Azevedo R.B. 1 , de Morais P.C. 2 and Felipe M.S. 11Instituto de Ciências Biológicas e 2 Física, Universidade de Brasília, UnB, Brasília, Brazil. 3 Ciências Genômicas e Biotecnologia,Universidade Católica de Brasília, UCB, Brasília, Brazil. 4 Instituto de Química, Universidade Federal de Goiás, UFG, Goiânia, Brazil.e-mail: amaralandre@yahoo.com.brIntroduction and aim: Functionalized magnetic nanoparticles can be used for several applications in biology,mainly because its ability to be driven in body by an external magnetic field (J. Biosci. Bioeng., 100(1):1-11, 2005).Properly functionalized these nanoparticles may aggregate biocompatible molecules and drugs to be deliverydirect to the site of infection and, once there, release the drug. Polymeric linear molecules, with reactive organicgroups at both ends, are used to functionalize nanoparticles. One group is attached to the nanoparticle surfaceand the other may be used to link polymers creating a shell in which the drug is added. After conjugated with thispreparation, the drug must be efficiently released or be able to interact with the pathogen (J. Nanob., 2:3, 2004).Here we report the in vitro tests using magnetic nanoparticles conjugate with amphotericin B (AMB) against P.brasiliensis Pb01 and Pb18 yeast cells. Methods: The Minimum Inhibitory Concentration (MIC) were determinedby incubation of the yeast cells in RPMI1640 medium containing AMB conjugated with magnetic fluid in blockpolymer (because of patent filled required, the type of polymers used are not detailed). After three days of exposuretimes to magnetic particles, 3 × 10 2 cells were plated in BHI supplemented with 4% horse serum, 5% P. brasiliensis192 culture filtrate and 40mg/L Gentamicin. The plates were incubated at 36 ºC and CFU was counted at 21 st daypost-plating to asses the cell viability. Results and discussion: AMB conjugate with magnetic nanoparticles inblock polymer was able to interact with the fungal cells. Also, the AMB did not loss its fungicidal efficacy oncethe MIC observed for both Pb01 and Pb18 was similar to that determined for the conventional D-AMB (0,5µg/ml).We can envision that AMB preparation tested here presents a promising potential to be used in the treatment ofparacoccidioidomycosis. Although AMB is successful used to treat PCM, its use is limited because of its toxicity.So, once conjugated with magnetic nanoparticle it could be delivered direct to the site of infection diminishing theirsevere adverse effects by reduction on drug amount. Financial support: CNPq.207

6-07PLGA-P10 conjugate improved 20-times the immunological protection of this peptide againstParacoccidioides brasiliensisA.C. Amaral 1, 3 , Marques A.F. 4 , Muñoz J.E. 4 , Bocca A.L. 1 , Simioni A.R. 6 , Titze-de-Almeida R. 1 , Tedesco A.C. 6 , de MoraisP.C. 2 , Taborda C.P. 4 , Travassos L.R. 5 and Felipe M.S.S. 11Instituto de Ciências Biológicas e 2 Física, Universidade de Brasília, UnB, Brasília, Brazil. 3 Ciências Genômicas e Biotecnologia,Universidade Católica de Brasília, UCB, Brasília, Brazil. 4 Departamento de Microbiologia, USP, São Paulo, Brazil. 5 Departamento deMicrobiologia, Imunologia e Parasitologia, Unifesp, São Paulo, Brazil. 6 Instituto de Química, Universidade de São Paulo, USP, RibeirãoPreto, Brazil. e-mail: amaralandre@yahoo.com.brIntroduction and aim: Because of the dramatic increase in the incidence of systemic mycosis, extensive researchhas been focused to design efficient adjuvant systems to develop vaccines to protect for fungal diseases (Nat RevMicrobiol, 5:13-28, 2007). Peptide antigens are especially promissory to trigger an effective immune-protectiveresponse against these infections. The P10 peptide, identified in the glycoprotein Gp43, the major antigen secreted byParacoccidioides brasiliensis, elicits an immunological protection against this fungus (Infect Immun, 66(2):786-793,1998). Encapsulation of peptides within polymeric systems, such as nano and microparticles, are suitable to provideits release at controlled rate under polymer degradation for a prolonged time. Polymers used in these nanocarrierscan be natural (e.g. chitosan and alginate) or synthetic (e.g. PLGA-poly(lacti-co-glicolic acid)) and they have beenchoosing mainly because of its biocompatibility and biodegradability (Trends in Biotech, 24(1):39-47, 2006). Herewe investigate the adjuvant effects of the peptide P10 encapsulated within PLGA-DMSA polymeric blends combinedwith sulfametoxazole to treat the paracoccidioidomycosis murine model. Methods: Balb/C mice were infected withthe virulent strain Pb18 of P. brasiliensis. After 30 days from fungal infection, the animals received 4 weekly doses ofthe peptide P10 (20µg) with complete Freud adjuvant (CFA) or within PLGA-DMSA blend containing 1, 5, 10, 20, or40µg of P10, followed by daily injections of 15mg/kg sulfametoxazole during 30 days. The animals were sacrificed ondays 30 and 60 from the beginning of treatments. Results and discussion: The results of fungal burden recoveryhave shown that 1µg of P10 entrapped within PLGA were more efficient than 20µg of P10 with CFA as adjuvantfor sulfametoxazole during 30 days of treatment. Also, in these two groups, the histological analysis revealed theformation of compact granulomas, probably as a consequence of the elevated levels of interferon-γ detected in thesegroups when compared with the others. In conclusion, the peptide P10 conjugated with PLGA-DMSA improved itsimmunological protection 20 times and proved to be an efficient aid to the sulfametoxazol chemotherapy to treatparacoccidioidomycosis in murine model. Financial support: CNPq.6-08Biological activity of the endophytic fungi UFMGCB 551 against ParacoccidioidesbrasiliensisF.F. Campos 1,2 , L.H. Rosa 3 , S. Johann 2 , B.B. Cota 2 , C.A. Rosa 1 , N.P. Sá 1 , P.S. Cisalpino 1 , C.L. Zani 2 .1Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil;2Laboratório de Química de Produtos Naturais, Centro de Pesquisas René Rachou, Fiocruz, Belo Horizonte, Brazil; 3 Laboratório deMicrobiologia, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, MG, Brazil.e-mail: fcampos@cpqrr.fiocruz.brEndemic mycoses caused by dimorphic fungi Paracoccidioides brasiliensis remain a major public healthproblem in several countries in Latin America, especially in Brazil, Venezuela, Colombia and Argentina. Somecompounds currently used for the control of P. brasiliensis infections are toxic and not efficacious, and infectionrelapses may occur. The aim of the present study was to evaluate the effects of endophytic fungus UFMGCB 551 onthree different clinical isolates of P. brasiliensis (Pb18, Pb01, Pb608). Thus, the Minimal Inhibitory Concentrations(MIC) of the crude extract, fractions, and two isolated compounds, were evaluated. The isolate UFMGCB 551 wasgrown in potato dextrose agar (PDA) and inoculated at the center of 150 Petri dishes containing malt extract agar(MEA). After incubating the plates for 14 days at 28 ± 2 ºC, their contents were pooled and extracted with ethylacetate to yield 1.6 g of crude extract. This extract was able to inhibit all three P. brasiliensis strains when testedat 6.3 µg mL -1 . One gram of this extract was subjected to high-speed co-current chromatography (HSCoCC) toafford 134 fractions of 15 mL, which were pooled into 25 groups after their analysis by thin layer chromathograpy(TLC). Group 18 completely inhibited fungal growth at 0.9 µg mL -1 . This group was then fractionated by reversedphaseHPLC to yield two pure compounds that inhibited all P. brasiliensis at concentration below 0.9 µg mL -1 .Thus, the bioassay-guided fractionation of the title fungus yielded compounds with strong inhibitory effect againstP. brasiliensis, for which new drugs are much needed. This work corroborates the potential of endophytic fungi assource of new leads for drug development.208

6-09Antagonistic effect of farnesol, a Candida albicans quorum sensing molecule, onParacoccidioides brasiliensis growth and morphogenesisL.S. Derengowski 1 , S.V. Braz 2 , C. De-Souza-Silva 1 , T.M. Mello-de-Sousa 1 , S.N. Báo 2 , C.M. Kyaw 3 & I. Silva-Pereira 1 .Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília/DF – Brazil. Laboratórios de 1 BiologiaMolecular, 2 Microscopia Eletrônica and 3 Microbiologia. e-mail: lorena.bio@gmail.comFarnesol, a quorum sensing molecule of Candida albicans, is a sesquiterpene produced by many otherorganisms, and also found in several essential oils, possibly protecting plants from parasitic induced damages.Recently, this sesquiterpene alcohol has been demonstrated to inhibit the growth of some microorganisms, suchas the human pathogens Staphylococcus aureus and Streptococcus mutans, and the plant pathogenic fungusFusarium graminearum, signaling its potential use as an antimicrobial agent. Farnesol also enhance microbialsusceptibility to antibiotics, indicating a putative application as an adjuvant therapeutic agent. This sesquiterpenoidprevents C. albicans transition from yeast to mycelium, and compromises its biofilm formation. A recent studyshowed that farnesol is employed by C. albicans in order to reduce competition with other microbes, since thiscompound mediated apoptosis in the filamentous fungus Aspergillus nidulans and inhibited biofilm formation inother Candida species.The potential use of farnesol as an antimicrobial agent was investigated, and here, we report its activityagainst the human pathogen Paracoccidioides brasiliensis. This isoprenoid was able to inhibit P. brasiliensisgrowth and, when employing concentrations that not compromise cell growth, it also affected the P. brasiliensisdimorphic transition. Electron microscopy shows that P. brasiliensis cells treated with farnesol exhibited a fullycytoplasm degeneration. However, the cell wall remained intact, and the cell permeability was not affected.However, no synergistic effect between farnesol and fluconazole was observed. Our results clearly showedthe in vitro antimicrobial activity of farnesol against P. brasiliensis. However, additional studies involving animalmodels of experimental paracoccidioidomycosis need to be performed to assess their potential use as an adjuvantchemotherapeutic agent.6-10Killing of Paracoccidioides brasiliensis yeast cells by magnetic hyperthermiaA.C. Amaral 1, 3 , Braun S 1 ., Nunes E.S. 4 , Bocca A.L. 1 , Lima E.C.D. 4 , Azevedo R.B. 1 , de Morais P.C. 2 and Felipe M.S.S. 11Instituto de Ciências Biológicas e 2 Física, Universidade de Brasília, UnB, Brasília, Brazil. 3 Ciências Genômicas e Biotecnologia,Universidade Católica de Brasília, UCB, Brasília, Brazil. 4 Instituto de Química, Universidade Federal de Goiás, UFG, Goiânia, Brazil. e-mail: amaralandre@yahoo.com.brIntroduction and aim: Magnetic fluids are colloidal systems in which the magnetic nanoparticles, usuallymagnetite or maghemite, are dispersed in a physiologic solution (J Mater Res, 13(10:)2975-2981, 1998). Theaverage size of the magnetic nanoparticle in a magnetic fluid varies between 3 and 20nm; and at this dimension(10 -9 part of the meter), matters show special characteristics, such as the ability to be directed to a body-sitespecific by an external magnetic field or respond to a time-varying magnetic field, which result in the nanoparticles’agitation, generating an increase on temperature up to 45-47 ºC. This is the principle of a technique calledmagnetic hyperthermia, used to treat some kinds of tumors (J. Phys. D. Appl. Phys, 36:R167-181, 2003). In thisstudy we have subjected the P. brasiliensis Pb18 to the magnetic hyperthermia. Methods: The yeast cells (3 × 10 4cells/mL) were incubated during 3 and 10 minutes under a time-variable magnetic field in YPD medium containingmaghemite nanoparticles functionalized with dimercaptosuccinic acid (10 14 particles/mL). After the exposure tothe magnetic field the cells (3 × 10 2 ) were plated in BHI supplemented with 4% horse serum, 5% P. brasiliensis192 culture filtrate and 40mg/L Gentamicin. The plates were incubated at 36 ºC and the Colony Forming Units(CFU) was counted at 21 st day post-plating to asses the cell viability. Results and discussion: Subjecting P.brasiliensis yeast cells, co-cultured with magnetic fluid, to an alternating magnetic field killed the fungus. Cellscultivated in YPD subjected to a magnetic field or incubated with magnetic fluid without magnetic field had notthe cell viability affected. These findings could be useful to evaluate the use of magnetic hyperthermia to treatparacoccidioidomycosis. Since the magnetic nanoparticles are sensitive to the action of an external magneticfield, they could be driven to the site of fungal infection and, once there, apply the alternate magnetic field. Asnoted for cancer treatment, the magnetic hyperthermia technique is used against tumor cells, which are sensible toincreases on temperature without or minor commitment of the health cells (J. Phys. D. Appl. Phys, 36:R167-181,2003). Financial support: CNPq.209

6-11Antifungal activity of plants extracts used in brazilian traditional medicine againstParacoccidioides brasiliensisS. Johann 1 , G.A. Watanabe 1 , B.B. Cota 3 , E.P. Siqueira 3 , C.L. Zani 3 ; M.G. Pizzolatti 2 , P.S. Cisalpino 1 , M.A. Resende 1, *1Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; 2Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil; 3 Laboratório de Química dos ProdutosNaturais, Centro de Pesquisas René Rachou, FioCruz, Belo Horizonte, Brazil. +Corresponding author: sjohann@cpqrr.fiocruz.brThe antifungal activity of extracts of plants employed in Brazilian traditional medicine was assayedagainst Paracoccidioides brasiliensis and their citotoxicity on murine macrophages was determined. Theextracts of ten plants were assessed for activity against three P. brasiliensis clinical isolates. Susceptibilitywas determined by the broth microdilution method. The hexanic fractions of Piper regnellii and Baccharisdracunculifolia have shown the lower inhibitory concentrations (MIC) (7.8-30 µg ml -1 and 7.8 µg ml -1 ,respectively).Additionally, neither exhibited cytotoxicity on murine macrophages. According to Gas chromatographymassspectrometry (GC-MS) analysis, the major components of the hexanic fraction of B. dracunculifolia were1H-cycloprop[e]azulen-7-ol (17.57%), cyclododecanol (11.77%) and α-farnesene (8.15%). The antifungalactivity of the hexanic fractions of P. regnellii and B. dracunculifolia against this pathogenic fungus, withoutevidences of cytotoxicity, supports further studies in search of new chemical structures with activity againstP. brasiliensis.6-12Treatment of murine paracoccidioidomycosis (PCM) with trimethoprim-sulfamethoxazolecombination (TMP-SMX)F. Moura 1 , C.R.G. Lima 1 , T.C. Moreto 1 , L.R. Carvalho 2 , R.A. Bueno 1 , D.V. Moris 1 , R.P. Mendes 1 .1 Tropical Diseases Área, Botucatu Medical School. 2 Department of Biostatistics, Biosciences Institute. São Paulo State University -UNESP. e-mail: tietemendes@terra.com.br (presenting Author)Introduction: Trimethoprim-sulfamethoxazole combination (TMP-SMX) has been widely used in thetreatment of paracoccidioidomycosis patients. However, only two experimental studies were carried out toverify its efficacy and its capacity in eradicating the infection. The objective of this study was the evaluationof the efficacy of different TMP-SMX regimens in the treatment of murine paracoccidioidomycosis.Methods: Different TMP-SMX regimens, 200 mg SMX/kg once a day by gavage, were used to treat 145male BALB/c mice infected with P. brasiliensis strain 18, comprising 6 groups: G1 – healthy controls; G2– Infected controls; G3 – early introduction and long course treatment (140 days); G4 – early introductionand short course (56 days); G5 – late introduction (at 28 th day post-infection) and long course (112 days);G6 – SMX serum levels evaluation. Treatment efficacy was evaluated by the number of colony-forming units(CFU) from lung and spleen fragments at 2 nd , 4 th , 8 th , 12 th , 16 th and 20 th weeks post-infection; endpoint was adecreased CFU. Kruskal-Wallis, Mann-Whitney, χ 2 , Fisher’s exact and Wilcoxon test made up the statisticalanalysis, with level of significance set at p < 0.05.Results: The cumulative mortalities of the mice revealed the following percentages of survival:G1=100.0%, G2=78.9%, G3=89.3%, G4=71.4%, and G5=88.2%. Survival was not different in G2 and G4(p>0.05), or G3 and G5 (p>0.05); however, survival rate was higher in G3+G5 than G2+G4 (p

6-13Therapeutic DNA vaccine in BALB/c and B10A mice against experimentalparacoccidioidomycosisG. M. Gomes Rittner 1 , J. E. Muñoz 1 , C. P. Taborda 1 and Travassos L. R. 21 Departamento de Microbiologia, Instituto de Ciências Biomedicas,USP, São Paulo, Brasil. 2 Departamento de Microbiologia, Imunologiae Parasitologia, UNIFESP, São Paulo, Brasil. e-mail: travassos@unifesp.brBackground: Paracoccidiodomycosis (PCM) is a systemic granulomatous disease caused by the thermo-dimorphicfungus Paracoccidioides brasiliensis. It is widespread in Latin America and found mainly in Brazil, Colômbia, Argentinaand Venezuela. Immunotherapy has been proposed as an adjuvant to PCM chemotherapy and to control relapses. ADNA vaccine is a promising approach to Ag-specific immunotherapy and was shown to be protective in mice, encodingthe major antigen gp43 (Pinto et al., 2000). Plasmid construction is easily prepared, stable, and can be repeatedlyadministered. Peptide 10 contains the T-cell epitope of gp43 and is protective against experimental infection in mice.The aim of this work was to analyze a DNA-based vaccine encoding P10 or IL-12 in mice intratracheally infected withP. brasiliensis. Methods: BALB/c and B10A mice were immunized with 100 µg of pCDNA3 encoding P10, IL-12 ora mixture of the two plasmids. Animals were infected intratracheally with 3x10 5 yeast cells of virulent P. brasiliensisPb18. After 30 days, they were immunized again, once a week for 4 weeks. Colony forming units (CFU) and cytokineproduction were measured in lungs after 1 week of treatment. Control groups of infected mice were immunized withpCDNA3 without insert. Results: Significant reduction of CFU in the lungs of mice immunized with plasmid encodingP10, IL-12 or plasmid mixtures encoding P10/IL-12 were observed. The cytokines in lungs showed enhanced levels ofIFN-β and IL-12. Conclusions: (I) DNA-based vaccine encoding P10 and/or IL-12 was very effective in reducing thefungal load; (II) therapy using a mixture of plasmids encoding P10 and IL-12 was the most effective formulation; (III)CFU reduction was associated with enhanced levels of IFN-β and IL-12, and reduced IL-4; (IV) DNA-P10 and DNA-IL-12 mixed therapeutic treatment induced a Th-1 immune response in BALB/c and B10A mice that was protective againstintratracheal challenge with virulent P. brasiliensis; (V) B10A mice treatment was more effective than that with BALB/cmice ending up with scarcely no lung CFU. Supported by Fapesp and CNPq.6-14Adrenal function in paracoccidioidomycosis patients treated with azolic derivatives: Resultsafter prolonged post-therapy follow-upTobón AM 1,2 , Restrepo CA 3 , Villa CA 3 , Agudelo CA 1,3,4 , Quiceno W 4 , Restrepo A 11Corporación para Investigaciones Biológicas (CIB), 2 Hospital La María, 3 Universidad Pontificia Bolivariana, 4 Hospital Pablo Tobón Uribe,Medellín, Colombia. e-mail: atobon@cib.org.coAdrenal insufficiency is a rather frequent sequela of paracoccidioidomycosis (PCM). The presence ofParacoccidioidomicosis brasiliensis in adrenal glands tissues has been previously reported in patients with this mycosiseven after their completion of specific therapy. Additionally, some of these patients exhibit reactive serologic tests andshow high antibody titers that persist after adequate treatment indicating continuity of an antigenic stimulus probablylinked to the presence of the fungus in these glands. This work attempted to study the situation of these glands afterprolonged post-therapy evaluation and tried to establish if a connection could exist between high anti-P. brasiliensisantibody titers and the diagnosis of adrenal insufficiency in these patients who had been followed years after terminationof their antifungal treatment. In this study we included 28 PCM patients who had been previously treated for this disorder;they were all males with a medium age of 55.3 years (SD+12.1) and were called for consultation after a mean of 13.32(SD+9.22) years after being diagnosed. From these patients, 17 (60.7%) had the chronic multifocal form of the disease,6 (21.4%) the subacute form, and 5 (17.8%) the chronic unifocal form. Eighteen (64.3%) patients had been treated withitraconazole, 4 (14.3%) with ketoconazole, and 6 (21.4%) with other antifungals alone or in combination. The media ofbasal cortisol was 13.45 (SD+4.81) µg/dl (N >5 µg/dl), after ACTH stimulation cortisol amounted to 34.36 (DS+7.41)µg/dl (N >18 µg/dl) with an increase of 20.47 (DS+6.35) µg/dl (N >16.5 µg/dl) after ACTH stimulus. In 2 (7.1%) patientsadrenal insufficiency was demonstrate while in 8 (28.6%) the response to ACTH was below normal. P. brasiliensis wasseen in a biopsy of adrenal glands from a patient with insufficiency. At diagnosis, most patients (71.4%) had shownhigh antibodies titers (≥1:32) which in 39.3% persisted up to the end of treatment and in 10.7% after the prolongedpost-therapy follow up. However, a statistically significant association between persistently high antibody titers at thefollow-up observation and low cortisol values post ACTH challenge was not demonstrated indicating that the fungalantigenic stimulus, as shown by the presence of specific antibodies, appeared not to originate in the adrenals implyingno relation between adrenal malfunction and antibody production. The proportion of adrenal insufficiency (7.1%) foundafter such an extended period of therapy cessation, as well as the subnormal response to ACTH stimulation in 28.6%of our cases, confirm that adrenal damage is an important marker of paracoccidioidomycosis. It would be advisable tocheck on this abnormality as soon as diagnosis is established so as to take the proper measures on time, thus avoidingfurther damage to vital organs such as the adrenal glands.211

6-15The association tuberculosis - paracoccidioidomycosis: Impact on the outcome oftheraphyTobón AM 1,2 , Agudelo CA 1,3,4 , Tabares AM 1 , Restrepo A 1.1Corporación para Investigaciones Biológicas, 2 Hospital La María, 3 Universidad Pontificia Bolivariana, 4 Hospital Pablo Tobón Uribe,Medellín, Colombia.e-mail: carlosagudelo@yahoo.comTuberculosis (TB) and paracoccidioidomycosis (PCM) are both chronic diseases with overlaping clinicalpresentations and which, additionally, may coexist in 10-13 % of the PCM cases. In patients with both diseasestreatment difficulties have been reported due to the induction of the P450 enzymatic system with increasing azolicclearance. This work evaluates the results of antifungal therapy in patients with the TB - PCM coinfection andcompares the findings with those in patients with PCM alone.The clinical records from 7 patients with dual infections who received simultaneously antifungal and anti-TBmedications were analyzed. Demographic data, serologic titers, and response to therapy based in a point systemevaluation, were analyzed. Their responses to therapy were compared with a weighted control group, formed byPCM patients whose clinical form and antifungal therapy were alike.The coinfected group consisted of 6 men and 1 woman, median age of 49.3 (SD+15.9) years with symptomshaving a duration of 232.1 (SD+184.3) days. All patients in the control group were men, with a median age of 42,0(SD+14.9) years and with symptoms duration of 167.9 (SD+114.8) days. At diagnosis, the point system evaluationscore was 21.1 (SD+6.5) in the group with TB and 14.6 (SD+6.2) in control group. The mean treatment durationwas 8,7 (SD+3.6) months and the medium itraconazole dosage was 228.6 (SD+125.6) mg/day in the group withTB and in the respectively of control group these figures were 7.3 (SD+2.2) months and 185.7 (SD+37.8) mg/dayof itraconazole. None of these features showed statistically significant differences between the two groups. Theresponse to therapy evaluated by point system and by serologic titers showed no statistically significant differencesbetween the TB coinfected and the PCM alone groups.These findings indicate that antiTB medication does not affect the response to antifungal therapy in patientswith PCM coinfected with tuberculosis. However, the small number of cases studied precludes more precisedeterminations.212

Poster Session7Other Mycoses

7-01Susceptibility to fluconazole and voriconazole of Candida species isolated from intensivecare units patients in several hospitals in Medellín, Colombia (2001 - 2007)A. Zuluaga 1 , C. Bedout 1 , CA. Agudelo 1,2,3 , H. Hurtado 1 , M. Arango 1,4 , A. Restrepo 1 and A. González. 1,5 .1 Corporación para Investigaciones Biológicas, CIB, Medellín – Colombia. 2. Universidad Pontificia Bolivariana, Medellín. 3. Hospital PabloTobón Uribe, Medellín. 4 Facultad de Medicina. Universidad de Antioquia. 5. Escuela de Microbiología. Universidad de Antioquia.e-mail: azuluaga@cib.org.coDisseminated candidiasis, an opportunist mycosis caused by different Candida species, has become an importanthealth problem that posses a threat for the lives of many patients, especially if they are immunosuppressed orhospitalized in Intensive Care Units (ICUs). From 2001 to 2007, a transversal cohort study was done aimingat determining the frequency and susceptibility to fluconazole and voriconazole of Candida species isolates sentto the CIB for susceptibility studies; they had been obtained from different ICUs patient. Based on the protocolsrecommended by the CLSI from the United States, document M44P, discs impregnated with these antifungal drugswere used in the agar diffusion technique. A bivaried statistical analysis was done to determine the associationbetween the isolated Candida species and their resistance to each antimycotic. From 337 isolated analyzed, 196corresponded to blood cultures, 111 to peritoneal fluids, 10 to deep organs biopsies, 9 to pleural fluids, 9 to closedabscesses, and 2 to cerebrospinal fluids. These isolates were speciated as follows: 147 (43,6%) C. albicans, 79(23,4%) C. tropicalis, 32 (9,5%) C. glabrata, 47 (13,9%) C. parapsilosis, 11 (3,3%) C. krusei and 12 (3,6%) C.guilliermondii. The remaining isolates (2,7%) were distributed in 5 different species (C. famata, C. lusitaniae, C.lipolytica, C. pelliculosa y Candida ssp). As it concerns fluconazole, 78,3% of all the above isolates were susceptible,11,9% susceptible dose dependent (SDD) and 9,8% resistant. Regarding voriconazole, we observed that 94,1% ofthe isolates were susceptible, 2,4% SDD and 3,6% resistant. The range of susceptibility of the various species tofluconazole, was as follows: C. albicans 95,2%, 86% C. tropicalis, 65,6% C. glabrata and 59,6% C. parapsilosis. As itconcerns voriconazole, susceptibility was shown for 100% of C. albicans isolates, 96,2% of C. tropicalis, 93,6% of C.parapsilosis and 84,4% of C. glabrata. The bivarieted analysis showed that the following isolates were significantlyassociated with susceptibility to fluconazole, thus, C. albicans (OR: 10,6, IC 95% 4,7 – 24,06, p < 0.01) and C.tropicalis (OR: 1,95, IC 95% 0,97 – 3,93, p 0,05), with SDD C. parapsilosis (OR: 3,2, IC 95% 1,50 – 6,88, p < 0,01)and C. guilliermondii (OR: 1,01, IC 95% 1,15 – 13,99, p 0.04) and C. guilliermondii (OR: 11,03, IC 95% 3,33 – 36,6,p < 0,01) and C. krusei (p < 0,01) with resistance. As for voriconazole, the isolations tested were significantlyassociated with susceptibility, in the cases of C. albicans (p < 0,01), with SDD C. krusei (OR: 29,3, IC 95% 6,45– 132,5, p < 0,01) and C. glabrata (OR: 7,88, IC 95% 2,34 – 26,52, p < 0,01) with resistance. These data indicate thatC. albicans is the species most frequently (43.6%) recovered from the different clinical samples; however, altogether,the group formed by other species different to C. albicans also represents an important proportion (56,4%) of theisolates recovered from the different clinical samples. In addition, these results reveal a remarkable change in thefrequency of non-albicans species, as well as the presence of new susceptibility patterns that demands the preciseidentification of the causative organism. There is need to determine the susceptibility pattern of these emergingisolates in order to take the correct therapeutical approach.7-02Therapeutic study of gomesin in mice infected with Candida albicansD. C.P. Rossi 1 , J. E. Muñoz 2 , C. P. Taborda 2 and Daffre S. 11 Departamento de Parasitologia; 2 Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, SãoPaulo – Brasil. e-mail: rossi.d@usp.brBackground: Gomesin is a cationic antimicrobial peptide produced by the hemocytes of the spider Acanthoscurriagomesiana. It has a molecular mass of 2270.4 Da, 18 amino acids and four cysteine residues forming two disulfidebridges. Gomesin strongly affects bacterial growth and the development of filamentous fungi and yeast. It alsoeffective against Leishmania amazonensis (Silva Jr et al. JBC 2000, 275: 33464) and different life stages of thePlasmodium spp. (Moreira et al., Exp Parasitol 2007, 116: 346). In addition, Gomesin causes death of Cryptococcusneoformans through membrane permeabilization. In association with Fluconazole, it kills fungi and enhances theactivity of brain phagocytes (Barbosa et al., FEMS 2007 Microbiol Lett 274: 279). In the present study we evaluatedthe efficacy of Gomesin on systemic candidiasis model. Methods: We used an experimental model of Balb/c miceinfected intravenously with 3 x 10 5 yeast cells of Candida albicans with high virulence (strain 78). Treatments withGomesin, Fluconazole, Anfotericin B and Gomesin in association with Fluconazole consisted of three doses (5mg/Kg)at 1, 3 and 6 days post infection. At the seventh day post infection, the kidneys, spleen and liver were removed andthe CFU were counted. Results: Gomesin has a dose-dependent effect and the treatment significantly decreased thenumber of CFU compared with control. Conclusions: Treatment with Gomesin is efficient in the systemic candidiasisexperimental model. This result suggests that Gomesin might be used as an alternative to the usual treatment,especially when treating resistant pathogens.215

7-03Study of genotypic and phenotypic characteristics among C. gattii serotype B - VGIImolecular type clinical isolates from Cúcuta, Colombia and isolates of the outbreak inVancouver, CanadaG.Torres, and P. Escandon.Grupo de Microbiología, Instituto Nacional de salud, INS, Bogotá D.C - Colombia. Tel. 2207700 ext. 445-446.e-mail: getorresr@unal.edu.coIntroduction: Cryptococcus gattii serotype B - VGII pattern emerged as a primary pathogen in Vancouver,Canada, causing a major epidemic of cryptococcosis in 2000 that affected immunocompetent humansand animals. In Cúcuta, Colombia isolates of C. gattii of the same serotype and molecular type have beenreported.Aim: Compare serotype B isolates from Cúcuta and isolates from the outbreak of Vancouver, with thepurpose of identifying similarities and differences in the virulence profile between the two groups of isolates.Methods: 20 isolates molecular type VGII – mating type α, from the outbreak of Vancouver and 11 Colombianisolates of C. gattii serotype B were studied, determining the molecular type by gene URA5 restriction. Factorsassociated with virulence evaluated were: mating type, characteristics of the colonies, phenotypic switching, celland capsular diameter, enzyme activity (phenoloxidase and phospholipase) and growing at 37 °C.Results: 6 / 11 Colombian isolates were molecular type VGII. These isolates were mating type a, and havemucoid colonies; 5 showed phenotypic switching. 19/20 isolates from Vancouver presented smooth coloniesand phenotypic switching. The cell and capsular diameter was higher in Colombian isolates (P = 0.02). Nodifference was observed in enzyme activity between the two groups of isolates (P = 0.083). The 26 VGII isolatesgrew at 37 ° C.Conclusion: The presence of C. gattii VGII molecular type in Colombia and the similarities observed insome of the factors associated with virulence between the two groups of isolates, shows the need to do asurveillance of the cases associated with this molecular type.7-04Microecology of Blastomyces dermatitidis: The ammonia hypothesisD. J. BaumgardnerCenter for Urban Population Health and Aurora University of Wisconsin Medical Group, Milwaukee, Wisconsin – USA.e-mail: dennis.baumgardner@fammed.wisc.eduBackground: The precise microecology of Blastomyces dermatitidis is unknown. The fungus has beenassociated with nitrogenous waste products and rapidly changing environmental conditions of water tension,temperature, pH and potential chemical inhibitors. Ammonia accumulates in certain microenvironments, is toxicto most fungi, yet may not be identified in processed soil samples.Methods: Ammonia tolerance of B. dermatitidis was investigated by growth of two Wisconsin strains(ATCC MYA-2585/6), a clinical and an environmental isolate, respectively, on phosphate and HEPES bufferedagar media supplemented with mineral salts, low (1 g/l) and high (20 g/l) dextrose and increasing amounts ofammonium sulfate, at pH 7, in gas-impermeable bags at 20 o C. Growth of soil fungi from 200 aqueous slurriesof fresh and frozen soil samples from the northern USA and Canada was tested on similar media.Results: Moderate mold growth and sporulation of both strains of B. dermatitidis was observed at calculatedammonia concentrations of 375-563 mmol/l when plates were inoculated with either mold or yeast forms. Fungalgrowth was inhibited in virtually all soil samples at these ammonia levels when low dextrose concentrations wereutilized.Conclusion: The ability of B. dermatitidis to survive and grow in organic carbon-poor, high ammoniamicroenvironments may be important to the competitive success of this fungus. Such microenvironmentsmay include animal droppings or guano on sand soils, animal burrow latrine chambers and runoff fromammonia fertilizers with nitrification inhibitors. This may have implications for other dimorphic fungi such asParacoccidioides brasiliensis.Financial Support: St. Luke’s Foundation Donation from Mr. and Mrs. Charles Goldsworthy, Eagle River, WI.216

7-05Evaluation of an antigen-capture ELISA to detect Histoplasma capsulatum antigenuria inimmunocompromised patientsC.M. Scheel 1 , B. Samayoa 2 L. Benjamin 1 , P. Riley 1 , M. Lindsley 1 , A. Herrera 2 , G. Raxcacoj 2 , S. Lima 2 , R. Miramontes 1 ,T.M. Chiller 1 , M. Brandt 1 , E. Arathoon 2 , B.L.Gómez 11Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA. 2 Clinica Familiar Luis Angel Garcia (CFLAG), HospitalGeneral San Juan de Dios, Guatemala City, Guatemala. e-mail: BGomez@cdc.govBackground: Histoplasma capsulatum infection causes significant morbidity and mortality in HIV-infectedindividuals, particularly those in developing countries without access to sophisticated diagnostics or highly-activeantiretroviral therapy. Furthermore, symptoms of histoplasmosis are non-specific, yet most deaths occur in thefirst 2 weeks after diagnosis. Current diagnostic methods can be complex, expensive and slow. A simple, rapidmethod to detect H. capsulatum infection would dramatically decrease time to diagnosis and treatment and reducemorbidity and mortality. We evaluated an antigen-capture ELISA to detect antigen in urine of HIV patients withhistoplasmosis in Guatemala.Methods: Urine samples were collected prior to treatment in HIV patients with culture-confirmed histoplasmosis(n = 48) or non-histoplasmosis fungal and non-fungal diseases (n = 113). Urine from healthy controls (n = 83)were used to define specificity. An antigen-capture ELISA was developed which utilizes polyclonal rabbit anti-H.capsulatum antibody as both capture and detection reagent. A standard curve was included in each assay plateto insure inter-assay reproducibility. Urine specimens were run twice on separate days and repeated a third timeif the coefficient of variance (CV) was greater than 20%.Results: The H. capsulatum antigen-capture ELISA demonstrates a sensitivity of 81% for confirmedhistoplasmosis and a specificity of 96% (all other disease controls, 95.0%; healthy controls, 97%) when testedagainst baseline urine specimens in these patient cohorts. Thirteen of the patients with follow-up urine specimensshowed decreased antigenuria during the course of antifungal chemotherapy.Conclusions: In this analysis, the antigen ELISA assay shows high sensitivity and specificity as a simple rapiddiagnostic test for histoplasmosis in HIV-infected individuals. This assay has the potential to be easily adaptedto laboratories across the world. A simple test using urine would allow for the rapid diagnosis and initiation oftreatment. Longitudinal analysis of H. capsulatum antigenuria in serial specimens during therapeutic interventionmay prove useful for monitoring patient recovery in a clinical setting.7-06An analysis of histoplasmosis in an endemic, resource poor area with a high HIV prevalencerate - Guatemala, 2007R. Miramontes 1 , B. Samayoa 2 , A. Herrera 2 , C. Scheel 1 , B. L. Gómez 1 , E. Arathoon 2 T. Chiller 1 .1Mycotic Diseases Branch, Centers for Disease Control and Prevention; Clinica Familiar Luis Angel Garcia (CFLAG), 2 Hospital GeneralSan Juan de Dios, Guatemala City, Guatemala. e-mail: TChiller@cdc.govBackground: Disseminated histoplasmosis is a serious opportunistic infection in AIDS, often representingthe first manifestation of the syndrome in endemic regions. Central America is a known endemic area forhistoplasmosis; however little is known about the true prevalence in persons with AIDS. In Guatemala, barriers topatient care such as lack of medication and extended travel to health facilities are common and rapid diagnosticsare not available.Methods: A prospective cohort study of hospital patients was conducted from February 2005 - December2007 in large public hospital in Guatemala City. Study criteria required that a patient be HIV-infected andhave three out of five of the following: Fever, pancytopenia, weight loss, radiological evidence consistent withhistoplasmosis, or skin/mucosal lesions suspicious for histoplasmosis. A histoplasmosis case was defined asa positive Histoplasmosis capsulatum culture from a clinical specimen, or positive tissue sample suggestive ofhistoplasmosis.Results: Of the 279 patients that met surveillance criteria, 217 (78%) were enrolled in the study. A total of63 (29%) of 217 patients met the case definition for histoplasmosis. There were 29 (46%) deaths among the 63case patients. The median time to death was 17 days. A total of 11 (17%) case patients were co-infected withtuberculosis and 4 of those were among reported deaths. The median CD4 cell count among case patients ondate of diagnoses was 25 (1-193).Conclusion: The incidence of histoplasmosis in this cohort of patients with HIV in Guatemala is high.Mortality occurred rapidly after admission and in more than a third of the patients. Patient care is complicated inthis setting by the lack of availability of a rapid diagnostic test. Rapid diagnosis is critical in this population. Thisstudy highlights the importance of histoplasmosis as an opportunistic infection in persons with AIDS in Guatemala.More work is needed to better define the burden of this disease in patients with HIV and its incidence among thosepatients co-infected with TB in order to provide guidance for better diagnosis and more rapid treatment.217

7-07Detection of DNA in peripheral blood from a patient with the ocular histoplasmosissyndrome: A case reportJ. Hernández 1, 3 , C. Muñoz 1, 2 , D. Hernández, 3, 4,5 C. Montoya , 5 Á. González 1,21Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas (CIB), 2 Microbiology School, Universidad deAntioquia 2 Medicine School Universidad Pontificia Bolivariana 3 , Hospital Pablo Tobón Uribe 4 , and Clínica Oftalmológica San Diego 5 ,Medellín Colombia. e-mail: jhernandez@cib.org.coThe ocular histoplasmosis syndrome (OHS) is a significant cause of vision loss in young and middle-agedadults. Despite the fact that the direct role of the fungus Histoplasma capsulatum var capsulatum in this conditionhas not been clearly demonstrated, some epidemiological studies appear to implicate the fungus in OHS. Wereport here a case of OHS in an immunocompetent individual in whom a nested PCR in peripheral blood for H.capsulatum turned out positive. A 37 years- old man indicated that previous to consultation he has had fever,malaise, headache and anterior cervical lymphadenopaty for one week after which he noticed gradual improvementof his symptoms but started to experience a sudden loss of his right eye visual acuity. The fluorescent angiographyand an optical coherent tomography informed the presence of a type II choroidal neo-vascular membrane inthe right eye. On the basis of these results, the ophthalmologist advanced a presumptive diagnosis of OHS.Conventional laboratory tests such as complement fixation and immunodiffusion tests for antibodies were negativeat the beginning of infection which became positive after 8 weeks with 1:8 titters - A peripheral blood sample wastested in a nested PCR for H. capsulatum var capsulatum using a set of primers known to amplify a DNA sequencecoding for the specific 100-kDa protein of this fungus (Hc100-PCR). The results were compared with a positivecontrol sample of H. capsulatum DNA and with a negative peripheral blood sample from a healthy individual.Additionally, the PCR products were sequenced to confirm the amplified product obtaining an identity of over 97%.The patient received intravitreal bevacizumab injection and Itraconazol therapy (200 mg 3 times daily for 3 daysand then 200 mg once or twice daily for 1 year). Median vision improves from 20/100 to 20/40 after 4-8 weeks oftreatment. In summary, we present a case of OHS with unusual manifestations. The molecular test used providedevidence linking the ocular lesions with an earlier infection by H. capsulatum. In addition, the Hc100-nested PCRrepresents an important and novel test for the diagnosis of histoplasmosis in human samples.218

Author IndexAAbadio AKR. 168Acorci MJ. 194, 195Adriana SG. 151Agudelo CA. 205, 212, 215Aguiar ESA. 141Aguiar JIA. 141Albe BP. 199Almeida AJ. 91, 165Almeida MA. 114Almeida RAMB. 147Alves BCA. 174Alves HY. 123, 179, 181, 193, 200, 207, 208, 209Andrade SR. 200Arango M. 171, 215Arathoon E. 101, 217Araújo SA. 156Arenas R. 142Arruda C. 174Azevedo RB. 181, 207, 209BBagagli E. 127, 137, 138, 147, 161, 162, 167, 176Bailão AM. 173, 175, 176, 178, 179Báo SN . 169, 170, 181, 209Barata LCB. 145Barbosa MS. 169, 170Barreto L. 172Barrezueta J. 127, 137, 150, 161, 162Baumgardner DJ. 216Bedout C. 215Benard G. 70, 127, 137, 145, 146, 147, 149, 151, 161,174, 182, 188Benjamin L. 217Bennett J. 121Bernardino S. 198Bertossi DRT. 189Bezerra CCF. 74Biondo GA. 195Blotta MHSL. 68, 187, 188, 189, 191Bocca AL. 181, 189, 191, 193, 196, 200, 201, 207,298, 209Bonfim CV. 191Bonifaz A. 142Bordon-Graciani AP. 194, 195Borges CL. 169, 173, 175, 179, 180Bosco SMG. 147, 162Braga CJM. 52Brandt ME. 95, 217Braun S. 181, 209Braz SV. 209Bredt Jr GL. 148, 150, 151, 157Bredt CSO. 148, 150, 151, 157Brito WA. 177Brown G. 53Bueno RA. 161, 210Burger E. 80, 199Burlandy-Soares LC. 68CCacere CR. 70Calich VGL. 50, 174, 192, 196, 197, 198Calvi SA. 147Camacho E. 170Campanelli AP. 71, 193Campos CLB. 173, 179Campos FF. 208Campos M. 150Cano LE. 62, 67, 82, 190, 199, 205Carmona JA. 91, 165Caro E. 67Carvalho A. 91, 165Carvalho CRR. 146, 152Carvalho LC. 103Carvalho LR. 141, 155, 156, 210Carvalho MJA. 168Casadevall A. 62Castro AG. 91Castro KP. 114Castro NS. 176, 177Castro-Silva MH. 144219

Catão E. 193Cavalcante RS. 103, 147, 156Cavassani KA. 71, 193Cazarín-Barrientos J. 142Chagas RF. 173Champion M. 43Chang MR. 141Chaves CCR. 147Chiller T. 95, 217Cisalpino PS. 208, 210Clemons KV. 49Coelho KYR. 152Coelho-Castelo AAM. 200Coimbra Jr CEA. 143Coitinho JB. 156Cole GT. 43, 55, 74Coltri KC. 194Costa AN. 146, 152Costa TA. 197Cota BB. 208, 210Coutinho ZF. 143Cunha C. 91, 165Cunha RV.141DDa Silva CJ. 201Daffre S. 215Dantas SFIM. 176, 179Datta SK. 53De Campos L. 148, 150, 151, 157De Morais PC. 181, 207, 208, 209De Souza-Silva C. 209Deepe Jr GS. 176, 179Derengowski LS. 209Di Benedette JPT. 179Dias-Melicio LA. 194, 195Diaz G. 143Diaz-Granados LR. 82, 190, 199, 205Diogo CL. 187Duarte AJS. 70, 188Duarte IX. 152Duarte MIS. 84, 116, 190Duarte PAD. 148, 150, 151, 157Duque JJ. 82, 190, 199, 205Duran LF. 143Escandon P. 216Eulálio KD. 74EFFaccioli LH. 180, 193Faffe DS. 191Fairbanks N. 147Falcomer CL. 207Faria FP. 170Feitosa LS. 169Felipe MS. 44, 123, 136, 165, 166, 168, 171,178,180,181, 182, 183, 189, 193, 196, 200, 207, 208, 209Félix CR. 170Felonato M. 197Fernandes CJCS. 152Fernandes L. 87, 165, 166, 171Fernandes VG. 156Ferraz DB. 194Ferreira LCS. 52Ferreria MC. 188Ferri PH. 180Fierer J. 53Figueiredo F. 191, 193, 196, 200, 201Filho AD. 74Fonseca CA. 189Fonseca JH. 156Fornazim MC. 187Fracon JF. 191Fraga CLF. 193, 196, 200Franca FOS. 151Furuchó CR. 189GGabetta CS. 187Galvãon MM. 151García AM. 89, 171, 181Gerolin GP. 144Giarolla I. 146, 149, 151Gil ML. 192Gimenes-Bosco SM. 138Goes AM. 156, 189, 200Goldman GH. 91Goldman WE. 59, 165Golim MA. 194Gomes Rittner GM. 211Gómez BL. 62, 113, 217Gómez CA. 143González A. 67, 192, 215, 218Gonzalez CR. 127, 162Goulart S. 145Gow NAR. 87Gozalbo D. 192Granzoto DS. 206220

Graybill JR. 101, 109Griva BL. 149, 152Gualtero S. 143Guedes HLM. 114Guilherme RB. 207Guimarães AJ. 114Heinsbroek SEM. 53Hernández D. 218Hernández JM. 218Hernández O. 89, 181Herrera A. 217Hidalgo JM. 82, 190Hooyakaas PJ. 165Hurtado H. 215HLazo R. 150Le T. 132Leão C. 91, 165Lenzi HL. 114Lima CRG. 210Lima DRA. 132Lima ECD. 181, 207, 209Lima S. 217Lindsley M. 217Lins TCL. 189Logarinho E. 91Lopera DE. 67, 82, 190, 199, 205Lopes DF. 141Lopes GP. 144Lopes JD. 79López-Martínez R. 142Loures FV. 50, 192Lozano VF. 189, 201Ludovico P. 91, 165Luiz AA. 151I, JIanhez LE. 151Jerônimo MS. 191Jiménez M del P. 53Jiménez RA. 82, 190, 205Johann S. 208, 210Johnson DI. 91Jukemura J. 149Junior SAD. 146Junqueira NM. 179Junta C. 180Kairalla RA. 146, 152Kibbler CC. 62, 124Kirkland T. 53Kohara VS. 147, 155Kono ASG. 146, 147, 149Kyaw CM. 209Lacava ZGM. 207Lazèra MS. 74Lazo J. 150KLMMacêdo RCI. 74Macoris SAG. 147, 162Madeiros PB. 207Malavazi I. 91Mamoni RL. 68, 187, 188, 191Mantilla JC. 148Mariano M. 79Mariano VS. 194Marques AF. 208Marques MEA. 155Marques SA. 127, 137, 147, 161, 162Martin MC. 107Martinez R. 71, 102, 193, 206Martins EMN. 200Martins LMS. 74Martins NF. 168Massafera-Tristão FS. 198Matos LF. 183Matute DR. 135, 167McEwen JG. 89, 167, 171, 181Medeiros AI. 180Mello de Souza TM. 209Mendes RP. 103, 127, 137, 141, 147, 149, 152, 155,156, 161, 162, 210Mendes-Giannin MJS. 60, 70, 174, 175, 176, 182,188Michelin MA. 144Milanezi CM. 193, 198Millington MA. 96Miramontes R. 217221

Molano VM. 143Molina RFS. 199Montoya C. 218Moraes DM. 150Moraes MPT. 152Morais FV. 173Moreira AP. 71, 193Moreira-Filho CA. 174Moreto TC. 155, 161, 210Moris DV. 103, 147, 155, 156, 161, 210Morris-Jones R. 62Motta-Castro ARC. 141Moura F. 210Muñoz C. 218Muñoz JE. 201, 208, 211, 215Myiamoto D. 146, 148, 151NNaranjo TW. 82, 190, 199, 205Negroni R. 73Neves FF. 144Niño-Vega G. 45, 169, 170, 172, 177, 206Nishikaku AS. 80, 199Nóbrega FG. 173Nogueira SV. 175Nosanchuk J. 62Nowill AE. 187Nunes ES. 181, 207, 209Nunes J. 193, 196, 200, 207OOdds F. 62OIiveira EXG. 143Oliveira AL. 141Oliveira CCU. 189Oliveira EB. 193Oliveira EF. 103Oliveira LL. 71, 193, 194, 198Oliveira MLSC. 155Oliveira RTD. 187Oliveria A. 166Oshiro TM. 187PPadilla-Desgarennes MC. 142Paes HC. 165, 171, 182, 183, 189Pagliari C. 84, 116, 190Panagio L. 71Paniago AMM. 141Panunto-Castelo A. 194Parente JA. 169, 178Passos AN. 155Passos EC. 187Passos GAS. 180Passos-Silva DG. 180Patarrollo ME. 130Patiño J. 82, 190Pavan D. 150Pedott F. 148, 150, 151, 157Pedroso EP. 156Pedroso SM. 173Pegorare AL. 141Peixoto DLG. 207Peraçoli MTS. 194, 195Pereira M. 173, 175, 176, 177, 178, 180Pereira NV. 84, 190Pereira RW. 189Pereira T. 148, 150, 151, 157Pereira M. 170Philippsen HK. 178Pina A. 196Pinto FA. 201Pinto FL. 207Pizzini CV. 114Pizzolatti MG. 210Polez VPP. 166Popi AF. 79Puccia R. 51, 92, 175, 201QQueiroz-Tellez F. 105, 129Quesada-Ocampo LM. 167Quiceno W. 211RRappleye CA. 165Rauscher JT. 167Ravanini JN. 145Raxcacoj G. 217Reis BS. 200Resende MA. 210Restrepo A. 62, 82, 89, 171, 181, 190, 199, 205, 211,212, 215Restrepo CA. 211Rezende RV. 180Rezende TCV. 114Rezende TCV. 176Ribeiro AA. 148, 151Ribeiro AM. 196, 200, 207222

Richart CAO. 169Richetti MA. 150Richini-Pereira V. 162Riley P. 217Rocha AA. 175Rocha FA. 71, 193, 198Rodrigues ACJ. 165Rodrigues F. 91, 165Rodrígues-Brito S. 206Romano CC. 188Roque-Barreira MC. 194Rosa CA. 208Rosa LH. 208Rossi DCP. 215Rossi M. 193Russo RC. 191SSá NP. 208Sadahiro A. 116, 187Saldanha CA. 207Salem-Izaac SM. 173, 179Salge JM. 152Salles BC. 200Salmito MA. 74Samayoa B. 101, 217Sampaio-Marques B. 91San-Blas G. 121, 169, 170, 172, 177, 182, 206Santana JM. 169, 170, 178Santana MS. 127, 162Santos MP. 179Santos RMM. 132Santos RS. 179Santos SC. 180Sato PK. 187Saul A. 142Scheel CM. 217Serrano A. 148Sharpton TJ. 43Shikanai-Yasuda MA. 116, 146, 147, 149, 151, 187,189Silva CL. 180, 200Silva JF. 174, 182Silva JR. 207Silva JS. 71, 193, 198Silva LFF. 145Silva MES. 137, 161Silva RP. 176Silva SS. 44, 166, 171Silva-Pereira I. 61, 209Silva-Vergara ML. 144Simioni AR. 207, 208Simoneide SS.178, 180Siqueira EP. 210Siqueira IM. 191, 196Soares AMVC. 68, 194, 195Soares CMA. 59, 114, 169, 170, 173, 175, 176, 177,178, 179, 180Soares MAS. 191Soliva Jr E. 148, 151Sorais F. 169, 172, 177Sotto MN. 84, 190Sousa MV. 169Souza ACO. 200Souza BS. 152Souza CR. 166Souza GND. 179Spago MC. 187Stajich J. 43Steensma HY. 91Suesada M. 152Suzin F. 157Tabares AM. 212Taborda CP. 51, 52, 201, 208, 211, 215Nóbrega YKM. 201Takagaki TY. 146Tavares AH. 44Tavares MG. 144Taylor JW. 43Tedesco AC. 207, 208Teixeira MM.136, 165, 168, 171, 182, 183, 189Teixeira SMR. 180Terçarioli GR. 127Theodoro RC. 138, 162, 167Titze de Almeida R. 207, 208Tobón AM. 117, 205, 211, 212Tomazett MV. 176Tomazett PK. 170Torres FA. 165Torres FAG. 166, 183Torres G. 216Torres I. 171Travassos CMR. 143Travassos LR. 51, 52, 201, 208, 211Trilles L . 74T223

Trinca LA. 167Tristão FSM. 71Urán ME. 62Urán ME. 67UVValente NYS. 147Valle ACF. 143Valpolini LC. 148, 151Vancim JO. 193Vargas J. 142Velasco Castrejón O. 142Veloso JMR. 156Vendruscolo PE. 194Viana DF. 165Vicentini-Moreira AP. 147, 155Vieira RG. 189Villa CA. 211Villalobos H. 172Viriyakosol S. 53Vitali LH. 206Walls L. 53Wanke B. 74, 141, 143Watanabe GA. 210Winters M. 175WYYañez A. 192Yoshida M. 116, 146, 147, 149, 151ZZambuzzi PF. 180Zancopé-Oliveira RM. 114Zani CL . 208, 210Zin WA. 191Zuluaga A. 215Zuluaga AF. 82, 190, 205224

Acknowledgment and GratitudeTo the following Institutions, Organizations and Enterprises that gave their support to theX International Congress on Paracoccidioidomycosis:Academic and Governmental Sponsors• Academia Colombiana de Ciencias Exactas, Físicas y Naturales• Alcaldía de Medellín – Secretaría de Salud Municipal• American Society for Microbiology (ASM)• Asociación Colombiana de Dermatología y Cirugía Dermatológica (ASOCOLDERMA)• Asociación Colombiana de Infectología, Capítulo Antioquia• Asociación Colombiana de Infectología, Capítulo Central• Asociación Colombiana de Parasitología y Medicina Tropical• Asociación Colombiana de Pneumología y Cirugía de Tórax, Capítulo Antioquia• Banco de la República - Fundación para la Promoción de la Investigación y la Tecnología• Biomédica, Revista del Instituto Nacional de Salud• Centers for Disease Control and Prevention, Department of Healt and Human Services (CDC)• Cooperación Técnica, Embajada de Brasil, Bogotá, D.C. – Colombia• Cooperativa Médica de Antioquia - COMEDAL• Empresas Públicas de Medellín – EPM• Federación Nacional de Cafeteros de Colombia• Gobernación de Antioquia – Dirección Seccional de Salud de Antioquia• Instituto Colombiano de Crédito y Estudios Técnicos en el Exterior – ICETEX• Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnología “Francisco José de Caldas”COLCIENCIAS• Instituto Nacional de Salud - INS• International Society for Human and Animal Mycology (ISHAM)• Medical Mycological Society of the Americas (MMSA)• Metrosalud• Metro de Medellín• Organización Panamericana de la Salud, Colombia• Pan American Health Organization/ World Health Organization ( PAHO/WHO)• Servicio Nacional de Aprendizaje - SENA• Springer Verlag, Mycopathologia• The Academy of Science for the Developing World (TWAS)• Universidad de Antioquia, Facultad de Medicina y Escuela de Microbiología• Universidad Pontificia Bolivariana, Facultad de Medicina225

Personal ContributorsDrs. Karl V. and Lynda ClemonsDrs. Steve and Maria CrokerDr. Juan Esteban Gutierrez C.Mrs. Catalina de Bedout G.Drs. Moisés and Maria Helena RobledoMembers of the Medical & Experimental Mycology and Molecular Biology Units,Corporación para Investigaciones Biológicas (CIB)Commercial SponsorsAM LimitadaARC AnálisisAviancaBioinstrumentalFiltración y AnálisisFundación SuramericanaInterconsulting S.A.Janssen-CilagMerck Sharp & DohmeMetroquirúrgicosPfizer S.A., IncSchering-Plough S.A.226

Notes