Memoria CD.indd - ISHAM

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