European Journal of Medical Research - Deutsche AIDS ...

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June 27, 2007 EUROPEAN JOURNAL OF MEDICAL RESEARCH 117 Results: In several rounds of bio-panning we selected the highest-affinity SH3-binders of Sam68 from the SH3-proteome-pool. These were in part members of the Src-family, consistent with earlier observations by several groups. 2 of the top-three-binders were identified as Sam68-binders for the first time. The specificity of the interactions was confirmed by pull-down assays with recombinant proteins and the binding-affinities were determined by a phage-ELISA. Conclusions: Using phage-display we could identify new high-affinity SH3-binders of Sam68 and confirm known ones, showing the general usability of the library as a tool for identifying SH3-interactions. Further detailed characterisation of these interactions will allow us to assess their importance for the role of Sam68 in the export of late viral transcripts, being the basis for design of novel therapeutic vaccines directed against cellular structures. E.9 (Poster) Is the transmembrane envelope protein gp41 involved in HIV induced immunopathogenesis? Results of cytokine arrays and microarrays Denner J. 1 , Behrendt R. 1 , Lau M. 1 , Schmidt C.-M. 1 , Kurth R. 1 1 Robert Koch Institut, Berlin, Germany Objective: Although the human immunodeficiency viruses (HIV) have long been known to be the cause of AIDS, the precise mechanism of disease induction by these viruses is still unclear. Other retroviruses such as the feline leukemia virus (FeLV) and the Koala retrovirus (KoRV) also induce immunodeficiencies in infected animals, which are clinically similar to AIDS. Reports showing inhibition of mitogen-induced lymphocyte proliferation by inactivated retroviral particles including HIV suggest that a viral protein may be involved in the retroviral pathogenesis. Methods: To study the influence of the viral transmembrane envelope (TM) protein on cytokine production by normal human lymphocytes, PBMCs from healthy blood donors were incubated with recombinant TM proteins of different retroviruses including HIV. In addition, synthetic peptides corresponding to a highly conserved domain in the TM proteins and designated immunosuppressive (isu-) peptides were also used. Cytokine release was measured using different methods, in addition a microarray analysis (Human Genome Survey Microarray V2.0, 29098 genes) was performed. Results: The expression of cytokines such as IL-10, IL-6, IL- 8, RANTES, MCP-1, MCP-2, TNF-alpha, MIP-1alpha, MIP- 1beta, MIP-3 increased upon exposure to the TM proteins and the corresponding isu-peptides. In contrast, the expression of IL-2 decreased and the expression of about 90 other cytokines measured remained unchanged. The extent of changes in the cytokine expression varied from donor to donor. The cytokine data were confirmed by a microarray analysis. Conclusion: These data indicate that retroviral TM proteins modulate the cytokine production of normal PBMCs and therefore may play an significant role in retrovirus-induced immunopathogenesis. It is important to note that elevated IL- 6, IL-10, IL-8, gro-alpha and TNF-alpha and decreased IL-2 values have been regularly observed in HIV-1 infected individuals. E.10 (Poster) Isolation of RNA aptamers directed against HIV-1 envelope-derived surface epitopes Müller I. 1 , Bunka D. 2 , Schult-Dietrich P. 1 , Brauer F. 1 , Adler A. 3 , Katinger H. 4 , Stockley P. 2 , von Laer D. 1 1 Georg-Speyer-Haus, Frankfurt am Main, Germany, 2 Astbury Centre for Structural Molecular Biology, Leeds, United Kingdom, 3 Darmstadt University of Technology, Darmstadt, Germany, 4 Institute for Applied Microbiology, Vienna, Austria Aim: As the limitations of anti-HIV drug therapy become evident, alternative therapeutic strategies are gaining interest. Peptides derived from the heptad repeats of the HIV-1 gp41 envelope glycoprotein have shown a strong potential to inhibit HIV-1 entry. To overcome the several disadvantages excluding a broad application, we have isolated RNA molecules, which were found to interact with the HIV-1 gp4, thus blocking membrane fusion. As another possibility RNA aptamers were selected against HIV-1 gp120. Method: The isolation of RNA aptamers was performed using the SELEX technology. Several different HIV-1 envelope peptides served as targets in manual or robot-driven selections. The starting DNA libraries contained a 30(50)-nucleotide random region flanked by defined primer binding sites. To ensure the selection of nuclease-resistant RNA aptamers, 2’-NH2 or -F-modified pyrimidines were included during all in vitro transcription reactions. Results: 10 rounds of selection were performed, followed by an affinity maturation step using neutralizing antibodies for binding competition. The most potent anti-gp41 RNA pool, 435UU, was able to bind to its target with a Kd » 750 nM. Moreover, the 435UU RNA pool demonstrated specific HIV neutralization with IC50 values approx. 250 nM. The selection of anti-gp120 aptamers using HIV pseudoviruses coupled to paramagnetic beads revealed an individual aptamer with an IC50 of about 500 nM. Analysis of individual sequences revealed for all selections still heterogenous pools. Furthermore, secondary structure prediction using the MFold algorithm displayed only a few defined structural motifs. Conclusion: In order to increase the selection stringency, new selection and maturation strategies are currently being established. E.11 (Poster) Investigation of HOCl-modified proteins which exhibit a high activity towards HIV-1 gp120 in vitro by inhibition of virus attachment van Yperen M. 1 , Schreiber M. 1 1 Bernhard-Nocht-Institut for tropical medicine (BNI), Virologie, Hamburg, Germany The high-affinity interaction of the HIV-1 envelope glycoprotein (gp120-gp41) and its receptor CD4 is important for viral entry into host cells. We have generated a novel viral envelope binding protein based on hypochlorite-modified HSA, BSA, MSA and ß-Gal which exhibit an antiviral activity directed towards NL4-3wt. Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes modifications in several amino acids. Antiviral tests were performed by X-Gal staining and counting of infected cells. The compounds revealed an inhibitory potential of rel. IC50 > 5 g/ml mHSA, > 3 g/ml mBSA, > 20 g/ml mMSA, and > 14 g/ml
118 EUROPEAN JOURNAL OF MEDICAL RESEARCH June 27, 2007 mß-Gal. Following this functionality assay, the cytotoxity was addressed by MTT proliferation assays. Modified proteins did not show any cytotoxic activity up to 200 g/ml. To determine whether the modified serum albumins attach to gp120 we used surface plasmon resonance (SPR) technology. Biotinylated mHSA binds to X4 monomeric glycoprotein 120 with a high binding affinity (Kd = 2,74 x 10-10 M). Furthermore, a biochemical characterization of hypochlorite-modified proteins has been started to define the binding features. The content of sulfhydryl groups was examined by colorimetric assays. The results show that the sulfhydryl content is decreased by 100% at a HOCl/Protein-Ratio molar ratio of 1:0,5. Together our findings make modified proteins interesting candidates for a novel antiviral therapy against HIV. E.12 (Poster) Differentiation of recent and chronic HIV-infections from filter dried-plasma-spots Loschen S. 1 , Bätzing-Feigenbaum J. 1 , Gohlke-Micknis S. 1 , Poggensee G. 1 , Jansen A. 1 , Hamouda O. 1 , Kuecherer C. 1 1 Robert Koch-Institute, Berlin, Germany Objective: HIV-1 incidence data in Germany are limited because only newly diagnosed infections of unknown duration are registered. The increase of HIV-1 specific antibody levels in the first year after infection is used by the commercial BED-CEIA to differentiate recent and chronic infections. The aim of this study was to compare the sensitivity and specificity of the BED-CEIA for the analysis of plasma samples and filter dried samples. Handling and transport of filter-dried samples is easier as compared to fresh blood and could facilitate HIV-incidence studies. Methods: The BED-CEIA was validated using a reference panel of 148 baseline and follow-up plasma samples from 81 HIV seroconverters with a documented date of seroconversion. The panel covers a time window from 0 to 222 weeks after seroconversion. Plasma (30 l) was dropped on filter disks (Whatman #903), dried for >12 h at room temperature (DPS) and eluted with PBS; 2% FCS; 0.05% TWEEN 20 (450 l). The optimal sensitivity and specificity of the BED-CEIA to differentiate truly incident and prevalent samples was calculated by varying the window period for recent infections. Results: Sensitivity and specificity (80% and 86%) of the BED-CEIA was optimal if samples were considered as incident until 20 weeks after seroconversion. 13 of 65 true-incident samples were classified as false-prevalent and 12 of 83 true-prevalent samples as false-incident. Applying the same cut-off, testing of DPS reached a sensitivity of 88% and specificity of 74% (n=8 false-prevalent, n=22 false-incident). By increasing the time window of incidence samples from 20 to 27 weeks after seroconversion the specificity was improved to 78% as the expense of the sensitivity (84%). Under these conditions 12 of 75 true incident samples tested false-prevalent and 16 of 73 true-prevalent samples false-incident. The difference between fresh and dried samples was not significant (pSE=0.346; pSP=0.158). Conclusion: Filter dried plasma samples can be used for the detection of incident infections in epidemiological studies. The results are comparable to testing plasma directly. An increase of the time span for incident infections to 27 weeks for DPS seems preferable because a lower proportion of samples was classified false-incident. E.13 (Poster) “Under-quantification” of HIV-1 RNA by the Cobas TaqMan HIV-1 assay may lead to delayed recognition of resistance development Weissbrich B. 1 , Winzer R. 2 , Guhl C. 2 , Schubert J. 1 , Langmann P. 2 , Heinz W. 2 , Klinker H. 2 1 Universität Würzburg, Institut für Virologie und Immunbiologie, Würzburg, Germany, 2 Universitätsklinik Würzburg, Medizinische Klinik II, Würzburg, Germany Objectives: The Cobas TaqMan HIV-1 Test (CTM HIV-1; Roche Diagnostics, Mannheim, Germany) is an assay for the quantification of HIV-1 RNA. It is intended for the use in the clinical management of HIV-1 infected patients. We report on a patient in whom a significant subquantification of HIV-1 RNA was observed when using the CTM HIV-1 assay. Methods: The CTM HIV-1 assay and the ultrasensitive version of the Cobas Amplicor HIV-1 Monitor v1.5 assay (CA HIV-1; Roche Diagnostics) were used for viral load testing. Resistance testing was performed with the ViroSeq HIV-1 genotyping system (Abbott, Wiesbaden, Germany). Results: A 54-year-old male patient with HIV-1 infection diagnosed in 1987 was successfully treated with an antiretroviral combination therapy (ART) consisting of protease inhibitors and two nucleoside reverse transcriptase inhibitors (NRTI) since 1997. HIV-1 RNA was below the detection limit (
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