P. Del Porto

Multispecific T cell response and negative HCV RNA tests during acute HCV infection are early prognostic factors of spontaneous clearance

Background/Aims: Hepatitis C virus (HCV) infection results in a high frequency of chronic disease. The aim of this study was to identify early prognostic markers of disease resolution by performing a comprehensive analysis of viral and host factors during the natural course of acute HCV infection. Methods: The clinical course of acute hepatitis C was determined in 34 consecutive patients. Epidemiological and virological parameters, as well as cell mediated immunity (CMI) and distribution of human leukocyte antigens (HLA) alleles were analysed. Results: Ten out of 34 patients experienced self-limiting infection, with most resolving patients showing fast kinetics of viral clearance: at least one negative HCV RNA test during this phase predicted a favourable outcome. Among other clinical epidemiological parameters measured, the self-limiting course was significantly associated with higher median peak bilirubin levels at the onset of disease, and with the female sex, but only the latter parameter was independently associated after multivariate analysis. No significant differences between self-limiting or chronic course were observed for the distribution of DRB1 and DQB1 alleles. HCV specific T cell response was more frequently detected during acute HCV infection, than in patients with chronic HCV disease. A significantly broader T cell response was found in patients with self-limiting infection than in those with chronic evolving acute hepatitis C. Conclusion: The results suggest that host related factors, in particular sex and CMI, play a crucial role in the spontaneous clearance of this virus. Most importantly, a negative HCV RNA test and broad CMI within the first month after onset of the symptoms represent very efficacious predictors of viral clearance and could thus be used as criteria in selecting candidates for early antiviral treatment.

Early impairment of hepatitis C virus specific T cell proliferation during acute infection leads to failure of viral clearance

Background and aims: Cellular mediated immunity (CMI) is thought to play a key role in resolution of primary hepatitis C virus (HCV) infection. However, CD4+ and CD8+ T cell responses are also generated during acute infection in individuals who become chronic, suggesting that they developed a defective CMI. The aim of this study was to verify if and when such immune dysfunction is established by measuring the breadth, magnitude, function, and duration of CMI in a large cohort of subjects during the natural course of acute HCV infection. Methods: CMI was comprehensively studied by prospective sampling of 31 HCV acutely infected subjects enrolled at the onset of infection and followed for a median period of one year. Results: Our results indicated that while at the onset of acute HCV infection a measurable CMI with effector function was detected in the majority of subjects, after approximately six months less than 10% of chronically infected individuals displayed significant CMI compared with 70% of subjects who cleared the virus. We showed that progressive disappearance of HCV specific T cells from the peripheral blood of chronic patients was due to an impaired ability to proliferate that could be rescued in vitro by concomitant exposure to interleukin 2 and the antigen. Conclusion: Our data provide evidence of strong and multispecific T cell responses with a sustained ability to proliferate in response to antigen stimulation as reliable pharmacodynamic measures of a protective CMI during acute infection, and suggest that early impairment of proliferation may contribute to loss of T cell response and chronic HCV persistence.

Human T cells with a type-2 cytokine profile are resistant to apoptosis induced by primary activation: consequences for immunopathogenesis

The mechanisms leading to a relative dominance of T cells producing type 2 cytokines in certain human immune disorders are still unclear. We investigated the relative susceptibility to apoptosis induced by primary in vitro activation of human type 1 (producing interferon‐gamma (IFN‐γ)) or type 2 (producing IL‐4) T cells. Peripheral blood lymphocytes were isolated from patients with immune disorders characterized by expansion of type 2 cells (four with AIDS and hyper‐IgE/hypereosinophilia, one with Churg–Strauss syndrome, and one with idiopathic hypereosinophilic syndrome) or from individuals with normal cytokine balances. Cells were stimulated for 16 h with ionomycin and phorbol ester, and apoptosis of cytokine‐producing cells was assessed by flow cytometry. T cells with a type‐2 cytokine profile, i.e. producing IL‐4 alone, were significantly more resistant to activation‐induced apoptosis than those producing IFN‐γ alone. This was observed in AIDS patients, whose type 2 cells were mostly CD8+, as well as in the patients with Churg–Strauss and with hypereosinophilic syndrome. CD4+ and CD8+ IL‐4‐producing cells were equally resistant to apoptosis. Lower susceptibility to apoptosis of type‐2 T cells was also observed in subjects with normal cytokine balances. Bcl‐2 expression was high in type‐2 cells and in viable type‐1 cells, whereas it was low in apoptotic type‐1 cells. Resistance to activation‐induced apoptosis may explain the expansion of cells producing type‐2 cytokines in certain immune disorders.

142 Development of an S/MAR based episomal vector for the CFTR gene delivery

Objectives The recent use of mutation-specific correctors in Cystic Fibrosis, reinforces the concept of gene therapy as a therapeutic treatment for all CF patients. Non-viral vectors are preferable to viral ones, particularly in treatment of chronic diseases, for their simpler composition, lack of immunogenicity and genotoxicity. Episomal vectors based on the S/MAR (scaffold/matrix attachment region) sequence have self-replicating capability and episomal persistence in different cell types, both in vitro and in vivo. To obtain long-term and episomal maintenance of the CFTR gene in target cells, we constructed a vector containing the S/MAR, GFP and the CFTR cDNA (pBQ-S/MAR). Methods pBQ-S/MAR was transfected in CF(DF508/DF508) airway epithelial cells and the cells were grown in non-selective conditions up to 14 days after transfection. The cells were evaluated for the episome maintenance and persistence as well as for CFTR gene expression (RT-PCR, immunofluorescence, western blot). Results The resulting data demonstrate that pBQ-S/MAR is maintained as an episome, with unchanged structure respect to the input vector, for at least 14 days after transfection, corresponding to about six cell duplications. CFTR expression analyses confirmed the presence of the wt transcript and the mature form of the protein. Additionally, the expression of a functional CFTR in pBQ-S/MAR-transfected cells was associated with an increase of Trans Epithelial Resistance respect to parental cells. Conclusion Our results suggest that the pBQ-S/MAR is stably maintained as an episome in airway epithelial cells and support expression of a functional CFTR.

56 Pseudomonas aeruginosa end-stage isolates: analysis of virulence traits and interaction with macrophages

56 Pseudomonas aeruginosa end-stage isolates: analysis of virulence traits and interaction with macrophages N. Cifani1,2, B. Pompili1, V. Ferri1, P. Fradiani3, E.G. Di Domenico1, F. Venuta4, M. Anile4, P. Del Porto1, F. Ascenzioni1. 1Sapienza University of Rome, Department of Biology and Biotechnology ‘C. Darwin’, Rome, Italy; 2Sapienza University of Rome, Department of Clinical and Molecular Medicine, Rome, Italy; 3Sapienza University of Rome, U.O.D. Microbiology, Sant’Andrea Hospital, Rome, Italy; 4Sapienza University of Rome, Department of Thoracic Surgery, Rome, Italy

109 Increased IL-8 production in human CFTR-deficient macrophages

108 Reduced effect of intravenous antibiotic treatment on sinonasal compared to pulmonary inflammatory markers J.G. Mainz1, J. Hentschel1, N. Fischer1, U.R. Markert2, K. Boer3, W. Pfister4, F. Doht1. 1Jena University Hospital, CF-Centre, Jena, Germany; 2Jena University Hospital, Department of Obstetrics, Placenta Laboratory, Jena, Germany; 3Jena University Hospital, Institute for Clinical Chemistry and Laboratory Diagnostics, Jena, Germany; 4Jena University Hospital, Institute of Medical Microbiology, Jena, Germany