Marco Artini

Long-lasting memory T cell responses following self-limited acute hepatitis B.

The molecular and cellular basis of long-term T cell memory against viral antigens is still largely undefined. To characterize anti-viral protection by memory T cells against non-cytopathic viruses able to cause acute self-limited and chronic infections, such as the hepatitis B virus (HBV), we studied HLA class II restricted responses against HBV structural antigens in 17 patients with acute hepatitis B, during the acute stage of infection and 2.2 to 13 yr after clinical resolution of disease. Results indicate that: (a) significant T cell proliferative responses to HBV nucleocapsid antigens were detectable in all patients during the acute phase of infection and in 14/17 also 2-13 yr after clinical resolution of disease; b) long-lasting T cell responses were sustained by CD45RO+T cells, predominantly expressing the phenotype of recently activated cells; c) limiting dilution analysis showed that in some patients the frequency of HBV-specific T cells was comparable to that observed in the acute stage of infection and, usually, higher than in patients with chronic HBV infection; d) the same amino acid sequences were recognized by T cells in the acute and recovery phases of infection; and e) HBV-DNA was detectable by nested-PCR in approximately half of the subjects. to conclusion, our results show that vigorous anti-viral T cell responses are detectable in vitro several years after clinical recovery from acute hepatitis B. Detection of minute amounts of virus in some recovered subjects suggests that long-term maintenance of an active anti-viral T cell response could be important not only for protection against reinfection but also for keeping the persisting virus under tight control.

Immunotherapy with interferon-alpha in patients affected by chronic hepatitis C induces an intercorrelated stimulation of the cytokine network and an increase in depressive and anxiety symptoms.

Immunotherapy with interferon-alpha (IFNalpha) may induce depressive symptoms, anxiety and major depression when administered for at least 1-3 months at a dose of 3-10 MUI daily, twice or three times a week. Previously, it has been shown that immunotherapy with interleukin-2 (IL-2) significantly induces the cytokine network, as measured by increases in serum IL-6, IL-10 and the IL-2 receptor (IL-2R), and that the immunotherapy-induced changes in the cytokine network are significantly correlated with the increases in depression ratings. The main aim of this study was to examine the effects of immunotherapy with IFNalpha on the cytokine network in relation to changes in depression and anxiety ratings. Fourteen patients, affected by chronic active C-hepatitis, were treated with IFNalpha (3-6 MUI s.c. three/six times a week for 6 months) and had measurements of serum IFN-gamma (IFNgamma), IL-2, IL-6, IL-6R, IL-8 and IL-10 before starting therapy and 2, 4, 16 and 24 weeks after immunotherapy with IFNalpha. Severity of depression and anxiety were measured with the Montgomery-Asberg Depression Rating Scale (MADRS) and the Hamilton Anxiety Rating Scale (HAM-A), respectively. Repeated measure (RM) design ANOVAs showed significantly higher MADRS and HAM-A scores 2-4 weeks and 4-6 months after starting IFNalpha-based immunotherapy than at baseline. RM design ANOVAs showed significantly higher serum IL-6 and IL-8 levels 2-4 weeks after starting IFNalpha-based immunotherapy and higher serum IL-10 levels 2-4 weeks and 4-6 months after starting therapy than at baseline. There were significant relationships between the IFNalpha-induced changes in serum IL-6 or IL-8 and the depression and anxiety scores. The findings show that IFNalpha-based immunotherapy induces the cytokine network and that IFNalpha-induced increases in IL-6 predicts the development of depressive symptoms. Depressive symptoms following IFNalpha treatment may be secondary to cytokine induction, including that of IL-6.

Induction of the DNA-binding activity of c-jun/c-fos heterodimers by the hepatitis B virus transactivator pX.

The hepatitis B virus (HBV) X protein (pX) is capable of activating transcription regulated by viral and cellular promoters containing binding sites for different transcription factors, including AP1. In this study we have analyzed the mechanisms of AP1 induction by pX. The hepatitis B virus transactivator was able to activate TRE (12-O-tetradecanoylphorbol-13-acetate response element)-directed transcription in different cell lines, including HepG2, HeLa, CV1, and PLC/PRF/5 cells. pX-induced AP1 activation in HepG2 cells was associated with an increase in the DNA-binding activity of c-Jun/c-Fos heterodimers, which was not dependent either on an increase in the overall amount of c-Fos and c-Jun proteins in the cells or on formation of dimers between pX and the two proteins, thus suggesting the involvement of posttranslational modifications of the transcription factor. The observation that the overexpression of c-Jun and c-Fos in the cells results in a strong augmentation of the effect of pX on TRE-directed transcription is additional evidence indicating the involvement of posttranscriptional modifications of c-Jun/c-Fos heterodimers. The increased AP1 binding observed in the presence of pX was unaffected by the protein kinase C inhibitors calphostin C and sphingosine and by the protein kinase A inhibitor HA1004, while it was almost completely blocked by staurosporine, a potent and nonspecific protein kinase inhibitor, suggesting that protein kinase C- and A-independent phosphorylation events might play a role in the phenomenon. The ability of pX also to increase TRE-directed transcription in cell lines in which AP1-binding activity is not increased (i.e., HeLa, CV1, and PLC/PRF/5 cells) suggests that pX can activate canonical TRE sites by different mechanisms as well.

Treatment with interferon-alpha (IFNα) of hepatitis C patients induces lower serum dipeptidyl peptidase IV activity, which is related to IFNα-induced depressive and anxiety symptoms and immune activation

We have shown that treatment with interleukin-2 (IL-2) or interferon-α (IFNα) may induce depressive symptoms and activation of the cytokine network and that IL-2 treatment may diminish serum dipeptidyl pepdidase IV (DPP IV) activity.1–3 DPP IV (EC 3.4.14.5) is a membrane bound serine protease which catalyzes the cleavage of some cytokines and neuroactive peptides which modulate T cell activity.4 The aims of the present study were to examine the effects of IFNα-based immunotherapy on serum DPP IV activity in relation to induction of the inflammatory response system. In 18 patients with chronic active hepatitis C, we determined the Montgomery and Asberg Rating Scale (MADRS),5 the Hamilton Anxiety Rating Scale (HAM-A),6 serum DPP IV activity, the kynurenine/tryptophan (K/T) quotient, which is an indicator of cytokine (in particular IFN)-induced catabolism of tryptophan,7 and serum interleukin-8 (IL-8) before starting therapy and 2, 4, 16 and 24 weeks after immunotherapy with IFNα. IFNα-immunotherapy significantly suppressed serum DPP IV 2–4 weeks and 16–24 weeks after starting IFNα-based immunotherapy. The reduction in serum DPP IV activity was more pronounced 16–24 weeks after starting immunotherapy than after 2–4 weeks. The IFNα-induced suppression of serum DPP IV activity was significantly correlated to IFNα-induced increases in the MADRS and HAM-A and increases in the K/T quotient and serum IL-8. In conclusion, long-term immunotherapy with IFNα suppresses serum DPP IV activity and the immunotherapy-induced changes in DPP IV are related to increases in severity of depression, anxiety and activation of the inflammatory response system.

Elevated serum levels of 90K/MAC-2 BP predict unresponsiveness to α-interferon therapy in chronic HCV hepatitis patients

BACKGROUND The clinical outcome of hepatitis virus infections is though to depend on the complex interplay between the host immune response profile and virus factors. 90K/MAC-2 BP is a novel member of the Scavenger Receptor Cysteine Rich protein superfamily that functions as a molecular alarm signal for the cellular immune system against both cancer cells and virus infections. METHODS To assess the significance and the potential clinical usefulness of testing for serum levels of 90K/MAC-2 BP in chronic viral hepatitis patients we studied 115 consecutive patients with chronic HCV hepatitis, 28 HBsAg chronic hepatitis patients, 12 asymptomatic HCV carriers and 11 asymptomatic HBV carriers. 103 out of the 115 HCV patients have been treated with recombinant alpha 2a-interferon at the dose of 3 Mega Units (MU) t.i.w. for 6 months followed by 1.5 MU t.i.w. for 6 months, and have been followed up for a further 12 months. Serum levels of 90K/MAC-2 BP were measured by an immunoradiometric assay based on the specific SP-2 monoclonal antibody. RESULTS AND CONCLUSIONS Serum 90K/MAC-2 BP levels are increased in chronic viral hepatitis patients, being significantly higher in HCV than in HBV patients. In chronic HCV hepatitis, serum 90K/MAC-2 BP levels are related to both the degree of disease severity and duration of infection. Moreover, elevated 90K/MAC-2 BP serum levels are an independent predictor of failure to respond to alpha-interferon treatment in a cohort of community-acquired chronic hepatitis C patients.

Distribution of three major hepatitis C virus genotypes in Italy. A multicentre study of 49 5 patients with chronic hepatitis C

SUMMARY. Different genotypes of hepatitis C virus (HCV) have been shown to have distinct geographical distribution and to associate with variable clinical features. To evaluate their role in chronic hepatitis in Italian patients, we studied 495 consecutive cases with chronic hepatitis C seen in nine sentinel centres homo geneously distributed over Italy. HCV genotyping was carried out using a dot‐blot hybridization assay with genotype‐specific probes. Four hundred and eleven patients were viraemic and could be evaluated: 57% were found to be infected with HCV‐1,31% with HCV‐2, 8% with HCV‐3. 1% showed mixed infection and 3% were ascribed to HCV‐2b or HCV‐4 by direct sequencing. Geographical distribution showed discrete territorial variations. A history of drug addiction was commoner in patients infected with HCV‐3. There were no signifi cant differences in activity of liver disease among different HCV genotypes but the response to interferon therapy was reduced in patients infected with HCV‐1 compared to HCV‐2 or HCV‐3.

Protease treatment affects both invasion ability and biofilm formation in Listeria monocytogenes

Listeria monocytogenes is a notably invasive bacterium associated with life-threatening food-borne disease in humans. Several surface proteins have been shown to be essential in the adhesion of L. monocytogenes, and in the subsequent invasion of phagocytes. Because the control of the invasion of host cells by Listeria could potentially hinder its spread in the infected host, we have examined the effects of a protease treatment on the ability of L. monocytogenes to form biofilms and to invade tissues. We have chosen serratiopeptidase (SPEP), an extracellular metalloprotease produced by Serratia marcescens that is already widely used as an anti-inflammatory agent, and has been shown to modulate adhesin expression and to induce antibiotic sensitivity in other bacteria. Treatment of L. monocytogenes with sublethal concentrations of SPEP reduced their ability to form biofilms and to invade host cells. Zymograms of the treated cells revealed that Ami4b autolysin, internalinB, and ActA were sharply reduced. These cell-surface proteins are known to function as ligands in the interaction between these bacteria and their host cells, and our data suggest that treatment with this natural enzyme may provide a useful tool in the prevention of the initial adhesion of L. monocytogenes to the human gut.

Long-term response to interferon alpha is unrelated to“interferon sensitivity determining region” variability in patients with chronic hepatitis C virus-1b infection

BACKGROUND/AIMS Contradictory data have been reported about the predictive value of the variability in interferon sensitivity determining region (ISDR) of hepatitis C virus (HCV) genotype-1b on response to interferon-alpha (IFN-alpha) therapy. The aim of this study was to examine this issue in a series of patients with long-term response to IFN treatment. METHODS We retrospectively analyzed 24 patients with chronic HCV genotype-1b infection treated with IFN-alpha (total dose median 677, range 216-1350 MU) selected in 6 Italian Liver Units. These patients were defined as true long-term responders (LTR) since they showed persisting biochemical and virological responses to IFN treatment (mean follow-up 38 months). HCV genomes from pretreatment serum samples were amplified and directly sequenced. The ISDR amino-acid sequences obtained were aligned and compared with the published sequence of HCV-J. RESULTS Amino-acid substitutions were found in 23 of the 24 patients, and 22 of them showed an H to R amino-acid change at codon 2218. Fourteen patients showed only one mutation (at codon 2218), two had 2, five had 3, one had 4 and one had 5 mutations. When we compared the ISDR sequences from the 24 LTR with those of non-responders (NR), we found no significant correlation between the number of mutations and the response to therapy. CONCLUSIONS Our results demonstrate that the persisting efficacy of IFN treatment in patients with chronic HCV is not related to the number of ISDR amino acid substitutions of the infecting viruses. Further studies are needed to verify whether other NS5A sequences outside the ISDR might be involved in the mechanisms of IFN resistance.

Primary effusion Burkitt's lymphoma with t(8;22) in a patient with hepatitis C virus related cirrhosis

Hepatitis C virus (HCV) infection may be complicated by non-Hodgkins lymphoma through yet unknown pathogenetic mechanisms. We describe the case of a patient with HCV-related cirrhosis who developed a primary effusion lymphoma (PEL) of Burkitts type confined to the peritoneal cavity, in the absence of immunodeficiency or autoimmunity. Paracentesis followed by immunophenotyping, karyotyping, and molecular studies allowed us to diagnose a small noncleaved B-cell lymphoma (CD20+, CD24+, CD10+, CD5-, CD23-, lambda+) with the t(8;22) (q24;q11) translocation and clonal rearrangement of the immunoglobulin heavy chain gene. HCV-RNA, Epstein-Barr virus and Kaposis sarcoma-associated herpesvirus were not identified within lymphoma cells. The finding of HCV-RNA in the ascitic fluid suggests a link between HCV and development of lymphoma with HCV playing the role of persistent antigenic stimulation to intraperitoneal B-cell clonal expansion(s).

Phosphorylcholine Impairs Susceptibility to Biofilm Formation of Hydrogel Contact Lenses

PURPOSE To compare silicone-hydrogel, poly(2-hydroxyethyl methacrylate) (pHEMA), and phosphorylcholine-coated (PC-C) contact lenses in terms of their susceptibility to biofilm formation by Staphylococcus epidermidis and Pseudomonas aeruginosa. DESIGN Laboratory investigation. METHODS Biofilm formation on colonized test lenses was evaluated with confocal microscopy and in vitro antibiotic susceptibility assays. The results of the latter assays were compared with those performed on planktonic cultures of the same organism. RESULTS For both microorganisms, sessile colonies on silicone-hydrogel and pHEMA lenses displayed lower antibiotic susceptibility than their planktonic counterparts. In contrast, the susceptibility of cultures growing on PC-C lenses was comparable with that for planktonic cultures. In particular, minimum inhibitory concentration for Tazocin (piperacillin plus tazobactam; Wyeth Pharmaceuticals, Aprilia, Italy; S. epidermidis) and gentamicin (P. aeruginosa) was identical, either in the presence of PC-C support or in planktonic cultures (Tazocin, </= 0.2 mug/ml; gentamicin, 0.4 mug/ml). Minimum inhibitory concentration for imipenem (P. aeruginosa) was two-fold higher for PC-C lenses (0.4 mug/ml) with respect to planktonic cultures (0.2 mug/ml). Confocal microscopy of lenses colonized for 24 hours with P. aeruginosa green fluorescent protein-expressing cells revealed a sessile colonization on silicone-hydrogel lens and a few isolated bacterial cells scattered widely over the surface of the PC-C lens. CONCLUSIONS An increase in antibiotic susceptibility of bacterial cultures was associated with diminished bacterial adhesion. Our results indicate that PC-C lenses seem to be more resistant than silicone-hydrogel and pHEMA lenses to bacterial adhesion and colonization. This feature may facilitate their disinfection.