Retreatment regimen of rituximab monotherapy given at the relapse of severe HCV-related cryoglobulinemic vasculitis: Long-term follow up data of a randomized controlled multicentre study.

OBJECTIVE: To evaluate the efficacy and safety in the long term of a retreatment regimen with Rituximab (RTX) alone administered at clinical relapse in cryoglobulinemic vasculitis (CV). METHODS: Thirty patients with severe HCV-related CV, previously enrolled in the multicentre Italian trial on RTX in the treatment of CV, were retrospectively evaluated after the end of the trial. All of them were managed with RTX alone at clinical relapse, if any. Disease activity at the last available follow up was defined as complete remission (absence of active disease), partial remission (response > 50% of at least one manifestation among glomerulonephritis, peripheral neuropathy or skin ulcers) or active disease. RESULTS: The mean follow up after the first RTX cycle was 72.6 (20.4) months. After the end of the trial, 21/30 (70%) patients showed an active follow up [81.7 (10.9) months)], 3/30 (10%) lost follow up and 6/30 (20%) died. 12/21 (57.1%) patients were in complete disease remission, 5/21 (23.8%) showed a partial response and 4/21 (19%) had an active disease. 17/30 (56.7%) patients needed retreatment for relapse with a mean time to retreatment of 22.3 (12.1) months. Treatment survival of this regimen was 7.6 (0.3) years. Recurrent non-severe infections occurred in 3/30, with chronic hypogammaglobulinemia in 2/3 patients. CONCLUSIONS: A long-term regimen of retreatment with RTX alone given at clinical relapse seems to be effective and safe in CV, with a low rate of infections and severe hypogammaglobulinemia.

CD4+/CD25+ T cells suppress autologous CD4+/CD25- lymphocytes and secrete granzyme B during acute and chronic hepatitis C.

We aimed to verify whether CD4(+)/CD25(+) T cells suppress CD4(+) T cells and secrete Granzyme B (GZB) during acute and chronic hepatitis C (CHC) infection. We enrolled 50 subjects: 20 patients with CHC (Group A), 15 healthy individuals (Group B), 10 patients with acute hepatitis C later evolved to persistent infection (Group C) and five patients who resolved hepatitis C virus infection during acute phase (Group D). We analysed, on enrolled subjects CD4(+)/CD25(+) T cells and related GZB production as well as Annexin V activity. Patients from Groups A and C had higher frequency and function of peripheral Treg cells than healthy individuals. Groups A and C showed an increase in spot-forming colonies (SFCs) of GZB compared with Group B (P < 0.01, Mann-Whitney U-test). CD4(+)/CD25(+) T cells in Group D had a lower number of GZB SFCs compared with Groups A and C but higher number than Group B (P < 0.01 Mann-Whitney U-test). Annexin V production was higher in Groups A and C than B or D. Patients having acute and chronic hepatitis C have a higher Treg frequency and function in peripheral blood than healthy controls or those resolving the infection in acute phase secreting GZB, probably inducing apoptosis.

Binding of hepatitis A virus to its cellular receptor 1 inhibits T-regulatory cell functions in humans.

BACKGROUND & AIMS: CD4+ T-regulatory (Treg) cells suppress immune responses and control self-tolerance and immunity to pathogens, cancer, and alloantigens. Most pathogens activate Treg cells to minimize immune-mediated tissue damage and prevent clearance, which promotes chronic infections. However, hepatitis A virus (HAV) temporarily inhibits Treg-cell functions. We investigated whether the interaction of HAV with its cellular receptor 1 (HAVCR1), a T-cell co-stimulatory molecule, inhibits the function of Treg cells to control HAV infection. METHODS: We studied the effects of HAV interaction with HAVCR1 on human T cells using binding, signal transduction, apoptosis, activation, suppression, cytokine production, and confocal microscopy analyses. Cytokines were analyzed in sera from 14 patients with HAV infection using bead arrays. RESULTS: Human Treg cells constitutively express HAVCR1. Binding of HAV to HAVCR1 blocked phosphorylation of Akt, prevented activation of the T-cell receptor, and inhibited function of Treg cells. At the peak viremia, patients with acute HAV infection had no Treg-cell suppression function, produced low levels of transforming growth factor-ß , which limited leukocyte recruitment and survival, and produced high levels of interleukin-22, which prevented liver damage. CONCLUSIONS: Interaction between HAV and its receptor HAVCR1 inhibits Treg-cell function, resulting in an immune imbalance that allows viral expansion with limited hepatocellular damage during early stages of infection-a characteristic of HAV pathogenesis. The mechanism by which HAV is cleared in the absence of Treg-cell function could be used as a model to develop anticancer therapies, modulate autoimmune and allergic responses, and prevent transplant rejection.

Preservation of nutritional-status in patients with refractory ascites due to hepatic cirrhosis who are undergoing repeated paracentesis.

BACKGROUND AND AIM: Refractory ascites in liver-cirrhosis is associated with a poor prognosis. We performed a prospective study to investigate whether aggressive nutritional-support could improve outcomes in cirrhotic patients. METHODS: Cirrhotic patients undergoing serial large-volume paracentesis for refractory-ascites were enrolled and randomized into three groups. Group A received post-paracentesis intravenous nutritional-support in addition to a balanced oral diet and a late-evening protein snack, group B received the same oral nutritional-protocol as the first group but without parenteral support, and group C (the control group) received a low-sodium or sodium-free diet. Clinical, anthropometric and laboratory nutritional parameters and biochemical tests of liver and renal function were reported for 12 months of follow-up. RESULTS: We enrolled 120 patients, who were randomized into three groups of equal size. Patients on the nutritional-protocol showed better preservation of clinical, anthropometric and laboratory nutritional parameters that were associated with decreased deterioration of liver function compared with patients on the low-sodium or sodium-free diet (group C). Groups A and B had lower morbidity and mortality rates than the control group (C). Mortality rates were significantly better in patients who were treated with parenteral-nutritional-support than for the other two groups. In patients who were on the nutritional-protocol, there was a reduction in the requirement of taps for the treatment of refractory ascites. CONCLUSIONS: Post-paracentesis parenteral-nutritional-support with a balanced oral diet and an evening protein snack appears to be the best care protocol for patients with liver-cirrhosis that has been complicated by refractory-ascites.

Oxidative stress and steatosis are cofactors of liver injury in primary biliary cirrhosis.

BACKGROUND: Factors responsible for the progression of primary biliary cirrhosis (PBC) are still poorly understood. In the present study, we investigated the associations between the stage of PBC and the immune reaction triggered by oxidative stress; the presence of obesity, steatosis,steatohepatitis; and other toxic, metabolic, or steatogenic factors. METHODS: We studied clinical, laboratory, and histological data for 274 untreated patients with serum antimitochondrial antibody (AMA)-positive PBC. Circulating IgG against human serum albumin adducted with malondialdeyde, the major product of lipid peroxidation, was measured in these patients and in a group of 98 sex-, age and body mass index (BMI)-matched controls. RESULTS: Steatosis was present in 40.5% of all patients. Steatohepatitis was present in 14.9% of all patients. There was a significant association between the frequencies of steatosis and steatohepatitis and the worsening of PBC. Circulating IgG against lipid peroxidation products was significantly higher in the PBC patients than in the controls. Titers of lipid peroxidation-related antibodies were significantly increased in patients with steatosis and inpatients at more advanced stages. Bivariate analysis revealed a significant association between indirect evidence of oxidative stress, steatosis, steatohepatitis, age, BMI, frequency of diabetes, alcohol intake, iron grade after Perl's stain, and PBC stage. Logistic regression analysis confirmed that the titers of antibodies against lipid peroxidation products (odds ratio 4.5, p< .001, 95% confidence interval 3.9­14.4), the presence of steatosis (odds ratio 4.1, 95% confidence interval 2.5­15.4, p< .001), higher BMI (odds ratio 3.9, p< .021, 95%confidence interval 1.4­9.5), and alcohol intake (males ≥ 30 g/day, females ≥ 20 g/day, odds ratio 4.5,95% confidence interval 1.3­19.8, p< .029) were independently associated with more advanced stages of the disease. CONCLUSIONS: The immune reactions triggered by oxidative stress, steatosis, obesity, and alcohol intake are independent predictors of PBC stage progression.

Immune signatures in human PBMCs of idiotypic vaccine for HCV-related lymphoproliferative disorders.

Hepatitis C virus (HCV) is one of the major risk factors for chronic hepatitis, which may progress to cirrhosis and hepatocellular carcinoma, as well as for type II mixed cryoglobulinemia (MC), which may further evolve into an overt B-cell non-Hodgkin's lymphoma (NHL). It has been previously shown that B-cell receptor (BCR) repertoire, expressed by clonal B-cells involved in type II MC as well as in HCV-associated NHL, is constrained to a limited number of variable heavy (VH)- and light (VL)-chain genes. Among these, the VK3-20 light chain idiotype has been selected as a possible target for passive as well as active immunization strategy. In the present study, we describe the results of a multiparametric analysis of the innate and early adaptive immune response after ex vivo stimulation of human immune cells with the VK3-20 protein. This objective has been pursued by implementing high-throughput technologies such as multiparameter flow cytometry and multiplex analysis of cytokines and chemokines.

Antiviral therapy in acute viral hepatitis B: why and when.

Correction to Morelli G, Perrella A, Sbreglia C, Bellopede P, Riccio V, Perrella O: Antiviral therapy in acute viral hepatitis B: why and when. Infectious Agents and Cancer 2009, 4:2.

Evidence of hepatitis C virus-specific interferon gamma-positive T cells in health care workers in an infectious disease department.

BACKGROUND: Few studies are available on possible hepatitis C virus (HCV)-specific T-cell immune response in health care workers (HCWs) involved in the care of patients with HCV infection. We aimed to investigate whether a HCV-specific interferon (IFN)-gamma T-cell response, known to be involved in infection resolution, was present in those HCWs involved in the management of patients with persistent HCV infection. METHODS: Our study involved 30 subjects, classified as group A (20 consecutive patients, 16 males and 4 females, with histologically proven chronic hepatitis), or group B (10 HCWs, 7 males and 3 females, with at least 7 years of health care experience and HCV-RNA and anti-HCV negative). As a control group, we used 10 blood samples from healthy donors at a blood donor center (group C). HCV-RNA was measured by real-time polymerase chain reaction. Blood samples (at least 35 mL) were collected from all group A and group B subjects in our hospital. Specific IFN-gamma was stimulated with HCV pool peptides (core, 2 microg/mL), with influenza Mp peptides used as a positive control. RESULTS: Levels of HCV-specific IFN-gamma-positive cells were higher in the HCWs (group B) compared with the infected patients (group A) and healthy blood donors (group C) (Mann-Whitney U test, P < .001). CONCLUSION: A clinically silent persistent exposure to HCV, through some as-yet undetermined mechanism, may induce a virus-specific IFN-gamma-producing CD8(+) T-cell response in healthy aviremic HCWs. This finding suggests that possible unapparent parenteral routes may stimulate host defenses with no evidence of hepatitis.

Antiviral therapy in acute viral hepatitis B: why and when.

Acute viral hepatitis B is cleared in more than 95% of patients, while the remainder ones may develop either chronic HBV infection or, rarely, fulminant hepatitis.Therefore there are elderly patients with severe acute HBV hepatitis caractherized by high serum bilirubin levels >15 mmole/dl, international normalized ratio (INR) with value more than 1.6; these patients are caractherized by a severe outcome of HBV infection.As known, outcome of infection and the pathogenesis of liver diseases are determined by viral and host factors, such as T reg lymphocytes.T regs may be associated with a negative immune response such as an inhibition of gamma- IFN secretion.The impact of viral load on antiviral T cell responses may play a critical role in these patients, influencing disease persistence and immune response.Antiviral drugs could be useful in these patients determing a possible down -regulation of T regs.

Molecular and phylogenetic analysis of HIV-1 variants circulating in Italy.

OBJECTIVE: The continuous identification of HIV-1 non-B subtypes and recombinant forms in Italy indicates the need of constant molecular epidemiology survey of genetic forms circulating and transmitted in the resident population. METHODS: The distribution of HIV-1 subtypes has been evaluated in 25 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the 1995-2005 period. Each sample has been characterized by detailed molecular and phylogenetic analyses. RESULTS: 18 of the 25 samples were positive at HIV-1 PCR amplification. Three samples showed a nucleotide divergence compatible with a non-B subtype classification. The phylogenetic analysis, performed on both HIV-1 env and gag regions, confirms the molecular sub-typing prediction, given that 1 sample falls into the C subtype and 2 into the G subtype. The B subtype isolates show high levels of intra-subtype nucleotide divergence, compatible with a long-lasting epidemic and a progressive HIV-1 molecular diversification. CONCLUSION: The Italian HIV-1 epidemic is still mostly attributable to the B subtype, regardless the transmission route, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes. Therefore, a molecular monitoring is needed to follow the constant evolution of the HIV-1 epidemic.

Impaired function of CD4+/CD25+ T regulatory lymphocytes characterizes the self-limited hepatitis A virus infection.

BACKGROUND AND AIM: Hepatitis A virus (HAV) causes a transient illness leaving permanent protection against reinfection. Few data are available on the regulatory mechanisms involved in the CD4+ T helper activation. We aimed to investigate the frequency and function of CD3+/CD4+/CD25+ T cells with regulatory function (Tregs) during acute HAV infection. METHODS: We enrolled 35 consecutive patients and 15 healthy donors, enumerated Tregs by flow cytometry assay and evaluated, after immunomagnetical sorting with magnetic beads, their ability to inhibit the proliferation of CD4+/CD25- T lymphocytes at different ratios (1:1, 1:10, 1:20). RESULTS: All patients had the usual course of infection. Our immunological analysis showed Tregs frequency in these patients (6.5% [range, 5-8.8%]; 36 [range, 10-87] cells) did not have any statistical difference compared with healthy donors (6% [range, 5-8%]; 48 (range, 23-71) cells), while their ability to suppress CD4+/CD25- was drastically reduced at different ratios (Mann-Whitney U-test; ratio 1:1, 93% vs 72%, z = -3.34, P < 0.0001; ratio 1:10, 86% vs 51%, z = -4.04, P < 0.001; ratio 1:20, 56% vs 30%, z = -3.43, P < 0.0001). After the seroconversion, CD4+/CD25+ frequency and function in HAV-infected patients did not differ from healthy individuals. CONCLUSION: CD4+/CD25+ T cells seem to be impaired in their function during the HAV acute infection. This evidence might help to determine an optimal T helper cell immune network that is a predisposing factor for a self-limiting disease.

Increase of granzyme B-positive cells in ascitic fluid of patients with spontaneous bacterial peritonitis.

Spontaneous bacterial peritonitis (SBP) occurs as a direct consequence of bacteria entering ascitic fluid (AF) from the intestinal lumen trough in several ways, including the hematogenous and mesenteric lymph nodes route. There are few studies on the cytokine profile of ascitic-derived mononuclear cells of patients with SBP, particularly on granzyme B (GZB). The aim of the present study was to verify whether patients with SBP have GZB-positive cells, whether they are increased in patients with aseptic ascites, and their trend after antibiotic treatment. We enrolled 36 consecutive patients (24 males and 12 females) with SBP on histologically-proven hepatitis C virus cirrhosis (group A) and 20 patients (11 males and nine females with ascites, but without evidences of SBP (group B). The diagnosis of SBP was made according to the following criteria: positive colture in AF or blood (at least two cultures) and neutrophils in AF (>250 mL polymorphonuclear leukocytes). For these patients we used ELISpot to assay GZB production on purified mononuclear cells in ascitesand peripheral blood, coupled with tumor necrosis factor-alpha tested using ELISA. A non-parametric statistical analysis was used to assess significant differences and correlations. We found positive culture in all of the patients with SBP (80% Escherichia coli; 20% Enterococcus faecium). Furthermore, the patients in group A had a higher number of GZB spot-forming colonies than the patients in group B (P < 0.001). GZB-positive cells were lower in the peripheral blood than those found in the AF of patients with SBP, while no differences were found between blood and AF in group B. Furthermore, after antibiotic treatment, GZB was reduced in the patients with SBP (P < 0.05). In conclusion, GZB may be an important mediator of the immune response towards bacteria in AF and could be used as a diagnostic tool.

Interleukin-10 and tumor necrosis factor-alpha: model of immunomodulation in multiple sclerosis.

OBJECTIVES: The mechanisms involved in the pathogenesis of relapsing-remitting multiple sclerosis are still unclear. The aim of the present study was to evaluate both cerebrospinal fluid (CSF) CD4+ CD7+ T cells and peripheral blood (PB) interleukin-10 (IL-10) as well as tumor necrosis-alpha (TNF-alpha) levels in patients with definite multiple sclerosis of the relapsing-remitting type. METHODS: To assess the above-mentioned cytokine levels we performed our test by the means of ELI-spot assay; the T-helper cell subset was assayed using flow cytometry. RESULTS: PB IL-10 levels of multiple sclerosis (MS) patients in remission were significantly (p<0.001) higher than in MS patients in the active phase. There was significant and increased evidence of TNF-alpha levels only in the MS patients in the active phase. CD4+ CD7+ T cells, characterized by a preferential Th1-like cytokine profile, were detectable only in seven patients in the active phase without evidence of a statistical significance with respect to cytokine levels. CONCLUSION: The data indicate that the production of different cytokines characterized the expression of relapsing-remitting MS. The data also suggest that is it possible to control MS using the regulatory cytokine balance.

Flow cytometry assay of myeloid dendritic cells (mDCs) in peripheral blood during acute hepatitis C: possible pathogenetic mechanisms.

AIM: To asses the expression of myeloid dendritic cells (CD11c+) subset during acute HCV hepatitis and its possible involvement in natural history of the infection. METHODS: We enrolled 11 patients with acute hepatitis C (AHC) (Group A), 10 patients with acute hepatitis A (AHA) (as infective control-Group B) and 10 healthy donors (group C) in this study. All patients underwent selective flow cytometry gating strategies to assess the peripheral number of the myeloid dendritic cells (mDCs) to understand the possible role and differences during acute hepatitis. RESULTS: Eight of 11 patients with acute HCV hepatitis did not show any increase of mDCs compared to healthy individuals, while a significant decrease of mDCs was found in absolute cell count (z = -2.37; P<0.05) and percentage (z = -2.30; P<0.05) as compared with AHA. On the contrary, The remaining three patients of the group A had a higher mDCs number and percentage as occur in group B. Interestingly, after six months, those patients did not show any increase of mDCs subset were chronically infected. while the three subjects with an increase of peripheral mDCs, as in HAV acute infection, resolved the illness. CONCLUSION: The lack of increase of mDCs during acute hepatitis C might be an important factor involved in chronicization of the infection.