Serena Ludovisi

Impaired intrahepatic natural killer cell cytotoxic function in chronic hepatitis C virus infection

Hepatitis C virus (HCV) persistence in the host results from inefficiencies of innate and adaptive immune responses. Most studies addressing the role of innate immunity concentrated on peripheral blood (PB) natural killer (NK) cells, whereas only limited information is available on intrahepatic (IH) NK cells. We therefore examined phenotypic and functional features of IH and PB NK cells in paired liver biopsy and venous blood samples from 70 patients with chronic HCV infection and 26 control persons subjected to cholecystectomy for gallstones as controls. Ex vivo isolated IH NK cells from HCV‐infected patients displayed unique phenotypic features, including increased expression of NKp46‐activating receptor in the face of reduced tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) and cluster of differentiation (CD) 107a expression, which resulted in impaired degranulation compared with controls. To gain insights into the effect of HCV on NK cells, we exposed peripheral blood mononuclear cells (PBMCs) from patients and healthy donors to cell‐culture–derived HCV (HCVcc) and measured NK cell degranulation, TRAIL, and phosphorylated extracellular signal‐regulated kinase 1/2 (pERK1/2) expression. Exposure of PBMCs to HCVcc significantly boosted NK degranulation, pERK1/2, and TRAIL expression in healthy donors, but not in patients with chronic HCV infection, a defect that was completely reversed by interferon‐alpha. Purified NK cells showed a minimal, though significant, increase in degranulation and TRAIL expression, both in patients and controls, after exposure to HCVcc. Conclusions: These findings indicate dysfunctional IH NK cell cytotoxicity associated with TRAIL down‐regulation in chronic HCV infection, which may contribute to virus persistence. PB NK cell impairment upon exposure to HCVcc suggests the existence of an accessory cell‐dependent NK cell lytic defect in chronic HCV infection predominantly involving the TRAIL pathway. (HEPATOLOGY 2012;56:841–849)

A phase II, single-arm multicenter study of low-dose rituximab for refractory mixed cryoglobulinemia secondary to hepatitis C virus infection.

Eradication of hepatitis C virus (HCV) by antiviral therapy is the treatment of choice for mixed cryoglobulinemia secondary to this infection, but many patients fail to achieve sustained viral responses and need second-line treatments. Several studies have demonstrated that the infusion of the anti-CD20 monoclonal antibody rituximab is highly effective for refractory mixed cryoglobulinemia, with a clinical response in approximately 80% of patients, although the relapse rate is high. Virtually all published studies employed a rituximab dosage of 375mg/m(2) given four times, a schedule used for treating non-Hodgkins lymphomas. Based on a prior pilot study, we designed a phase II single-arm two-stage study (EUDRACT n. 2008-000086-38) to evaluate the efficacy of a lower dosage of rituximab, 250mg/m(2) given twice, for refractory mixed cryoglobulinemia. We present here the preliminary results in the first 27 patients enrolled. The overall response rate in 24 evaluable patients was 79%, and the mean time to relapse was 6.5months, similar to the 6.7months reported in studies with high-dose rituximab. Side effects were comparable to those seen in patients treated with high-dose. Increase of HCV viral load, reported in some high-dose studies, was not observed in our patients. Low-dose rituximab may provide a more cost/effective and possibly safer alternative for treating refractory HCV-associated mixed cryoglobulinemia.

Performance of Real-Time Strain Elastography, Transient Elastography, and Aspartate-to-Platelet Ratio Index in the Assessment of Fibrosis in Chronic Hepatitis C

OBJECTIVE The purpose of this article is to evaluate the diagnostic performance of transient elastography, real-time strain elastography, and aspartate-to-platelet ratio index in assessing fibrosis in patients with chronic hepatitis C by using histologic Metavir scores as reference standard. SUBJECTS AND METHODS Consecutive patients with chronic hepatitis C scheduled for liver biopsy were enrolled. Liver biopsy was performed on the same day as transient elastography and real-time strain elastography. Transient elastography and real-time strain elastography were performed in the same patient encounter by a single investigator using a medical device based on elastometry and an ultrasound machine, respectively. Diagnostic performance was assessed by using receiver operating characteristic curves and area under the receiver operating characteristic curve (AUC) analysis. RESULTS One hundred thirty patients (91 men and 39 women) were analyzed. The cutoff values for transient elastography, real-time strain elastography, and aspartate-to-platelet ratio index were 6.9 kPa, 1.82, and 0.37, respectively, for fibrosis score of 2 or higher; 7.3 kPa, 1.86, and 0.70, respectively, for fibrosis score of 3 or higher; and 9.3 kPa, 2.33, and 0.70, respectively, for fibrosis score of 4. AUC values of transient elastography, real-time strain elastography, aspartate-to-platelet ratio index were 0.88, 0.74, and 0.86, respectively, for fibrosis score of 2 or higher; 0.95, 0.80, and 0.89, respectively, for fibrosis score of 3 or higher; and 0.97, 0.80, and 0.84, respectively, for fibrosis score of 4. A combination of the three methods, when two of three were in agreement, showed AUC curves of 0.93, 0.95, and 0.95 for fibrosis scores of 2 or higher, 3 or higher, and 4, respectively. CONCLUSION Transient elastography, real-time strain elastography, and aspartate-to-platelet ratio index values were correlated with histologic stages of fibrosis. Transient elastography offered excellent diagnostic performance in assessing severe fibrosis and cirrhosis. Real-time elastography does not yet have the potential to substitute for transient elastography in the assessment of liver fibrosis.

Performance of liver stiffness measurements by transient elastography in chronic hepatitis

AIM To compare results of liver stiffness measurements by transient elastography (TE) obtained in our patients population with that used in a recently published meta-analysis. METHODS This was a single center cross-sectional study. Consecutive patients with chronic viral hepatitis scheduled for liver biopsy at the outpatient ward of our Infectious Diseases Department were enrolled. TE was carried out by using FibroScan™ (Echosens, Paris, France). Liver biopsy was performed on the same day as TE, as day case procedure. Fibrosis was staged according to the Metavir scoring system. The diagnostic performance of TE was assessed by using receiver operating characteristic (ROC) curves and the area under the ROC curve analysis. RESULTS Two hundred and fifty-two patients met the inclusion criteria. Six (2%) patients were excluded due to unreliable TE measurements. Thus, 246 (171 men and 75 women) patients were analyzed. One hundred and ninety-five (79.3%) patients had chronic hepatitis C, 41 (16.7%) had chronic hepatitis B, and 10 (4.0%) were coinfected with human immunodeficiency virus. ROC curve analysis identified optimal cut-off value of TE as high as 6.9 kPa for F ≥ 2; 7.9 kPa for F ≥ 3; 9.6 kPa for F = 4 in all patients (n = 246), and as high as 6.9 kPa for F ≥ 2; 7.3 kPa for F ≥ 3; 9.3 kPa for F = 4 in patients with hepatitis C (n = 195). Cut-off values of TE obtained by maximizing only the specificity were as high as 6.9 kPa for F ≥ 2; 9.6 kPa for F ≥ 3; 12.2 kPa for F = 4 in all patients (n = 246), and as high as 7.0 kPa for F ≥ 2; 9.3 kPa for F ≥ 3; 12.3 kPa for F = 4 in patients with hepatitis C (n = 195). CONCLUSION The cut-off values of TE obtained in this single center study are comparable to that obtained in a recently published meta-analysis that included up to 40 studies.

Efficacy of low-dose rituximab for the treatment of mixed cryoglobulinemia vasculitis: Phase II clinical trial and systematic review

OBJECTIVE To evaluate whether rituximab at a low dose of 250 mg/m(2) × 2 may be as effective as at higher dosages, most commonly 375 mg/m(2)×4, used in previous studies on the treatment of patients with refractory mixed cryoglobulinemia (MC) vasculitis associated with hepatitis C virus (HCV) infection. METHODS We conducted a phase 2, single-arm two-stage trial (EUDRACT n. 2008-000086-38) of low-dose rituximab in 52 patients with HCV-associated MC who were ineligible/intolerant or non-responder to antiviral therapy. The primary outcomes were response of vasculitis evaluated by the Birmingham Vasculitis Activity Score (BVAS) at months 3, 6 and 12, rate of relapses and time to relapse, and rate of adverse events. Our data were compared with those reported in 19 published studies selected among 291 reviewed in a literature search. RESULTS The cumulative response rate (complete and partial) at month 3 was 81% in our patients, and 86% in 208 patients from studies using high-dose rituximab. The relapse rate and median time to relapse were, respectively, 41% and 6 months in our study, and 32% and 7 months in high-dose studies. Treatment-related adverse events were 11.5% in our study and 19.9% in high-dose studies. None of these differences was statistically significant. CONCLUSION Rituximab at a low dosage of 250 mg/m(2) × 2 is as effective as at higher dosages for treating MC vasculitis. This low-dose regimen may improve the cost/benefit profile of rituximab therapy for MC.

Lack of Siglec-7 expression identifies a dysfunctional natural killer cell subset associated with liver inflammation and fibrosis in chronic HCV infection

Objective Sialic-acid-binding immunoglobulin-like lectin-7 (Siglec-7) is a natural killer (NK) cell inhibitory receptor associated with NK phenotypic and functional abnormalities in HIV-1 infection. We investigated the significance of NK-expressed and serum soluble Siglec-7 in relation to NK functional ability and parameters of liver necroinflammation and fibrosis in chronic HCV infection. Design NK-expressed and serum Siglec-7 were evaluated in 130 and 166 HCV-infected individuals by flow cytometry and ELISA, respectively. NK cell degranulation and cytokine secretion were determined by flow cytometry. 65 patients with chronic HBV infection, 84 with chronic biliary disorders and 168 healthy donors served as controls. Results Expression of Siglec-7 was significantly decreased on NK cells from HCV-infected and HBV-infected patients and, conversely, serum Siglec-7 was significantly increased in these patients compared with controls. The frequency of Siglec-7pos NK cells was significantly higher at baseline in sustained virological responders to pegylated interferon-α/ribavirin treatment than in non-responders. Activating receptor expression was significantly higher in Siglec-7pos NK cells and was associated with increased degranulation and cytokine secretion compared with Siglec-7neg cells. In chronic HCV infection, there was an inverse correlation between Siglec-7 expression and serum aminotransferases, γ-glutamyl transpeptidase, liver stiffness, aspartate aminotransferase to platelet ratio index and fibrosis-4 scores, and a positive correlation between serum Siglec-7 and the same clinical parameters, including histological staging. Conclusions These findings identify Siglec-7neg NK cells as a dysfunctional subpopulation associated with severe liver disease in chronic HCV infection.

Hepatitis C virus-induced NK cell activation causes metzincin-mediated CD16 cleavage and impaired antibody-dependent cytotoxicity

BACKGROUND & AIMS The Fc receptor family for immunoglobulin (Ig)G type III (FcγRIII, CD16) is an activating receptor on natural killer (NK) cells and an essential mediator of antibody-dependent cellular cytotoxicity (ADCC). There is only limited information on its role during chronic hepatitis C virus (HCV) infection. We studied CD16 expression in relation to NK cell functional activity in HCV-infected patients and sought mechanistic insights into virus-induced modulation. METHODS NK cell CD16 expression and activation status were evaluated ex vivo by flow cytometry in HCV-infected patients and healthy controls (HC) as well as in vitro after co-culture with HCV-infected HuH7.5 cells. Rituximab-mediated ADCC was assessed in HC and HCV-infected patients using Daudi cells as a target. The role of metzincins in CD16 down-modulation was assessed using specific inhibitory molecules and by evaluating intracellular mRNA levels. RESULTS HCV-infected patients exhibited increased frequencies of ex vivo activated NK cells and a concomitantly decreased NK CD16 expression, which resulted in impaired ADCC activity. Moreover, exposure of NK cells to culture-derived HCV recapitulated the ex vivo findings of decreased CD16 expression and increased NK cell activation. Importantly, blockade of metzincin-mediated shedding activity, including selective a disintegrin and metalloproteinase 17 (ADAM-17) inhibition, restored NK CD16 expression. Successful treatment with direct-acting antivirals partially improved NK ADCC function despite delayed CD16 reconstitution. CONCLUSION Chronic HCV infection induces NK cell activation resulting in ADAM-17-dependent CD16 shedding and consequent impaired ADCC function. Altered ADCC may contribute to failure to eradicate HCV-infected hepatocytes. LAY SUMMARY We show here that hepatitis C virus (HCV) activates natural killer (NK) lymphocytes which, as a consequence, loose their Fc receptor for IgG (CD16), an essential molecule for antibody binding. We show that this occurs through the action of enzymes named metzincins, resulting in altered NK-mediated antibody-dependent killing (ADCC) of target cells. This mechanism may contribute to HCV persistence and may represent a general phenomenon whereby some viruses can escape hosts immune responses.

Hepatitis C virus inhibits CD4 T cell function via binding to Toll-like receptor 7

Toll-like receptor 7 (TLR7) is a ssRNA receptor that activates dendritic cells and macrophages upon ssRNA binding; however, little is known of its role in CD4+ T cells. We show here that hepatitis C virus (HCV) induces a dose dependent inhibition of cytokine production and expression of activation markers in CD4 T cells, which were restored by a TLR7-specific antagonist. These findings indicate that HCV induces CD4 T cell impairment via TLR7 which may contribute to failure of virus eradication, casting doubts on the use of TLR7 agonists to boost innate immunity in chronic RNA virus infections.

HCV intergenotype 2k/1b recombinant detected in a DAA-treated patient in Italy

Direct-acting antiviral (DAA) combinations are potent and effective drugs currently recommended for treatment of chronic HCV infection. Difficult to treat genotypes are the most important predictors of treatment failure. We report a case of DAA treatment failure in an HCV-infected patient carrying a recombinant genotype 2k/1b. This strain, first isolated from a Russian patient in 2002, has now been observed for the first time in Italy.