Addressing barriers to the prevention, diagnosis and treatment of hepatitis B and C in the face of persisting fiscal constraints in Europe: report from a high level conference.

In the WHO-EURO region, around 28 million people are currently living with chronic viral hepatitis, and 120,000 people die every year because of it. Lack of awareness and understanding combined with the social stigma and discrimination exacerbate barriers related to access to prevention, diagnosis and treatment services for those most in need. In addition, the persisting economic crisis has impacted on public health spending, thus posing challenges on the sustainable investment in promotion, primary and secondary prevention, diagnosis and treatment of viral hepatitis across European countries. The Hepatitis B and C Public Policy Association in cooperation with the Hellenic Center for Disease Prevention and Control together with 10 partner organizations discussed at the Athens High Level Meeting held in June 2014 recent policy developments, persisting and emerging challenges related to the prevention and management of viral hepatitis and the need for a de minimis framework of urgent priorities for action, reflected in a Call to Action (Appendix S1). The discussion confirmed that persisting barriers do not allow the full realisation of the public health potential of diagnosing and preventing hepatitis B and C, treating hepatitis B and curing hepatitis C. Such barriers are related to (a) lack of evidence-based knowledge of hepatitis B and C, (b) limited access to prevention, diagnosis and treatment services with poor patient pathways, (c) declining resources and (d) the presence of social stigma and discrimination. The discussion also confirmed the emerging importance of fiscal constraints on the ability of policymakers to adequately address viral hepatitis challenges, particularly through increasing coverage of newer therapies. In Europe, it is critical that public policy bodies urgently agree on a conceptual framework for addressing the existing and emerging barriers to managing viral hepatitis. Such a framework would ensure all health systems share a common understanding of definitions and indicators and look to integrate their responses to manage policy spillovers in the most cost-effective manner, while forging wide partnerships to sustainably and successfully address viral hepatitis.

Hepatocellular carcinoma risk in HBeAg-negative chronic hepatitis B patients with or without cirrhosis treated with entecavir: HepNet.Greece cohort.

Hepatocellular carcinoma (HCC) may still develop in chronic hepatitis B (CHB) patients treated with lamivudine. Whether HCC rates are comparable in patients treated with the current first-line antivirals remains uncertain. We estimated the incidence and evaluated predictors of HCC in a large nationwide prospective cohort (HepNet.Greece) of HBeAg-negative CHB patients treated with entecavir. HBeAg-negative CHB patients from the same cohort who were initially treated with lamivudine were used as controls. We included 321 patients treated with entecavir for a median of 40 months and 818 patients treated initially with lamivudine for a median of 60 months. In the entecavir group, HCC developed in 4 of 321 (1.2%) patients at a median of 1.5 (range: 1.0-4.5) years, while the cumulative HCC incidence was significantly higher in cirrhotics than noncirrhotics (1, 3, 5 years: 0%, 3%, 9% vs 1%, 1%, 1%; P = 0.024) and in older patients (P = 0.026). Entecavir compared with lamivudine group patients had lower HCC incidence (1, 3, 5 years: 0.3%, 1.2%, 2.8% vs 0.7%, 3.8%, 5.6%; P = 0.024). However, in multivariable Cox regression analysis, the HCC risk was independently associated with older age (P < 0.001), male gender (P = 0.011) and cirrhosis (P = 0.025), but not with the initial agent. In conclusion, our large nationwide study indicates that the HCC risk remains increased in entecavir-treated HBeAg-negative CHB patients with cirrhosis, particularly of older age, at least for the first 5 years. The HCC risk does not seem to be significantly reduced with entecavir compared with antiviral therapy starting with lamivudine.

Activated lymphocytes in the peripheral blood of asymptomatic HBsAg carriers.

Proportions of T lymphocyte subsets and activated cells bearing receptors for transferrin, Il-2 and HLA-DR antigens were studied in 15 asymptomatic HBsAg carriers and 10 subjects with negative markers of hepatitis B virus infection. T lymphocyte subsets ranged within normal values in HBsAg carriers whereas a significant increase in activated cells was noted (P less than 0.001). These data suggest that abberations of T cell subsets are not responsible for the failure of HBV clearance. Furthermore, the increased levels of activated lymphocytes support the view that other factors, possibly in the serum, are modulating the immune response to HBV thus playing an important role in the pathogenesis of the carrier state.

The effect of loratadine on activated cells of the nasal mucosa in patients with allergic rhinitis.

The effect of loratadine on the numbers of activated cells--cells expressing interleukin-2 receptors(IL-2R), HLA-DR antigens and proliferating cell nuclear antigen (PCNA)--in the nasal mucosa was studied in 48 patients with allergic rhinitis. Patients were treated with either loratadine (10 mg) or placebo for 1 month. At the end of treatment, a significant decrease in the symptom scores was noted in both groups of patients. However, the clinical score was significantly lower in the loratadine group compared to the placebo group. At the end of treatment, the numbers of IL-2R+, HLA-DR+ and PCNA+ cells were significantly decreased only in the group on loratadine. An almost significant correlation was also observed between the numbers of IL-2R+ cells and symptoms in the loratadine group. Our results show that loratadine exerts its beneficial effect possibly by inhibiting both the action of histamine and immune activation.

Interleukin 2-dependent immunoregulatory function in hemodialysis patients with chronic hepatitis C.

To investigate the state of activation of T lymphocytes in hemodialysis patients with chronic hepatitis C, serum levels of soluble interleukin-2 receptors (sIL-2R), interleukin-2 (IL-2) and expression of interleukin-2 receptors (IL-2R) on peripheral T lymphocytes were measured in 30 hemodialysis patients with chronic hepatitis C, 14 hemodialysis patients without hepatitis, 30 patients with chronic hepatitis C and 41 normal subjects. A significant increase in sIL-2R levels was noted in all patients with chronic hepatitis compared to normal controls. This increase was even more significant in hemodialysis patients, while a less significant increase was also found in hemodialysis patients without hepatitis C viral infection. Expression of IL-2R on T lymphocytes was increased only in hemodialysis patients with chronic hepatitis. All patients had low levels of serum IL-2. These findings show that a T-cell activation involving the IL-2 system is present in chronic hepatitis C and that this activation is more prominent in hemodialysis patients, probably due to additional extrahepatic factors.

alpha-Interferon therapy in patients with chronic active hepatitis B: immunological profile.

The effect of interferon-alpha 2b (IFN) on viral markers, liver function and immunological parameters (CD3, CD4, CD8, B, NK, II-2 receptor and HLA-DR positive cells in blood and T cell proliferation) was studied in 9 patients with HBsAg(+), HBeAg(-) chronic active hepatitis (CAH). Three patients were HBV-DNA(+) and 6 also had complications of cirrhosis of the liver (LC). IFN was given at a dose of 2.5 mil IU x 3 weekly for 6 months. One patient with LC developed hepatic coma and died 2 months later. Severe leukopenia limited duration of treatment to 2 and 4 months in another 2 patients. By the end of treatment, the 8 patients were in good clinical status, SGOT, SGPT levels and prothrombin time were decreased, HBV-DNA became negative in 2 out of 3 patients and proportions of CD3, CD4, B, NK and activated cells were significantly decreased. When compared to controls, NK and activated cells were significantly increased in patients before and were gradually decreased by the end of treatment. In contrast, T transformed cells were significantly decreased before and ranged in normal levels by the end of treatment. These findings suggest that immunomodulatory activity possibly contributes to the beneficial effect of IFN therapy.

Genotype 4 HCV infection is difficult to cure with pegylated interferon and ribavirin. Results from a Greek Nationwide Cohort Study.

BACKGROUND AND AIM: Patients with genotype 4 (G4) chronic hepatitis C (CHC) are considered a difficult to treat population, although current data on G4 treatment responsiveness and duration are controversial. Greece represents a country with an intermediate prevalence of G4 infections, offering an opportunity to compare treatment outcomes by genotype and to identify potential prognostic factors for sustained virologic response (SVR). METHODS: All CHC patients from the HepNet.Greece, an ongoing nationwide cohort study on viral hepatitis, with known hepatitis C virus (HCV) genotype who received treatment with Peg-IFNa and ribavirin were analyzed. RESULTS: From 4443 patients, 951 (61.7% males, 78.4% Greeks, median age 40.6 years, 10% cirrhosis) fulfilled the inclusion criteria. G4 was found in 125 (13.1%) patients. Genotype distribution was not significantly different between Greeks and immigrants. Patients with G4 had similar odds of SVR compared to G1 but significantly lower compared to G2/G3. Age, treatment discontinuation, presence of cirrhosis and previous history of HCV-treatment were associated with lower probabilities of SVR. Ethnicity did not affect SVR for all genotypes while response to treatment was similar between Greek and Egyptian patients groups (35.7% vs 40.9%, p=0.660%) with G4 infection. The relation between SVR and genotype did not substantially change after adjustment for age, gender, cirrhosis, treatment interruption and history of HCV-treatment. CONCLUSIONS: The findings of this large cohort of CHC patients with a well balanced genotype distribution further supports the idea of considering G4 as a difficult to treat genotype. Further investigation is needed to identify genotype specific prognostic factors.

Hepatitis B reactivation in a patient with rheumatoid arthritis with antibodies to hepatitis B surface antigen treated with rituximab.

Hepatitis B virus (HBV) can still be found within the hepatocytes after its clearance and the control of viral replication depends on the immune response. However during immunosuppression, seroconversion of HBsAg has been described followed by disease reactivation. Hepatitis B virus reactivation represents an emerging cause of liver disease in patients undergoing treatment with biologic agents and in particular, by the use of rituximab (anti-CD20) and alemtuzumab (anti-CD52) that cause profound and long-lasting immunosuppression. We describe a case of a 64-year old female patient with rheumatoid arthritis and resolved HBV infection, who experienced a severe hepatitis B reactivation after the administration of rituximab.

Virologic response and resistance to lamivudine in patients with chronic hepatitis B: a ten-year retrospective analysis.

BACKGROUND: Emergence of resistance was recognized shortly after the introduction of lamivudine. This 10 year retrospective study investigates resistance to lamivudine and the modifications of antiviral strategies required. PATIENTS AND METHODS: Two hundred and nine patients were treated with lamivudine. Sixty seven out of 209 patients were excluded from the present study. HBVDNA was tested using the PCR assay and genotypic resistance was performed using the direct PCR sequencing. RESULTS: In the 125 patients initially treated with lamivudine monotherapy: Α) 48 (38.4%) patients with a mean time of 63.6±26.2 months under lamivudine treatment have normal ALT levels with negative (19%) or low (<1X102) HBVDNA levels, 10% developed cirrhosis, 1 HCC and 6% cleared HBsAg. Β) Resistance was developed in 61.60% patients within 45±23.84 months of lamivudine treatment. These patients were: 1) either switched to adefovir (9), entecavir (2) or tenofovir (2) or adefovir was added to lamivudine (21) for a short time and then they were switched to adefovir alone. Six out of 34 patients developed cirrhosis and 4 HCC while on treatment. 2) or adefovir was added-on to lamivudine (43). In 39 out of 43 treatment is ongoing while on virological response. No one developed cirrhosis or HCC. C) Seventeen patients received de novo combination therapy with lamivudine and adefovir and 2 out of 17 (11.7%) showed resistance to adefovir after 24 months of therapy. CONCLUSIONS: Our results showed that a) approximately 38.4% of patients maintain viral suppression more than 5 years of lamivudine treatment and b) rescue therapy with add-on adefovir to ongoing lamivudine, seems to be a better treatment strategy associated with long term benefit regarding disease complications.

Cytokine mRNA expression in hepatitis C virus infection: TH1 predominance in patients with chronic hepatitis C and TH1-TH2 cytokine profile in subjects with self-limited disease.

Many determinants of the immune response have been implied in the pathogenesis of chronic hepatitis C. TH1 and TH2 cytokines play a prominent role in viral infections and a dysregulation of these cytokines could account for viral persistence and evolution of chronic disease. To explore a possible TH1 and TH2 cytokine dysregulation resulting in the inability to terminate hepatitis C virus (HCV) infection, we studied TH1 [interferon (IFN)-gamma, interleukin (IL)-2] and TH2 (IL-4, IL-10) mRNA expression of peripheral blood mononuclear cells (PBMC) in response to NS3 HCV antigen stimulation, in 31 untreated patients with chronic hepatitis C and 29 subjects with self-limited disease. After a 48 h culture of PBMC, total RNA isolation was performed and complementary DNA was prepared by reverse transcription. mRNA levels were quantified by real-time polymerase chain reaction using a standard curve formed after cloning each cytokine gene and a reference gene using recombinant DNA technology in a specific plasmid vector. In the patients group, mRNA expression of IFN-gamma, IL-2 and IL-4 but not IL-10 was detected, IFN-gamma being the predominant cytokine expressed. All four cytokines were expressed in subjects with self limited disease, however levels of IFN-gamma were lower and a significant higher expression of IL-10 compared to patients was found. There was a significant correlation between IFN-gamma mRNA expression levels and stage of fibrosis. Our findings show that in chronic hepatitis C, TH1 cytokines predominate and correlate to liver immunopathology. Furthermore, subjects with self-limited disease, maintain the ability to respond to HCV antigens for a long time after disease resolution.

Adamantiades-Behcet disease (ABD) in northern Greece patients: experience from a single center.

UNLABELLED: OBJECTIVE-METHODS: Adamantiades-Behcet disease (ABD) is a multi-systemic vasculitis of unknown origin, with a characteristic geographic distribution, that affects vessels of all kinds and sizes and is characterized by recurrent mucosal, skin and ocular lesions. In the present study, a series of 36 patients from Northern Greece is analyzed retrospectively in regard to the epidemiological, clinical and immunological parameters. RESULTS: All patients had recurrent oral ulcerations (36/36, 100%), while 23/36 (63.9%) experienced genital ulcerations and 22/36 (61.1%) developed ocular disease. Skin manifestations were observed in 23/36 patients (63.9%) and pathergy test was found positive in 14/36 patients (38.9%). Other manifestations included central nervous system involvement, recurrent genitourinary inflammations, arthralgias and superficial thrombophlebitis. Laboratory findings were not specific, partly reflecting the severity of inflammation. Ocular disease was more often observed in HLA-B51 (+) patients (20/31, 64.5%) than in HLA-B51 (-) patients. Standard of care (SOC) treatment consisted of cyclosporine A, azathioprine, methylprednisolone and aspirin, whereas refractory disease was treated with intravenous pulses of methylprednisolone and cyclophosphamide. Occasionally, anti-TNF agents (infliximab) were applied to treat refractory ocular disease. CONCLUSION: The findings of the present study come in agreement with those reported for other Mediterranean series. HLA-B51 seems to predispose to more severe disease, while early therapeutic intervention is beneficial for these patients.

CD80/CD28 co-stimulation in human brucellosis.

Despite treatment, 10-30% of brucellosis patients develop chronic disease, characterized by atypical clinical picture and/or relapses. A defective T helper 1 (Th1) response and a low [corrected] percentage of CD4(+)/CD25(+) cells have been described in chronic brucellosis patients. CD80/CD28 co-stimulation is critical for an efficient Th1 response and has not been studied previously in human brucellosis. In order to investigate the role of CD80/CD28 co-stimulation, 13 acute brucellosis patients (AB), 22 chronic brucellosis patients (CB, 12/22 relapsing type-CB1 and 10/22 atypical type-CB2), 11 'cured' subjects and 15 healthy volunteers (controls) were studied. The percentage of CD4(+)/CD28(+) T lymphocytes and CD14(+)/CD80(+) monocytes were analysed by flow cytometry both ex vivo and after phytohaemagglutinin (PHA)-stimulation with or without heat-killed Brucella abortus (HkBA). Ex vivo analysis showed no differences between all groups studied. PHA stimulation up-regulated the percentage of CD80(+) monocytes in AB compared to 'cured' subjects and controls (P < 0.001), although the proportion of CD4(+)/CD28(+) cells did not alter. A higher percentage of CD80(+) monocytes was observed in the CB1 subgroup, compared to AB, 'cured' subjects and controls (P = 0.042, < 0.001 and < 0.001, respectively). CB2 was characterized by a lower percentage of CD80(+) monocytes in comparison to CB1 (P = 0.020). HkBA in PHA cultures down-regulated the percentage of CD80(+) monocytes compared to PHA alone in all groups, especially in AB and CB patients (P < 0.001 and P = 0.007, respectively). In conclusion, the diminished percentage of CD4(+)/CD25(+) T cells in CB is not associated with inadequate CD80/CD28 co-stimulation. We speculate that differential frequency of CD80(+) monocytes after PHA stimulation could serve as a qualitative parameter of disease status, related to the different clinical forms of chronic brucellosis.

The effect of interferon on activated peripheral blood T lymphocytes in patients with chronic active hepatitis B.

The effect of interferon-alpha 2b treatment on activated T lymphocytes -T cells expressing interleukin-2 receptors-(IL-2R) was studied in 18 patients with chronic active hepatitis B. Blood samples were taken before, on the 2nd, 4th and 6th month of treatment. Patients were divided in 3 groups according to their response to therapy (complete, partial, no response). At the end of treatment, IL-2R+ cells were decreased in the group of patients with complete response, unchanged in patients with partial response and increased in patients with no response. These results confirm the immunomodulatory effect of interferon and reflect the effect of treatment in the management of the disease.

Possible mechanisms underlying peripheral lymphocyte activation in chronic liver disease and asymptomatic HBsAg carriers.

Proportions of T lymphocyte subsets (OKT3, OKT4, OKT8) and expression of interleukin-2 (IL-2), transferrin (TFR) receptors and HLA-DR antigens were studied in the peripheral blood of 21 healthy HBsAg carriers, 10 patients with chronic active hepatitis B (CAH), 10 patients with alcoholic liver cirrhosis (ALC) and 10 subjects with negative markers of previous hepatitis B virus (HBV) infection. T lymphocyte subsets ranged within normal levels in CAH and carriers, whereas a significant decrease of OKT4 was noted in ALC possibly involved in the pathogenesis of this disorder. Significantly elevated numbers of activated cells (cells expressing IL-2, TFR receptors and HLA-DR antigens) were observed in all three groups. A significant increase in OKT8 cells within the TFR population was noted in CAH and ALC, whereas proportions of T subsets in the TFR population were normal in carriers. These findings possibly suggest a common pathogenetic mechanism in the activation of lymphocytes in CAH and ALC leading to liver damage and an immune response against HBV in healthy carriers.

Levamisole in the treatment of chronic brucellosis.

Ten patients with chronic brucellosis were treated with levamisole, 150mg daily for 3 consecutive days every week for 6 months. Patients were followed up for another 6 months after the end of treatment. By the end of the treatment period, patients improved clinically, their E rosettes showed a significant increase and specific leukocyte inhibition migration factor (LIF) was produced. By the end of the follow up period, all patients except one became symptom free, antiglobulin titers were very low or negative, E rosettes showed a further increase ranging to normal levels and specific LIF activity was not detected to most of the patients. Our results suggest that levamisole might prove to be beneficial in the treatment of chronic brucellosis.

Serum inhibitory factors and E rosettes in chronic renal failure.

The inhibiting effect of uraemic serum on both normal and uraemic lymphocytes capacity to form E rosettes was studied in 10 uraemic patients and 14 healthy controls. Uraemic serum was found to significantly inhibit E rosette forming cells of normal and uraemic lymphocytes. On the other hand, E rosettes of uraemic patients ranged in normal values after incubation in normal serum. These findings suggest that rosette inhibiting factors are present in uraemia and that uraemic T lymphocyte sheep erythrocyte rosette function is intact.

Lymphocyte subpopulations in gastric disease; effect of histamine and cimetidine on immunoregulatory T cell subsets.

The numbers of T lymphocytes, helper and suppressor T lymphocytes, were measured in peripheral blood of 10 patients, 13 patients with gastric cancer and 20 normal controls. T lymphocyte subpopulations were enumerated by the use of monoclonal antibodies OKT3 (pan-T lymphocytes), OKT4 (helper/inducer lymphocytes) and OKT8 (cytotoxic/suppressor lymphocytes) in an indirect immunofluorescence technique. Furthermore, the possible pharmacological modulation of 10(-4) histamine and 10(-4), 10(-6) M cimetidine of T lymphocyte subsets was investigated. Lymphocyte subpopulations were found to range in normal values in patients with ulcer and chronic gastritis. A marked decrease of OKT3 and OKT8 positive lymphocytes was noted in patients with gastric cancer, whereas OKT4 lymphocytes from the three groups of patients to histamine and cimetidine resulted in no significant changes of lymphocyte subsets.

Reevaluation of the effect of levamisole in chronic brucellosis: in vitro and in vivo effect on monocyte phagocytosis.

The in vitro effect of levamisole on peripheral blood monocyte (P.B.M.) phagocytosis was studied in 32 patients with chronic brucellosis and 20 normal subjects. It was shown that levamisole enhances P.B.M. phagocytic capacity, not reaching however normal levels. A subgroup of 11 patients were treated with levamisole for 6 months and the drug effect on cellular and humoral immunity and monocyte phagocytosis was also studied. By the end of the 6 month treatment-study period, the following results were obtained: 1. six patients were symptom free while five had significantly improved. 2. T lymphocyte number and monocyte phagocytosis reached normal values. 3. Significant specific cellular immunity against both brucella antigens was noted. 4. B. lymphocytes showed no significant changes. 5. Antiglobulin titers varied. These findings suggest that the good therapeutical effect of levamisole in patients with chronic brucellosis could probably be a attributed to the enhancement of both T-cell function and monocyte phagocytosis.

A study of cell-mediated immunity to gastric antigens in patients with atrophic type A and B gastritis.

The leucocyte migration in the presence of gastric antigens was studied in 10 patients with type A gastritis, 38 patients with type B gastritis (28 with atrophic and 10 with superficial gastritis) and 10 healthy controls. A positive leucocyte migration was found in a significant proportion of patients with both types of gastritis, whereas no difference between the two types was observed. These results indicate that cellular immunity is implicated in the aetiology of both types of gastritis, is of greater importance than auto-antibody production and is associated with the severity of the atrophic lesion.

Peripheral blood monocyte phagocytosis in patients with chronic brucellosis.

Peripheral blood monocyte phagocytosis of SRBC was studied in 32 patients with chronic brucellosis and 20 normal controls. It was found that phagocytosis was significantly decreased in patients with chronic brucellosis. Our findings suggest that disfunction of this cell-system in chronic brucellosis could be responsible for the chronicity of the disease.