The combined molecular adjuvant CASAC enhances the CD8+ T cell response to a tumor-associated self-antigen in aged, immunosenescent mice.

BACKGROUND: Ineffective induction of T cell mediated immunity in older individuals remains a persistent challenge for vaccine development. Thus, there is a need for more efficient and sophisticated adjuvants that will complement novel vaccine strategies for the elderly. To this end, we have investigated a previously optimized, combined molecular adjuvant, CASAC (Combined Adjuvant for Synergistic Activation of Cellular immunity), incorporating two complementary Toll-like receptor agonists, CpG and polyI:C, a class-II epitope, and interferon (IFN)-γ in aged mice. FINDINGS: In aged mice with typical features of immunosenescence, antigen specific CD8+ T cell responses were stimulated after serial vaccinations with CASAC or Complete/Incomplete Freund's Adjuvant (CFA/IFA) and a class I epitope, deriving either from ovalbumin (SIINFEKL, SIL) or the melanoma-associated self-antigen, tyrosinase-related protein-2 (SVYDFFVWL, SVL). Pentamer analysis revealed that aged, CASAC/SIL-vaccinated animals had substantially higher frequencies of H-2K(b)/SIL-specific CD8+ T cells compared to the CFA/IFA-vaccinated groups. Similarly, higher frequencies of H-2K(b)/SVL-pentamer+ and IFN-γ+ CD8+ T cells were detected in the aged, CASAC + SVL-vaccinated mice than in their CFA/IFA-vaccinated counterparts. In both antigen settings, CASAC promoted significantly better functional CD8+ T cell activity. CONCLUSION: These studies demonstrate that functional CD8+ T cells, specific for both foreign and tumour-associated self-antigens, can be effectively induced in aged immunosenescent mice using the novel multi-factorial adjuvant CASAC.

Management of patients with decompensated cirrhosis.

During the assessment of a patient with liver disease, finding the patient has decompensated cirrhosis, as defined by the presence of jaundice, ascites, variceal haemorrhage or hepatic encephalopathy, has major implications regarding management and prevention of cirrhosis-related complications, as well as consideration for a referral for liver transplantation evaluation. Prognosis is markedly worse in patients with decompensated compared with compensated cirrhosis. In general, any patient with decompensated cirrhosis should receive evaluation and medical care by a hepatologist. Since patients frequently present with more than one facet of liver decompensation, such cases pose a complex management challenge requiring input from a multidisciplinary team and close liaison with a liver transplant centre.

Hepatitis delta virus.

Hepatitis delta virus (HDV) is a small, defective RNA virus that can infect only individuals who have hepatitis B virus (HBV); worldwide more than 15 million people are co-infected. There are eight reported genotypes of HDV with unexplained variations in their geographical distribution and pathogenicity. The hepatitis D virion is composed of a coat of HBV envelope proteins surrounding the nucleocapsid, which consists of a single-stranded, circular RNA genome complexed with delta antigen, the viral protein. HDV is clinically important because although it suppresses HBV replication, it causes severe liver disease with rapid progression to cirrhosis and hepatic decompensation. The range of clinical presentation is wide, varying from mild disease to fulminant liver failure. The prevalence of HDV is declining in some endemic areas but increasing in northern and central Europe because of immigration. Treatment of HDV is with pegylated interferon alfa; however, response rates are poor. Increased understanding of the molecular virology of HDV will identify novel therapeutic targets for this most severe form of chronic viral hepatitis.

Early predictors of corticosteroid treatment failure in icteric presentations of autoimmune hepatitis.

UNLABELLED: Autoimmune hepatitis (AIH) typically responds to treatment in 90% of patients. Early prediction of treatment outcome would be advantageous in clinical practice. We evaluated whether parameters at initiation of therapy or changes in these parameters at day 3 and day 7 following corticosteroid initiation predicted treatment failure. Treatment-naive, jaundiced patients presenting to our tertiary unit between 1999-2009 were identified and mathematical models of prognosis in liver disease scores calculated at day 0, day 3, and day 7. Overall, 72 patients were identified (48 women, 24 men). Treatment failure occurred in 18% (13/72) of patients. At diagnosis, higher median bilirubin (451 µmol/L versus 262 µmol/L, P = 0.02), INR (1.62 versus 1.33, P = 0.005), model for endstage liver (MELD) score (26 versus 20, P = 0.02), MELD-sodium (Na) score (27 versus 22, P = 0.03) and United Kingdom endstage liver disease score (UKELD) score (59 versus 57, P = 0.01) significantly correlated with treatment failure. Analysis of area under the receiver operator characteristic curve (AUROC) values at day 7 identified change (Δ) bilirubin (AUROC 0.68), Δ creatinine (0.69), Δ MELD (0.79), Δ MELD-Na (0.83) and Δ UKELD (0.83) best predicted treatment failure. Specifically, a fall in UKELD of less than 2 points predicted treatment failure with a sensitivity of 85% and specificity of 68%. Of 13 treatment failures, nine required second-line immunosuppression, three required emergency transplant, and one died of sepsis. In total, four patients died in the treatment failure group compared with one in the responder group (4/13 = 31% versus 1/59 = 1.7%, P = 0.003). CONCLUSION: Approximately 20% of icteric AIH presentations fail corticosteroid therapy. This is associated with significant mortality and the need for emergency transplantation. Treatment failure is best predicted by change in MELD-Na and UKELD at day 7. Early identification of nonresponders may allow timely escalation of immunosuppression to prevent clinical deterioration.

Pancreatic pseudocysts in children: treatment by endoscopic cyst gastrostomy.

AIM: The aim of the present study was to review the use of endoscopic cyst gastrostomy (E-CG) as a treatment option for pancreatic pseudocysts referred to a tertiary paediatric surgical centre. METHODS: Retrospective review during a 10-year period (January 2001-December 2010). Cyst gastrostomies were performed using 1 or 2 double pigtailed Zimmon stents (7-10 Fr) under general anaesthesia. Data are quoted as median (range). RESULTS: E-CG was performed in 7 (5 males) children (median age at presentation 11.7 [8.2-15.8] years). Pancreatic pseudocysts were caused by acute pancreatitis in 5 (gallstones n = 1, hereditary pancreatitis n = 1, pancreatic divisum n = 1, asparaginase induced n = 1, and idiopathic n = 1) and pancreatic trauma in 2 (motor vehicle accident n = 1, and handlebar injury n = 1). All of the cases were associated with a rise in serum amylase level, median 1028 (276-2077) IU/L at the peak of symptoms. Three children had pancreatic duct stent placement during endoscopic retrograde cholangiopancreatography as the initial therapeutic intervention, but went on to have E-CG later. One who had a huge pseudocyst at presentation had already undergone an open cyst gastrostomy, which had recurred at 1 month. Rescue E-CG was performed 38 days later. All of the stents were removed endoscopically at 8 (6-40) weeks. E-CG was uncomplicated and pseudocysts resolved completely in 5. One required repeat placement at 15 days due to catheter slippage with later full resolution. One child required open cyst gastrostomy due to reaccumulation two months following removal of the stent. Median hospital stay post E-CG was 3 (1-23) days. There has been no recurrence at median follow-up of 18 (5-108) months. CONCLUSIONS: Endoscopic cyst gastrostomy is a safe and effective alternative for the management of pancreatic pseudocysts in children and should now be considered as treatment of choice.

Non-invasive assessment of fibrosis in liver grafts due to hepatitis C virus recurrence.

Hepatitis C virus (HCV) infection has become the most common indication for liver transplantation in the United States and Europe. Recurrence of HCV is universal in all liver graft recipients, but the natural history of the disease is variable with some patients displaying slowly progressive liver injury, while approximately 30% become cirrhotic within five yr of surgery. Currently, liver biopsy is the reference standard to assess liver injury in the post-transplant setting. But biopsy is associated with complications such as bleeding and pain, as well as the risk of sampling error and discordance in reporting between histopathologists. Thus, as in the pre-transplant setting, there is increasing attention being drawn to the use of non-invasive tests of liver fibrosis. This review examines the role of non-invasive assessment of hepatic fibrosis in the post-transplant setting including simple tests such as aspartate aminotransferase-to-platelet ratio index, the Benlloch formula, London Transplant Centre score, and finally transient elastography. The authors assess the respective advantages and disadvantages of the tests and consider how non-invasive tests of liver fibrosis can be utilized in the future management of post-transplant HCV infection.

Evaluation of risk factors in the development of hepatocellular carcinoma in autoimmune hepatitis: Implications for follow-up and screening.

UNLABELLED: Hepatocellular carcinoma (HCC) has traditionally been considered a rare complication of cirrhosis secondary to autoimmune hepatitis (AIH), yet the true incidence remains unknown due to a lack of published data. Consequently, some professional guidelines do not mandate routine surveillance for HCC in this condition. Our aims were to evaluate the rate at which HCC develops among a large, prospectively obtained cohort of patients with AIH at a single center. Demographic, clinical, and laboratory indices associated with the development of HCC were also identified. HCC was discovered in 15 of 243 patients with AIH, all of whom had type 1 AIH equating to 1090 cases per 100,000 patient follow-up years. HCC occurred in the same proportion of females as males, 6.1% versus 6.4%, P = 0.95. HCC occurred more frequently in patients who had cirrhosis at presentation, 9.3% versus 3.4%, P = 0.048, or who had a variceal bleed as the index presentation of AIH, 20% versus 5.3%, P = 0.003. The median duration from time of confirmed cirrhosis to a diagnosis of HCC was 102.5 months, range 12-195 months. Median survival in patients whose HCC was diagnosed on surveillance was 19 months (range 6-36 months) compared with 2 months (range 0-14 months) for patients presenting symptomatically (P = 0.042). CONCLUSION: Cirrhosis in AIH is the sine qua non for HCC development, which subsequently occurs at a rate of 1.1% per year and affects men and women in equal proportions.

Selective use of endoscopic retrograde cholangiopancreatography in the diagnosis of biliary atresia in infants younger than 100 days.

OBJECTIVES: We investigated the role and safety of endoscopic retrograde cholangiopancreatography (ERCP) in diagnosing biliary atresia (BA) in prolonged neonatal cholestasis, when standard workup was inconclusive. PATIENTS AND METHODS: We reviewed notes of 48 cholestatic infants younger than 100 days undergoing ERCP from 1997 to 2007. RESULTS: Amongst approximately 3300 infants evaluated for liver disease during the study, 224 (6.8%) were diagnosed with BA. Forty-eight children underwent ERCP. Findings at liver biopsy (n=47) included nonspecific cholestasis (n=19, 40%), giant-cell hepatitis (n=12, 26%), "large bile duct obstruction" (n=9, 19%) in the presence of pigmented stools, and mixed cholestatic/hepatitic features (n=7, 15%). ERCP demonstrated a patent biliary tree in 20 infants (42%). BA was confirmed at exploratory laparotomy in all 3 infants (6%) in whom cannulation failed. The remaining 25 infants (52%) also proceeded to exploratory laparotomy, in which BA was confirmed in 22 (46%). Amongst the 20 children in whom ERCP ruled out BA, 8 (17%) had normal biliary anatomy, whilst 12 (25%) had an abnormal biliary tree, including 6 (12.5%) with neonatal sclerosing cholangitis. After ERCP none developed clinical pancreatitis or peritonitis. CONCLUSIONS: ERCP is a safe procedure for diagnosing BA even in the smallest infants with high positive and negative predictive values.

Transcriptional regulation of the hepatocyte growth factor gene by pyrrolidine dithiocarbamate.

Hepatocyte growth factor (HGF) mediates cancer cell invasion and metastasis. This study characterised the down-regulation of HGF expression by pyrrolidine dithiocarbamate (PDTC), which markedly reduced HGF mRNA expression and protein production in MRC-5 cells. Reporter gene studies revealed that PDTC inhibited HGF gene transcription and that the response element is located in the region -75 to +42 bp flanking the transcription initiation site. Electrophoretic mobility shift assay identified three specific protein complexes binding in this region, which were abrogated by exposure of cells to PDTC. PDTC deserves further investigation as a novel therapeutic agent for HGF-driven cancers.

Chronic mesenteric venous thrombosis: evaluation and determinants of survival during long-term follow-up.

BACKGROUND & AIMS: The natural history of chronic portomesenteric (PM) and portosplenomesenteric (PSM) venous thrombosis is defined poorly. Therapeutic options are limited, and are directed at the prevention of variceal bleeding and the control of abdominal pain related to gastrointestinal hyperemia. METHODS: Patients with extensive PM and PSM thrombosis were reviewed retrospectively to evaluate the efficacy of medical therapy and to determine which clinical variables had prognostic significance regarding long-term survival. RESULTS: Sixty patients, with a median age at diagnosis of 44 years (range, 18-68 y), were assessed. The median follow-up period was 3.5 years (range, 0.2-32.0 y). The overall survival rate was 73.3%, with 1- and 5-year survival rates of 81.6%, and 78.3%, respectively. One- and 5-year survival rates, excluding patients who died from malignancy-related causes, were 85.7% and 82.1%, respectively. Factors associated with improved survival included treatment with beta-blockers (P = .02; odds ratio [OR], .09; 95% confidence interval [CI], 0.01-0.70) and anticoagulation (P = .005; OR, 0.01; 95% CI, <0.01 to 0.26). Eighteen patients in total were anticoagulated, including 8 patients who had variceal bleeding, all of whom underwent endoscopic band ligation of esophageal varices before anticoagulation. By using Cox regression analysis, variables associated with reduced survival were the presence of ascites (P = .001; OR, 42.6; 95% CI, 5.03-360), and hyperbilirubinemia (P = .01; OR, 13.8; 95% CI, 1.9-100) at presentation. Six patients died of variceal hemorrhage. CONCLUSIONS: Patients with chronic PM and PSM venous thrombosis without underlying malignancy have an acceptable long-term survival. Treatment with beta-blockers and anticoagulation appears to improve outcome.

Controversies in the management of primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) remains a rare but significant disease, which affects mainly young males in association with inflammatory bowel disease. There have been few advances in the understanding of the pathogenesis of the condition and no therapeutics with proven mortality benefit aside from liver transplantation. There remain areas of controversy in the management of PSC which include the differentiation from other cholangiopathies, in particular immunoglobulin G4 related sclerosing cholangitis, the management of dominant biliary strictures, and the role of ursodeoxycholic acid. In addition, the timing of liver transplantation in PSC remains difficult to predict with standard liver severity scores. In this review, we address these controversies and highlight the latest evidence base in the management of PSC.

The role of the immune system in the control of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is common, and current therapies may not be suitable for the majority of patients with advanced disease. Cases of spontaneous regression suggest that immune mechanisms are important in the control of HCC. Experiments in animal models have shown that tolerance to HCC associated antigens can be overcome and using a number of different techniques researchers have been able to prevent the growth of implanted tumours. The most promising of these techniques is based on the use of dendritic cells, which are able to process and present antigens to activate naive T cells and, when loaded with tumour antigens, can stimulate a specific and durable anti-tumour response. The success of animal studies has led to interest in the clinical applicability of HCC immunotherapy. Non-specific adoptive immunotherapy has been successful in preventing disease recurrence after resection and cellular vaccines based on dendritic cells are now entering clinical trials. The use of dendritic-cell vaccination raises exciting possibilities of preventing the formation of HCC in high-risk individuals such as those with cirrhosis.

Frequency and nature of cytokine gene polymorphisms in hepatocellular carcinoma in Hong Kong Chinese.

BACKGROUND: The genetic basis of susceptibility to hepatocellular carcinoma (HCC) is poorly understood. Genomic DNA was available from 98 patients with HCC, 77 familial controls, 97 controls from Hong Kong and 96 Northern European controls (NECs). METHODS: Polymorphisms of interleukin (IL)-1beta; IL-1 receptor antagonist (IL-1RN); IL-10 promoter (positions -1082, -819, and -592); and tumor necrosis factor (TNF)-alpha promoter (TNF-a -238 and -308) were investigated. RESULTS: There was marked restriction in the distribution of the IL-1beta and IL-1RN genotypes among Chinese subjects with a predominance of the IL-1beta*1,1 and IL-1RN*1,1 (for unrelated controls compared with NECs only for IL-1beta: chi2 = 15.32, Pc = 0.000091 and for IL-1RN: chi2 = 16.08, Pc = 0.000061). For IL-10, the distribution of alleles was reversed in Chinese vs NECs. The TNFA*2 allele (TNFA -308 A), which is associated with high TNF-alpha production both in vivo and in vitro, was found in <10% of Chinese but was present in 33% of NECs (chi2 = 21.52, Pc <0.0000035). CONCLUSION: Though this study failed to highlight specific associations between any of the polymorphisms tested and the HCC in Hong Kong Chinese, the differences in the distribution of tested alleles may, in part, account for the increased susceptibility of the Chinese population to develop HCC.

Development and validation of an efficient in-house real-time reverse transcription polymerase chain reaction assay for the quantitative detection of serum hepatitis delta virus RNA in a diverse South London population.

Hepatitis delta virus (HDV) causes both acute and chronic hepatitis, always in the presence of hepatitis B. Analysis of global HDV isolates has shown that at least eight genotypes exist. HDV RNA quantitation and genotyping are important tools in the diagnosis and management of infected individuals. There is, as yet, no commercially available quantitative HDV RNA assay. Several laboratories have developed in-house assays, but equivalent detection and quantitation across all HDV genotypes has not been demonstrated. In this study, the development of an in-house real-time reverse transcription polymerase chain reaction (RT PCR) assay is described to quantify HDV RNA in serum or plasma. Its efficiency was validated by testing 99 samples from patients with known chronic HDV infection, along with 22 samples from individuals without HDV. The assay has a dynamic range of 6.4×10(2) to 6.4×10(8)copies/mL. Amplicons of the quantitative PCR can be directly used for sequence analysis and genotyping. HDV-1, HDV-5 and HDV-6 were identified, reflecting the areas of origin of our cohort of patients. The ability to genotype and to accurately quantify HDV RNA levels in the more recently discovered African genotypes will be important for investigating the natural history of HDV in this group, compared to those with genotype 1 disease.

Generation of functional CD8+ T cells by human dendritic cells expressing glypican-3 epitopes.

BACKGROUND: Glypican 3 (GPC-3) is an oncofoetal protein that is expressed in most hepatocellular carcinomas (HCC). Since it is a potential target for T cell immunotherapy, we investigated the generation of functional, GPC-3 specific T cells from peripheral blood mononuclear cells (PBMC). METHODS: Dendritic cells (DC) were derived from adherent PBMC cultured at 37 degrees C for 7 days in X-Vivo, 1% autologous plasma, and 800 u/ml GM-CSF plus 500 u/ml IL-4. Immature DC were transfected with 20 microg of in vitro synthesised GPC-3 mRNA by electroporation using the Easy-ject plus system (Equibio, UK) (300 V, 150 microF and 4 ms pulse time), or pulsed with peptide, and subsequently matured with lipopolysaccharide (LPS). Six predicted GPC-3 peptide epitopes were synthesized using standard f-moc technology and tested for their binding affinity to HLA-A2.1 molecules using the cell line T2. RESULTS: DC transfected with GPC-3 mRNA but not control DC demonstrated strong intracellular staining for GPC-3 and in vitro generated interferon-gamma expressing T cells from autologous PBMC harvested from normal subjects. One peptide, GPC-3522-530 FLAELAYDL, fulfilled our criteria as a naturally processed, HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope: i) it showed high affinity binding to HLA-A2, in T2 cell binding assay; ii) it was generated by the MHC class I processing pathway in DC transfected with GPC-3 mRNA, and iii) HLA-A2 positive DC loaded with the peptide stimulated proliferation in autologous T cells and generated CTL that lysed HLA-A2 and GPC-3 positive target cells. CONCLUSIONS: These findings demonstrate that electroporation of GPC-3 mRNA is an efficient method to load human monocyte-derived DC with antigen because in vitro they generated GPC-3-reactive T cells that were functional, as shown by interferon-gamma production. Furthermore, this study identified a novel naturally processed, HLA-A2-restricted CTL epitope, GPC-3522-530 FLAELAYDL, which can be used to monitor HLA-A2-restricted CTL responses in patients with HCC. Further studies are required to investigate whether anti-GPC-3 immunotherapy has a role in the treatment of GPC-3 dependent tumours, such as HCC.

The importance of steatosis in chronic hepatitis C infection and its management: A review.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease with approximately 180 million people infected worldwide. Hepatic steatosis is a frequent histological finding in chronic hepatitis C (CHC) infection and is 2- to 3-fold more common than would be expected by chance alone. A high body mass index with excess visceral fat distribution is associated with steatosis in patients infected with HCV genotype 1 but not genotype 3, re-enforcing the concept that in patients with CHC, some have "metabolic steatosis", predominantly HCV genotype 1, and others "viral steatosis", mainly HCV genotype 3. Accumulating evidence suggests that steatosis may contribute to progression of fibrosis in CHC. Hepatic insulin resistance appears to play a role through the pro-fibrogenic effects of compensatory hyperinsulinemia. The aim of this review was to assess the effect host and viral factors play in steatosis development in patients with CHC infection and its possible relationship with hepatocellular carcinoma. The review examines the mechanisms by which CHC infection causes hepatic steatosis, the impact hepatic steatosis has on the natural history of the disease and finally, explores if treatments leading to a reduction in the amount of steatosis might lead to improved treatment outcomes. The basic medical science of steatosis in CHC will be discussed including proposed models of steatogenesis and the influence of viral and metabolic factors at the molecular level and how these might impact on current and future therapies.

Monotherapy versus combination therapy for the treatment of chronic hepatitis B.

BACKGROUND: Nucleos(t)ide analogues, active against hepatitis B polymerase, suppress viral replication and improve clinical outcome. However, the emergence of drug-resistant mutants can result in treatment failure. OBJECTIVES: We describe how the choice of first-line therapy is critical to long-term treatment success. METHODS: A review of current drug therapies is provided. RESULTS/CONCLUSIONS: Monotherapy with early-generation drugs (lamivudine or adefovir) was associated with a high rate of viral drug resistance and combination therapy with these agents was shown to reduce the incidence of resistance. The latest-generation drugs (entecavir and tenofovir) are potent inhibitors of viral replication and, in treatment-naive subjects, viral resistance to entecavir is uncommon and is not yet reported to tenofovir. Therefore, monotherapy with either entecavir or tenofovir is the current preferred option in treatment-naive patients. Combination therapy is appropriate in those with drug-resistant HBV infection, where drug choice is guided by the viral drug-resistance genotype/phenotype. Although combination therapy has been advocated in other patient groups (e.g., those with decompensated cirrhosis and following liver transplantation), there are, as yet, no data to mandate the use of combination therapy in such patients and any perceived benefit must be weighed against increased cost and risk for toxicity.