Maria Luisa Manzano

Effects of delayed freezing of liver biopsies on the detection of hepatitis C virus RNA strands

BACKGROUND/AIMS There are no data about the influence of handling conditions of liver biopsies on the integrity of viral RNAs. We studied the influence of the time delay between obtaining and freezing the liver biopsy on the stability of intrahepatic positive and negative hepatitis C virus RNA (HCV-RNA) strands. METHODS Liver samples from 30 anti-HCV patients were included. For each case, one portion of the liver biopsy (first sample) was immediately frozen (20-28 s), while the other section (second sample) was kept at room temperature (1-30 min) before freezing. Each experimental time point was performed in triplicate using liver samples from three different patients. Semi-quantitative analysis of the positive and negative HCV-RNA strands and of the al-antitrypsin mRNA was performed by a Tth-based reverse-transcription polymerase chain reaction. RESULTS A significant time-related decrease in both positive (r=-0.8412, p=0.001) and negative (r=-0.8539, p=0.001) HCV-RNA strand titres was found in the second liver fractions. There were no appreciable changes in RNA titres in those samples frozen after less than 3 min. The RNA titres decreased in all but two samples incubated for 4-30 min. Thus, 3/15 (20%) and 7/11 (64%) of these samples lost positive and negative HCV-RNA strands, respectively. Alpha-1-antitrypsin mRNA titres decreased significantly (r=-0.8935, p=0.01) in those samples kept at room temperature for more than 4 min. CONCLUSION Freezing of liver samples immediately after extraction is crucial to avoid false negative HCV-RNA detection results, especially for the antigenomic RNA strand.

Clinical outcomes of patients undergoing antiviral therapy while awaiting liver transplantation

BACKGROUND & AIMS Antiviral therapy for the treatment of hepatitis C (HCV) infection has proved to be safe and efficacious in patients with cirrhosis awaiting liver transplantation (LT). However, the information regarding the clinical impact of viral eradication in patients on the waiting list is still limited. The aim of the study was to investigate the probability of delisting in patients who underwent antiviral therapy, and the clinical outcomes of these delisted patients. METHODS Observational, multicenter and retrospective analysis was carried out on prospectively collected data from patients positive for HCV, treated with an interferon-free regimen, while awaiting LT in 18 hospitals in Spain. RESULTS In total, 238 patients were enrolled in the study. The indication for LT was decompensated cirrhosis (with or without hepatocellular carcinoma [HCC]) in 171 (72%) patients, and HCC in 67 (28%) patients. Sustained virologic response (SVR) rate was significantly higher in patients with compensated cirrhosis and HCC (92% vs. 83% in patients with decompensated cirrhosis with or without HCC, p=0.042). Among 122 patients with decompensated cirrhosis without HCC, 29 (24%) were delisted due to improvement. No patient with baseline MELD score >20 was delisted. After delisting (median follow-up of 88weeks), three patients had clinical decompensations and three had de novo HCC. Only two of the patients with HCC had to be re-admitted onto the waiting list. The remaining 23 patients remained stable, with no indication for LT. CONCLUSIONS Antiviral therapy is safe and efficacious in patients awaiting LT. A quarter of patients with decompensated cirrhosis can be delisted asa result of clinical improvement, which appears to be remain stable in most patients. Thus, delisting is a safe strategy that could spare organs and benefit other patients with a more urgent need. LAY SUMMARY Antiviral therapy in patients awaiting liver transplantation is safe and efficacious. Viral eradication allows removal from the waiting list of a quarter of treated patients. Delisting because of clinical improvement is a safe strategy that can spare organs for patients in urgent need.

Enfermedad celíaca y síndrome de Budd-Chiari: una asociación infrecuente

Resumen La enfermedad celiaca puede presentar una enorme heterogeneidad clinica y se asocia con una frecuencia superior a la normal con una serie de enfermedades intestinales y extraintestinales, mediadas inmunologicamente o no. Presentamos un paciente diagnosticado de enfermedad celiaca y de un sindrome de Budd-Chiari, este ultimo con una etiologia desconocida. Esta asociacion solo se ha descrito previamente en casos aislados del norte de Africa. La aparicion de este caso en Espana hace poco probable que la coexistencia de ambos procesos en un mismo paciente sea debida a factores ambientales o geograficos.

A pilot study of β-interferon for treatment of patients with chronic hepatitis B who failed to respond to α-interferon

Abstract Background/Aims : Alpha-interferon achieves persistent loss of hepatitis B virus (HBV) in about 30–40% of patients with chronic hepatitis B. In non-responder patients, the disease may progress leading to complications such as cirrhosis and hepatocellular carcinoma. The aim of the current study was to evaluate the efficacy of beta-interferon in patients with chronic hepatitis B who did not respond to one course of alpha-interferon. Methods : Twenty nine alpha-interferon-non-responder patients with chronic hepatitis B (11 hepatitis B e antigen, HBeAg-positive; 18 HBeAg-negative) were treated with 6 million units beta-interferon five times a week for 24 weeks. The post-treatment follow-up lasted for 48 weeks. Results : At the end of treatment, 38% of patients (18% HBeAg-positive; 50% HBeAg-negative) had normal serum aminotransferase levels and negative serum HBV DNA. At the end of follow-up, HBV DNA was no longer detectable in serum in 21% of patients (18% HBeAg-positive; 22% HBeAg-negative). Beta-interferon was well tolerated and safe. Conclusions : This pilot study suggests that beta-interferon therapy is effective and safe in the retreatment of patients with chronic hepatitis B who had not responded to a previous alpha-interferon cycle.

Randomized prospective study evaluating tenofovir disoproxil fumarate prophylaxis against hepatitis B virus reactivation in anti-HBc-positive patients with rituximab-based regimens to treat hematologic malignancies: The Preblin study

Background Hepatitis B virus (HBV) reactivation in patients with resolved HBV infection (HBsAg negative, antiHBc positive) is uncommon, but potentially fatal. The role of HBV prophylaxis in this setting is uncertain. The aim of this study was to compare the efficacy of tenofovir disoproxil fumarate (TDF) prophylaxis versus close monitoring in antiHBc-positive, HBsAg-negative patients under treatment with rituximab (RTX)-based regimens for hematologic malignancy. Methods PREBLIN is a phase IV, randomized, prospective, open-label, multicenter, parallel-group trial conducted in 17 hospitals throughout Spain. Anti-HBc-positive, HBsAg-negative patients with undetectable HBV DNA were randomized to receive TDF 300 mg once daily (Group I) or observation (Group II). The primary endpoint was the percentage of patients showing HBV reactivation during 18 months following initiation of RTX treatment. Patients with detectable HBV DNA (Group III) received the same dose of TDF and were analyzed together with Group I to investigate TDF safety. Results Sixty-one patients were enrolled in the study, 33 in the TDF treatment group and 28 in the observation group. By ITT analysis, HBV reactivation was 0% (0/33) in the study group and 10.7% (3/28) in the observation group (p = 0.091). None of the patients in either group showed significant differences in liver function parameters between baseline and the last follow-up sample. TDF was generally well tolerated and there were no severe treatment-related adverse events. Conclusion In patients with hematological malignancy and resolved hepatitis B infection receiving RTX-based regimens, HBV reactivation did not occur in patients given TDF prophylaxis.

Asociación entre hiperhomocisteinemia y esteatosis hepática en pacientes con hepatitis crónica C

BACKGROUND AND OBJECTIVES Liver steatosis in chronic hepatitis C (CHC) is related to viral and metabolic factors and likely to genetic factors. The aim of this study was to know if hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR)-C677T polymorphisms are associated with liver steatosis in nonalcoholic patients with CHC. PATIENTS AND METHOD In 54 consecutive patients with CHC, alcohol consumption less than 40g/week, and no other causes of liver disease, a liver biopsy was performed. All variables were obtained at the time of biopsy. MTHFR-C677T was also performed in 128 healthy subjects, with age and gender similar to the patients. RESULTS Liver steatosis was found in 33 patients (61%), 30 of them having a mild degree. Hyperhomocysteinemia was more prevalent in patients with steatosis (61% vs 24%; p=0.008) and overweight tended to be more prevalent in the same patients (61% vs 33%; p=0.05). All patients with virus C genotype 3 had steatosis. Viral load, liver inflammatory and fibrosis score were not different in patients with and without steatosis. MTHFR-C677T polymorphism was similar in controls and cases and in cases with and without steatosis. A multiple logistic regression showed that hyperhomocysteinemia was associated with liver steatosis after adjustment for age and sex (OR: 3.94; 95% CI: 1.09-14.29), and adjustment for overweight (OR: 4.43; 95% CI: 1.27-15.51). CONCLUSIONS In nonalcoholic patients with CHC mild liver steatosis is frequent, and is associated with hyperhomocysteinemia. An association between steatosis and MTHFR-C677T polymorphism was not found.

OriginalAsociación entre hiperhomocisteinemia y esteatosis hepática en pacientes con hepatitis crónica CAssociation of hyperhomocysteinemia with liver steatosis in patients with chronic hepatitis C

BACKGROUND AND OBJECTIVES Liver steatosis in chronic hepatitis C (CHC) is related to viral and metabolic factors and likely to genetic factors. The aim of this study was to know if hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR)-C677T polymorphisms are associated with liver steatosis in nonalcoholic patients with CHC. PATIENTS AND METHOD In 54 consecutive patients with CHC, alcohol consumption less than 40g/week, and no other causes of liver disease, a liver biopsy was performed. All variables were obtained at the time of biopsy. MTHFR-C677T was also performed in 128 healthy subjects, with age and gender similar to the patients. RESULTS Liver steatosis was found in 33 patients (61%), 30 of them having a mild degree. Hyperhomocysteinemia was more prevalent in patients with steatosis (61% vs 24%; p=0.008) and overweight tended to be more prevalent in the same patients (61% vs 33%; p=0.05). All patients with virus C genotype 3 had steatosis. Viral load, liver inflammatory and fibrosis score were not different in patients with and without steatosis. MTHFR-C677T polymorphism was similar in controls and cases and in cases with and without steatosis. A multiple logistic regression showed that hyperhomocysteinemia was associated with liver steatosis after adjustment for age and sex (OR: 3.94; 95% CI: 1.09-14.29), and adjustment for overweight (OR: 4.43; 95% CI: 1.27-15.51). CONCLUSIONS In nonalcoholic patients with CHC mild liver steatosis is frequent, and is associated with hyperhomocysteinemia. An association between steatosis and MTHFR-C677T polymorphism was not found.

Higher proliferative capacity of T lymphocytes from patients with Crohn disease than from ulcerative colitis is disclosed by use of Herpesvirus saimiri-transformed T-cell lines

Background: T lymphocytes play a crucial role in the pathogenesis of inflammatory bowel disease. Achieving stable T-cell lines, rather than continuous bleeding of patients, is desirable in order to dissect their implication in the disease. Methods: Long-lasting T-cell lines from patients with Crohn disease and ulcerative colitis and from healthy volunteers have been obtained by transformation of T lymphocytes using the lymphotropic Herpesvirus saimiri. Lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. Results: Fresh cells revealed only minor differences between patients and controls, with regard to phenotype and proliferative capacity. In contrast, the use of T-cell lines showed that cells from Crohn disease patients, but not ulcerative colitis patients, over-responded to several membrane or cytoplasmic stimuli when compared to control T-cell lines. Thus, higher responses were found when stimulated with αCD3 and IL2, αCD3 and αCD28, IL2 alone, phorbol esters (PMA) and αCD3 and, finally, PMA and αCD2 (P < 0.05 in all instances). Further, lines from patients with Crohn disease responded more vigorously to αCD3 and αCD28 or αCD3 and PMA when compared to ulcerative colitis (P < 0.05 in both instances). Conclusions: The data obtained with these lines suggest that T cells from patients with Crohn disease differ in vivo in their proliferative capacity, as compared with those from ulcerative colitis patients, a finding that may reflect the clear Th-1 phenotype found in the former and absent in the latter.

Herpesvirus saimiri (HVS)-transformed T-cell lines: A method to study mucosal T cells in inflammatory bowel disease

Alterations in the mechanisms regulating the immune response are key elements in inflammatory bowel disease (IBD, comprising Crohn’s disease (CD) and ulcerative colitis (UC)). Although the precise aetiology remains unknown, T lymphocytes play a crucial role in its pathogenesis. Initially, descriptions of the immunological features in IBD focused on peripheral blood lymphocytes. Since these data do not necessarily reflect the situation at the mucosal milieu, it is important to assess the phenotypic profile and activation state of immune cells at this level. We have used a common lymphotropic virus of squirrel monkeys, Herpesvirus saimiri (HVS), to immortalize T cells of intestinal mucosal origin from patients and healthy individuals as controls. HVS-transformed T lymphocytes become antigenand mitogen-independent for their continuous growth and upon stimulation with membrane or transmembrane stimuli, these cells show normal downstream functional responses [1,2].

290 Relevance of DNA Repair Polymorphisms As Genetic Markers of Gastric Cancer Susceptibility and Prognosis in Caucasians

Colorectal cancer (CRC) screening has been shown to reduce CRC incidence and mortality through the endoscopic detection and removal of colorectal adenomas. Still, these patients are at increased risk for developing metachronous adenomas or even cancer, with the recurrence rate reaching the 50%. The pleiotropic effects of higher levels of PGE2 contribute to key steps of cancer development, including cell proliferation, angiogenesis, invasiveness and migration, inhibition of apoptosis and immunosurveillance as a refletion of deregulation of ATP-binding cassete sub-family c member 4 (ABCC4) and solute carrier organic anion transporter family, member 2A1 (SLCO2A1) genes responsable for carrying PGE2 accross the membrane. To evaluate the influence of genetic polymorphisms in ABCC4 and SLCO2A1 on the risk and time for colorectal adenoma recurrence a retrospective case-cohort study was designed gathering 195 patients diagnosed with colorectal adenomas. Adenoma reccurence was defined has the diagnosis of an adenoma after a total normal colonoscopy at least one year after the initial diagnosis. Thirty-three tagSNPs were characterized using the MassARRAY iPLEX Gold technology based on multiplex amplification followed by mass-spectrometric product separation. Three tagSNPs were identified as susceptibility biomarkers for colorectal adenoma recurrence after a bootstrap analysis. The rs1131598GG homozygous genotype of SLCO2A1 gene was associated with an enhanced risk of 6.3 (95%CI:1.31-30.0, P=0.021). In contrast and under a dominant model of inheritance, the rs1751031 and rs9524821 polymorphisms in ABCC4 gene displayed a protective behaviour (OR=0.29, 95%CI:0.12-0.72, P=0.007 and OR=0.42, 95%CI:0.19-0.93, P=0.033, respectively). Furthermore, when stratifying patients considering the endoscopic findings at baseline colonoscopy, low-risk individuals carriers of rs2274403AA genotype in ABCC4 gene had a lower interval until recurrence (85 (29140) vs 122 (109-135), P=0.011) with 44% of metachronous tumors developing by 36 months (vs 23% for AG/GG). This study demonstrates for the first time the involvement of genetic variants in PGE2 transporters in colorectal adenoma recurrence. The incorporation of genetically-based approaches might allow an optimization of current risk models for the development of metachronous colorectal adenomas or even more advanced lesions possible laeding to a decrease in CRC burden and mortality.