MicroRNAs in myocardial ischemia: identifying new targets and tools for treating heart disease. New frontiers for miR-medicine.

MicroRNAs (miRNAs) are natural, single-stranded, small RNA molecules which subtly control gene expression. Several studies indicate that specific miRNAs can regulate heart function both in development and disease. Despite prevention programs and new therapeutic agents, cardiovascular disease remains the main cause of death in developed countries. The elevated number of heart failure episodes is mostly due to myocardial infarction (MI). An increasing number of studies have been carried out reporting changes in miRNAs gene expression and exploring their role in MI and heart failure. In this review, we furnish a critical analysis of where the frontier of knowledge has arrived in the fields of basic and translational research on miRNAs in cardiac ischemia. We first summarize the basal information on miRNA biology and regulation, especially concentrating on the feedback loops which control cardiac-enriched miRNAs. A focus on the role of miRNAs in the pathogenesis of myocardial ischemia and in the attenuation of injury is presented. Particular attention is given to cardiomyocyte death (apoptosis and necrosis), fibrosis, neovascularization, and heart failure. Then, we address the potential of miR-diagnosis (miRNAs as disease biomarkers) and miR-drugs (miRNAs as therapeutic targets) for cardiac ischemia and heart failure. Finally, we evaluate the use of miRNAs in the emerging field of regenerative medicine.

Strategies for successful vaccination against hepatocellular carcinoma.

Current therapies against hepatocellular carcinoma (HCC) are not curative in the majority of patients. In the past, immunotherapy approaches aimed to non-specifically stimulate immune response were quite ineffective. New treatments based on stimulation of specific anti-tumor immune response are currently proposed and appear more promising. Tumor-specific antigens identified in HCC demonstrated immunogenicity both in preclinical and clinical trials. Effectiveness in animal studies raised interest in the clinical applicability of non-specific adoptive immunotherapy that prevented disease recurrence after tumor resection. Dendritic cell (DC)-based tumor vaccines achieved encouraging results, and cellular vaccines based on DCs have already entered clinical trials. Preventive and therapeutic DNA vaccination have been proposed, all based on tumor-associated antigens (TAAs), either modified or not, an example being alpha-fetoprotein (AFP). The concomitant expression of co-stimulatory molecules and cytokines was used to increase tumor immunogenicity. Syngeneic or nude mice models indicated that immunotherapy for HCC could stimulate an anti-tumor T-cell response leading to clinical benefit devoid of significant toxicity. The use of DNA-based vaccination raises exciting possibilities in preventing HCC in high-risk individuals such as those with cirrhosis. Novel immunotherapy strategies may contribute in the future to prevention and treatment of HCC.

Sub-epitopic dissection of HCV E1315-328HRMAWDMMMNWSPT sequence by similarity analysis.

Our labs are focused on identifying amino acid sequences having the ability to react specifically with the functional binding site of a complementary antibody. Our epitopic definition is based on the analysis of the similarity level of antigenic amino acid sequences to the host proteome. Here, the similarity profile to the human proteome of an HCV E1 immunodominant epitope, i.e. the HCV E1(315-328)HRMAWDMMMNWSPT sequence, led to i) characterizing the immunoreactive HCV E1 315-328 region as a sequence endowed with a low level of similarity to human proteins; ii) defining 2 contiguous immunodominant linear determinants respectively located at the NH(2) and COOH terminus of the conserved viral antigenic sequence. This study supports the hypothesis that low sequence similarity to the host's proteome modulates the pool of epitopic amino acid sequences in a viral antigen, and appears of potential value in defining immunogenic viral peptide sequences to be used in immunotherapeutic approaches for HCV treatment.

[Gastroenterology outpatient clinic of the Molinette Hospital (Turin, Italy): the 2003-2006 report]. / Poliambulatori specialistici di gastroenterologia: l'esperienza 2003-2006 dell'Ospedale Molinette di Torino.

AIM: Given the demographic shifts and needs of cost rationalization, it is of high priority to organize health care on the basis of ambulatory outpatients models. The aim of this study was to examine activity at the gastro-hepatology outpatients clinic of the Molinette Hospital. In this facility, the management is based on a work team organization that follows cohorts of patients with specific pathologies. METHODS: All services, consultations and urea breath test (UBT) for the diagnosis of Helicobacter pylori infection, carried out from January 2003 to December 2006, were extrapolated from the computerized system. Consultations were divided into first examination and controls. Furthermore, the destination of the patients after each consultation was considered. RESULTS: During the year 2003, 8 842 consultations and 4 071 UBT were carried out, in the year 2004, 11 342 consultations and 2 409 UBT, in the year 2005, 12 474 consultations and 2 510 UBT, in the year 2006, 12 249 consultations and 2 357 UBT. No further specialistic management was required for 25% of patients, while 2% had been hospitalized in the bed unit, 3% in the short hospitalization unit or the day-hospital. The remaining 70% were included in work teams or monitored thereafter. The comparison with consultations from 1994 shows an increase due to both first examination (+300%) and controls (+83%). CONCLUSIONS: The burden of the requests from the population and primary care structures addressed to the outpatients clinic of gastro-hepatology is relevant. The activity of this facility leads to a low rate of hospitalization as well as of cost reduction.

Detection of helicobacter candidatus suis by PCR in oesophagogastric ulcers of swine in Italy.

The aim of this study was to evaluate by PCR the presence of Helicobacter spp. in gastric mucus from the fundic region of the stomach and to investigate its role in oesophagogastric ulcers in swine bred and regularly slaughtered in Piedmont (Northern Italy). Stomachs from 595 regularly slaughtered swine were subjected to gross pathological examination in order to evaluate the presence of gastric ulcers (revealed in 75 cases, 12.6%). Histopathological examination was performed to better characterise erosions and ulcers. DNA extracted from gastric mucus collected from all the ulcer-affected and from 25 normal stomachs was submitted to PCR using Helicobacter spp. 16S rRNA gene target primers. Sixty-three percent (47/75) of the affected stomachs was positive as well as 24% (6/25) of the non-affected ones. Sequence analysis from 5 positive samples showed 99% homology with Helicobacter candidatus suis 16S ribosomal RNA gene.

How accurate is the culture of Helicobacter pylori in a clinical setting? An appraisal.

AIM: The trend towards increasing prevalence of Helicobacter pylori (H. pylori) antibiotic resistance may jeopardize the efficacy of most regimens. Culture of the bacterium, the useful method able to address therapy, is influenced by various factors. Thus, validation of the procedure is fundamental. Most studies have been carried out in microbiological settings, while only few have been conducted in clinical frames. We evaluated the accuracy of culture for detection of H. pylori in a clinical dedicated laboratory. METHODS: Forty-six patients (28 females, 18 males, mean age 56+/-4.7 years) were included. Thirty experienced failure to H. pylori eradication after at least 3 courses of treatment. The control group included 16 subjects suffering from gastroesophageal reflux disease and negativity for H. pylori infection. Diagnostic strategy was based on histology, culture testing, serology and 13C-urea breath test. A patient was considered infected if 2 tests were positive. A commercial culture medium in microaerophilic atmosphere was utilized. RESULTS: Out of 30 positive specimens, culture correctly identified 29. In 1 case, no growth of micro-organisms occurred. In the control group, bacterial culture accurately identified all negative samples. One of them indicated growth but neither aspect nor confirmation tests identified H. pylori. Sensitivity was 96.7%, specificity 100%, and accuracy 97.8%. Positive and negative predictive values were 100% and 94.1%, respectively. CONCLUSIONS: Culture of H. pylori is a feasible method and provides a good level of diagnostic accuracy even in a clinical setting by following international guidelines combined with training of specialized personnel.

HLA-DR5 antigen. A genetic factor influencing the outcome of hepatitis C virus infection?

BACKGROUND: Prognosis may be quite different among individuals infected with hepatitis C virus (HCV): a chronic liver disease is believed to occur in half the patients while in the other half there are no signs of histologic progression of liver damage. The host immune response might play an important role in such different outcomes. A relationship has been shown between HLA genes and immune response to viral hepatitis B, but to our knowledge, no evidence of an association with HCV has been reported so far. We investigated whether HLA class II alleles might influence the outcome of HCV infection. METHODS: Eighty-seven individuals, positive for anti-HCV by second-generation enzyme-linked immunosorbent assay and recombinant immunoblot assay tests, enrolled from May 1, 1991, to June 31, 1992, were evaluated. Thirty-six were symptom-free subjects found to have HCV antibodies when screened for blood donation: they all had normal results of liver function tests, normal results of physical examination, and normal hepatobiliary ultrasonography. Fifty-one were patients diagnosed as having a chronic liver disease by percutaneous liver biopsy specimen; histologic assessment was chronic persistent hepatitis in 15, chronic active hepatitis in 28, and liver cirrhosis in eight. A group of 231 donors of platelets and bone marrow, negative for anti-HCV, was used as a control population. All participants were typed for HLA class II antigens (DR and DQ) using National Institutes of Health recommended microlymphocytotoxicity test and were followed up by means of alanine aminotransferase and HCV testing for at least 1 year. RESULTS: Frequency of HLA-DR5 antigen was higher in symptom-free anti-HCV-positive individuals (52.8%) than among HCV-related patients with chronic liver disease (13.7%). The difference was statistically significant (corrected P value = .005; 95% confidence interval, 19.6% to 58.6%); between DR5 and long-term evolution of hepatitis C, there was a negative association (relative risk = 0.142). Moreover, frequency of HLA-DR5-positive subjects appeared to be inversely proportional to severity of liver disease (52.8% in symptom-free patients, 26.6% in patients with chronic persistent hepatitis, 10.7% in patients with chronic active hepatitis, and 0% in patients with liver cirrhosis, P < .001). CONCLUSIONS: Our results point to a strict relationship between HLA haplotype and ability of immune response to influence the outcome of HCV infection. Presence of HLA-DR5 antigen appears as a protective factor against a severe outcome of chronic infection, being correlated with a benign evolution of the infection, often asymptomatic, or a less severe chronic liver disease.

[Intestinal metaplasia, dysplasia, gastric cancer and Helicobacter pylori: epidemiological observations]. / Metaplasia intestinale, displasia, cancro gastrico e Helicobacter pylori: considerazioni epidemiologiche.

Gastric cancer (GC) is the world's second leading cause of cancer-related mortality, and carries a bad prognosis. In 1994, Helicobacter pylori (H. pylori) has been classified by the International Agency for Research on Cancer as a group I carcinogen. There are increasing indications that this infection is associated with both the initiation and progress of gastric carcinoma and lymphoma. Evidence supporting a causal association has been demonstrated by epidemiological data and in experimental animal models. Despite this, there is still lack of final conclusion regarding the association between the infection and the malignancy due both to marked geographic variations and heterogeneity in study designs. Given the high rate of morbidity and mortality associated with GC, any means of reducing the occurrence of the disease or increase its early detection is most desirable. In this paper, the epidemiological aspects on the evidence of a causal relationship between H. pylori and GC are discussed. Prospective cohort studies and interventional trials focused on the effects of H. pylori eradication on lesions predisposing to GC should be performed in order to provide further data.

Helicobacter pylori and stomach cancer: a case-control study in Venezuela.

The role of Helicobacter pylori infection in gastric cancer was evaluated in a high-risk population in Venezuela using serological assays in a study of 302 cases and 483 neighborhood controls. To investigate the claim that assays for H. pylori should use antigens derived from local strains, four different assays derived from Venezuelan and European strains were used. Prevalence of IgG H. pylori antibodies in controls was very high, with estimates between 72 and 92%. Prevalence was similar in cases and controls. However, cases had lower antibody titers. This effect was observed only in subjects with low pepsinogen (PG) levels PGI/PGII <3.0), which suggested that extensive atrophy in cases causes a loss of H. pylori infection, with a consequent reduction in antibody titer. In addition, advanced cases (stage II or higher) had lower antibody titers than less advanced cases, which indicated that the lower antibody titers in cases may be attributable partially to a diminished immune response. All of the four assays for anti-H. pylori antibodies gave similar results. No evidence was found for the superiority of the assay based on Venezuelan strains. These results are consistent with other case-control studies in high-risk populations and highlight the difficulties of investigating H. pylori infection in retrospective studies.

A solid-phase enzyme immunoassay (EIA) for detection of HBeAg and anti-HBe.

A sensitive, reproducible and easily performed enzyme immunoassay (EIA) based on a sandwich technique is described for serological detection of HBeAg and anti-HBe. EIA appears to be 600 times more sensitive than immunodiffusion and counterimmunoelectrophoresis and its performance compares well with available radioimmunoassays.

Radioimmunoassay and characterization of woodchuck hepatitis virus core antigen and antibody.

Solid-phase radioimmunoassays for woodchuck hepatitis virus core antigen (WHcAg) and antibody (anti-WHc) were developed. WHcAg in woodchuck liver homogenates was characterized by ultracentrifugation in CsCl gradients; both heavy (1.35 g/cm3) and light (1.31 g/cm3) cores were obtained from the liver of an animal during acute WHV infection, which is consistent with observations in hepatitis B virus infection in man. Endpoint titers of anti-WHc were higher in chronic WHV carriers than in animals recovered from acute infections. Both IgM and IgG anti-WHc antibodies were produced by infected woodchucks. A survey of colony woodchucks demonstrated that 88/89 animals having one or more markers of past or ongoing WHV infection were positive for anti-WHc. Thus, serum anti-WHc appears to be a sensitive marker of WHV infection.

Histogenesis of primary liver carcinomas: strengths and weaknesses of cytokeratin profile and albumin mRNA detection.

To assess the utility of cytokeratin (CK) profile and albumin mRNA detection (as revealed by in situ hybridization) in the differential diagnosis of primary liver carcinomas (PLCs) we evaluated a series of surgically resected PLCs, comprising 20 "pure" hepatocellular carcinomas (HCCs) (10 well-differentiated, 10 poorly differentiated), 15 cholangiocarcinomas (CCs) (6 peripheral, 5 hilar, and 4 major duct ones) and 10 hepatocholangio-carcinomas (HCC-CCs). 11 of 20 (55%) of the pure HCCs expressed CKs of pure hepatocytic lineage (CK 8 and CK 18); 2 of 10 (20%) of the HCC-CCs displayed only hepatocytic profile, whereas 12 of 15 (80%) of the CCs evidenced mature bile duct cell phenotype (CK 8, CK 18, CK 7, CK 19). All HCCs expressed varying distributions of albumin mRNA, whereas 4 of 6 (67%) peripheral CCs showed cells with focal positivity for albumin mRNA. This suggests that the phenotypic expression of PLC cells are often not fixed, and in particular: (1) peripheral CCs have a different phenotype from hilar and large duct ones; (2) the CK profile and albumin mRNA expression in peripheral CCs show many similarities with those of some HCCs. Furthermore, the results show that a mixed biological phenotype (ie, CK 8, CK 18 and CK 7 and/or CK 19) can be found both among morphologically pure HCCs and peripheral CCs, suggesting that these two forms could share a common histogenesis. We think that special attention should be given to cases in which CK profile and albumin mRNA reveal mixed phenotype, as these tumors could have different biological behavior and respond differently to therapy.

Susceptibility of human liver cell cultures to hepatitis C virus infection.

To develop a cell culture system susceptible to infection by hepatitis C virus (HCV), human fetal hepatocytes, grown in serum-free medium, were inoculated with serum samples from two HCV-infected patients. Viral RNA sequences were detected by polymerase chain reaction, using primers specific for the 5' noncoding region of HCV, in extracts prepared from the hepatocyte cultures as early as 5 days after inoculation. Virus was also released from the infected cells into the medium. The HCV strains could be serially passaged three times into fresh liver cell cultures using intracellular virus as inoculum. Evidence that HCV replication really took place in primary human fetal hepatocytes was also obtained by detection of minus-strand viral RNA (replication intermediate) in cell extracts and of viral antigens in the infected cells.

Extended lymph node dissection for gastric cancer: results of a prospective, multi-centre analysis of morbidity and mortality in 118 consecutive cases.

This study reports interim data on post-operative morbidity, hospital mortality and duration of hospital stay of Italian patients undergoing extended lymph-node dissection combined with a pancreas-preserving technique for gastric cancer. Of the 218 patients admitted to one of eight general and/or university hospitals in North Italy, 118 were enrolled in the trial. Eligible patients presented with proven primary adenocarcinoma of the stomach without clinical evidence of distant, peritoneal and/or liver metastasis, or metastasis in para-aortic and retropancreatic nodes at intraoperative biopsy. Patients underwent the extended procedure as described by the Japanese Research Society for the Study of Gastric Cancer, following the Maruyama pancreas-preserving technique. A strict quality control system was used to ensure the performance of a standard surgical treatment. A surgeon of the reference centre (M.D.), who stayed at the National Cancer Center Hospital in Tokyo to learn the D2 technique from a specialist Japanese surgeon, became the trial supervisor and assisted each surgeon in all the Italian participating centres. The patients were staged according both to the TNM system and to the General Rules for the Gastric Cancer Study in Surgery and Pathology. Post-operative surgical complications developed in 21 patients (17.8%). The non-surgical complication rate was 2.5%. Reoperation was necessary in six patients (5%), all of whom survived. The 30-day mortality rate for the eligible group was 2.5%. The overall hospital mortality was the same. Total gastrectomy was associated with a slightly higher operative mortality (4.5% vs 1.3%). Only one patient died from an anastomotic leak. The rate of leakages was higher after total than after distal gastrectomy (15.9 vs 5.4%); the association of splenectomy and pancreatectomy worsened the morbidity rate. D2 lymphadenectomy with pancreas-preserving technique, when performed at experienced centres, seems a feasible and safe technique for the radical treatment of gastric cancer in selected Western patients.

Helicobacter pylori: from the stomach to the heart.

A surprising number of extra-gastrointestinal diseases have been reported to be associated with Helicobacter pylori infection, including coronary heart disease and stroke. Since coronary heart disease is the principal cause of death in western countries, and since the known risk factors cannot fully explain the pathogenic mechanisms of the disease, the exploration of the role of possible causal agents has stimulated intense research. Infectious agents have been linked to coronary heart disease on epidemiological and pathogenic grounds. In 1994, H. pylori infection was reported to be one of them. Since then, a number of studies have been published with controversial results. Studies performed thus far show a high degree of heterogeneity in the selection of patients and also in the type of disease studied, i.e. coronary heart disease in general or acute myocardial infarction. Since the pathogenic development is most likely different for each of these two conditions (one chronic and the other acute) they should be studied separately. H. pylori infection can cause platelet aggregation and induces a procoagulant activity. H. pylori can also contribute to atherosclerosis, through increased concentration of homocysteine in the blood, caused by decreased levels of folic acid and cobalamin, or to an autoimmune process. Prospective cohort studies and interventional trials focusing separately on the chronic and acute phases of coronary heart disease and H. pylori infection should be performed in order to provide firm epidemiological data for a causal relationship.

Helicobacter pylori infection and infertility.

OBJECTIVE: To determine (1) the prevalence of Helicobacter pylori infection in male and female patients with reproductive disorders and controls; (2) the presence of anti-H. pylori antibodies in samples of follicular fluid, vaginal secretions and sperm; and (3) the existence of a structural homology between a major spermatozoa protein, tubulin, and H. pylori proteins. PATIENTS AND METHODS: Serum samples from 167 patients with infertility and 837 age- and gender-matched controls (blood donors) were examined by enzyme-linked immunosorbent assay (ELISA) and Western blotting to determine the seropositivity for H. pylori infection. The presence of anti-H. pylori antibodies in samples of follicular fluid, vaginal secretions and sperm was determined using the same techniques. The possible cross-reactivity with spermatozoa of anti-H. pylori hyperimmune sera and human antibodies was studied by immunofluorescence. The N-acid homology of human tubulin with the principal H. pylori proteins was assayed by the WU-blastp program available on the Internet. RESULTS: The prevalence of infection was significantly higher in patients than controls (49.1% v. 33.5%, P < 0.001). Follicular fluids from infected patients contained specific antibodies in all cases, sperm samples in about 50% of cases, and vaginal secretions in a minority of cases. Sera to H. pylori whole antigens and VacA reacted with the tails and the pericentriolar area of human spermatozoa (which are rich in tubulin); sera to urease and heat-shock protein (Hsp) did not. Follicular fluids with anti-H. pylori antibodies immune reacted with spermatozoa. A linear homology was found between beta-tubulin and three H. pylori proteins, flagellin, VacA and CagA. CONCLUSIONS: H. pylori infection may increase the risk of developing reproductive disorders or worsen the clinical expression of this syndrome.

The journey from hepatitis to hepatocellular carcinoma. Bridging role of Helicobacter species.

Hepatocellular carcinoma (HCC) is a long-term consequence of chronic liver disease, whose aetiology could result from viral, environmental and hereditary causes. Viral infection, by itself, could only partially explain the pathogenesis of cirrhosis and HCC. A new aetiologic agent capable of inducing chronic active hepatitis and hepatocellular tumours was discovered: it is a bacterium belonging to the genus Helicobacter, and named H. hepaticus. Presence of sequences belonging to the 16S rRNA of Helicobacter species (spp.) has been demonstrated in liver of most patients with cirrhosis and HCC. H. pylori and related bacteria, such as H. hepaticus, produce toxins that kill hepatocyte by a granulating effect on liver cell lines. In vivo, such toxins might reach the liver through the portal tract, thereby causing hepatocellular damage. The recognition of Helicobacter spp. as a possible risk factor for cirrhosis and HCC might have a practical impact on the general population: the treatment of this infection is easy and far less expensive than liver transplantation or any long term treatment for the other risk factors of HCC. Any confirmation of the involvement of Helicobacter in liver disease would eventually come from the success of culturing the bacterium from liver tissues. Future research is needed to clarify the importance of Helicobacter spp. in respect to the other pathogens already known as causative agents of chronic inflammation of the liver and its long term sequelae, namely cirrhosis and HCC.