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.

HCV infection by cell-to-cell transmission: choice or necessity?

In vitro models of HCV infection have allowed for the clarifying of molecules and mechanisms involved in the main steps of virus cell-entry. HCV entry and neutralization appear to be closely related. Neutralizing antibodies inhibit the E2-CD81 binding, therefore CD81 is considered to be a major target of immune response. The tight-junction proteins are also implicated in E2-binding to CD81 and successive steps of virus entry, in cooperation with several co-receptors, whose involvement has still to be elucidated. Increasing evidence has emphasized the importance of cell-to-cell HCV-transmission in chronic infection. This route for infection could favour virus-escape from host-neutralization though its CD81-dependency is still debated. The main reasons which have delayed our understanding of HCV-infection are here critically reviewed, as are the challenges faced by investigators in the field. A deeper insight into the different pathways involved could help to elucidate some crucial features of HCV infection mechanisms and disclose important implications in its pathogenesis, which could help in suggesting new targets for successful immune-prophylactic/therapeutic strategies.

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.

[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.

Effect of HCV infection on THP-1 monocytoid cells.

Hepatitis C virus (HCV) is the main cause of hepatocellular carcinoma in industrialized countries. HCV-HIV-1 co-infection occurs frequently among users of illicit intravenous drugs, thereby increasing the severity of HIV disease and the evolution of chronic active hepatitis towards cirrhosis and hepatocellular carcinoma. The present work shows that THP-1 monocytoid cells are susceptible to HCV infection, of strain 1b, and that this strain can induce cellular modifications in this cell line. Infection of HCV was demonstrated by positivity for the E2 antigen within THP-1 cells and by indirect immunofluorescence; moreover, HCV-RNA was detected in supernatants of THP-1 cells from day 7 post-inoculation. Cell shape and membrane surface antigens varied upon viral infection, which is also capable of inducing oxygen radicals. In particular we underline the relevant intracellular accumulation of ferritin that paralleled an increase of cell surface expression of the transferrin receptor. Evaluation of cellular events upon HCV infection in THP-1 cells may represent a useful tool with which to identify alteration in monocytes metabolism and to study therapeutic approaches for such alterations.

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.

Gastro-oesophageal reflux disease and asthma: would be possible to improve therapy on the basis of what is now known?

Gastro-oesophageal reflux disease (GORD) is a chronic and relapsing clinical condition, associated or not to histopathologic alteration resulting from repeated contact of oesophageal mucosa with gastric content. This condition occurs with high prevalence in the general population and represents one of the most frequent reasons for health care-seeking addressed to primary care physician and gastroenterologists. A plethora of extra-oesophageal manifestations have been described in patients suffering from GORD and a causal relationship has been postulated by many investigators. A large cohort of studies has focused on the possible cause-effect interaction between GORD and asthma. However, despite the improvement in both asthma symptoms and medication requirements after anti-secretory therapy, no change in pulmonary function is evident. Furthermore, the pathogenetic mechanism, a vagally mediated reflex, microaspiration or increasing reactivity to the stimuli, is yet unclear. Since conflicting conclusions and the failure to find a causal relationship are generally due to the heterogeneity of the studies, further research is needed to clarify the role of GORD in asthma pathogenesis, or viceversa, and whether a medical or surgical anti-secretory treatment may be an approach to curing the asthma patients non-responders to the classical therapy.

HCV infective virions can be carried by human platelets.

It has been previously demonstrated that platelets (PLTs) can bind and transport HIV-1 infectious virions. Hepatitis C virus (HCV)-HIV-1 co-infection occurs frequently among users of illicit intravenous drugs, thereby increasing the severity of HIV disease and the evolution towards chronic active hepatitis and hepatocellular carcinoma of HCV-related hepatitis. In the present study we investigated whether or not PLTs can carry HCV, and studied the binding mechanisms. Purified PLTs, obtained from healthy donors, HCV negative and HIV negative, were adsorbed with HCV-containing serum and then employed to infect a THP-1 monocytoid cell line. Replication of HCV was observed as shown by positivity for the E2 antigen within THP-1 cells, by indirect immunofluorescence; moreover, HCV-RNA was detected in supernatants of THP-1 cells at day 7 post-incubation with HCV-adsorbed PLTs. The binding of HCV to PLTs seems to involve fibronectin (FN), as already shown in the case of HIV-1. Indeed, treatment with RGD (Gly-Arg-Gly-Asp-Ser), the key oligopeptide of FN binding, inhibits the ability of HCV to be carried by PLTs in infective forms; the same phenomenon occurs with Mabs to FN. Moreover the infection of THP-1 cells seems to increase FN surface expression, as demonstrated by immunofluorescence tests.

[The diagnosis of helicobacter pylori infection: guidelines from the Maastricht 2-2000 Consensus Report]. / La diagnosi dell'infezione da Helicobacter pylori: raccomandazioni dal Maastricht 2-2000 Consensus Report.

The European Helicobacter pylori Study Group (EHPSG), during the Maastricht 2-2000 Workshop, revised and updated the original guidelines on the management of Helicobacter pylori (H. pylori) infection. The present review focuses on the diagnostic approach for patients referred to the primary care as well as to the specialist. Currently, two diagnostic methods can be used to detect H. pylori: invasive (urease test, histological detection, culture, polymerase chain reaction, smear examination, string test) or non-invasive (serology, urea breath test, antigen stool assay, ''doctor's tests'') tests. These methods vary in their sensitivity and specificity, and the choice depends on the situation, for example, whether the aim is to detect infection or the success of eradication treatment. Urea breath test (UBT) and antigen stool assay are recommended from EHPSG in patients without alarm symptoms or under 45 years of age, at low risk of malignancy in the ''test and treat strategy''. Confirmation of H. pylori eradication following treatment should be tested by UBT; a stool antigen assay is the alternative if the former is not available. Important added value can be gained from other tests: histology allows evaluation of the status of the mucosa while culture allows strain typing and tests for antibiotic susceptibility.

Helicobacter pylori infection in patients with Hepatitis C Virus positive chronic liver diseases.

BACKGROUND AND GOALS: One-third of patients with liver cirrhosis suffers from acute peptic ulcer, a disease strongly correlated with Helicobacter pylori (H. pylori) infection. We report the seroprevalence of antibodies to H. pylori in 179 patients with Hepatitis C Virus (HCV)-related chronic active hepatitis and cirrhosis. MATERIALS AND METHODS: Among patients, 135 (86 males and 49 females, mean age 51.2 +/- 13.28, range 27-77 years) had chronic active hepatitis (CAH) and 44 cirrhosis (28 males and 16 females, mean age 62.4 +/- 9.2, range 37-77 years). Serum antibodies to H. pylori were tested using a commercial enzyme immunosorbent assay. The control population consisted of 619 consecutive blood donors (523 males, 96 females, mean age 47 +/- 5.3 years, range 18-65). RESULTS: The overall prevalence of antibodies to H. pylori was 73.1% (131/179) among patients and 47% (291/619) among blood donors (p<0.0001; OR 3.08 [95%CI, 2.10-4.51]). 70.5% (24/34) of patients aged less than 40 years were seropositive for H. pylori versus 34.2% (90/263) of controls (p<0.0001; OR 4.61[95%CI, 2.0-10.85]). Among cirrhosis patients, the prevalence of antibodies to H. pylori was 79.5% (35/44) versus 47% (291/619) of controls (p<0.0001; OR 4.38 [95%CI, 1.98-9.98]). Overall seroprevalence among CAH patients was 71.1% (96/135) versus 47% (291/619) of blood donors (p<0.0001; OR 2.77 [95%CI, 1.82-4.24]). CONCLUSIONS: The high seroprevalence of antibodies to H. pylori in patients with HCV-positive liver diseases explains the elevated incidence of peptic ulcer, and warrants studies on the pathogenic role in human liver diseases of Helicobacter spp which is known to cause chronic hepatitis and hepatocellular carcinoma in mice.