Endothelial nitric oxide synthase (eNOS) is not upregulated in gastric mucosa of Helicobacter pylori (H. pylori)-positive patients with type 2 diabetes mellitus.

AIM: To evaluate the expression of eNOS and CD34 in gastric mucosa of Helicobacter pylori (H. pylori) positive diabetic patients, in correlation with glycaemic control and diabetic autonomic neuropathy (DAN). METHODS: We prospectively studied 49 diabetic type 2 patients (29 women, mean age 65.32+/-8.56 years) and 30 control subjects (15 women, mean age 58.47+/-12.40) that underwent endoscopy. Biopsies from the body and antrum were evaluated for H. pylori-gastritis, eNOS and angiogenic marker CD34 expression. Statistical analysis in correlation with mean glycosylated haemoglobin (HbA1c) of the last 3 years, and DAN was performed. RESULTS: The two groups were matched for age (p=0.144), sex (p=0.335), H. pylori-infection (p=0.617) and degree of gastritis (p=0.78). eNOS and CD34 attenuated expression correlated with diabetes mellitus (DM) in the corpus (p=0.009 and 0.02, respectively). eNOS and CD34 expression was upregulated in H. pylori-positive controls but not in H. pylori-positive diabetic patients (p=0.010 and 0.007 for the corpus and p=0.036 and 0.047 for the antrum, respectively). eNOS expression correlated with good glycaemic control (GGC) in the gastric corpus (p<0.001) and antrum (p=0.0037) and with absence of DAN (p=0.009 and 0.036, respectively for the corpus and antrum). CONCLUSION: Chronic glycaemic control affects eNOS expression and angiogenesis in the gastric mucosa of patients with type 2 DM. eNOS expression is not upregulated in H. pylori-positive diabetic patients.

Acute hepatitis C in patients receiving hemodialysis.

Hepatitis C virus (HCV) infection is frequent in patients with end-stage renal disease treated by chronic dialysis, with a prevalence varying from 10-65% according to the geographical data. The prevalence is significantly associated with the duration of dialysis and the number of transfused blood products[1,2] and has dramatically declined with efficient blood screening.[3] We studied patients with acute HCV infection in a dialysis unit. The diagnosis was based on both anti-HCV detection and HCV-RNA detection. Other virological tools including HCV genotype determination was also used to tailor treatment to the individual patient and determine its efficacy for a one-year follow-up period. Seventeen patients (7 male and 10 female, mean age: 63.7 +/- 11.6 SD) with acute hepatitis C were enrolled to our study. All of them were followed up for a period of one year after the diagnosis was established. Phylogenetic analysis distinguished two separate HCV subtypes 1b, which were both responsible for this acute infection (see Figure 1). These types did not differ in their behavior on the clinical situation of our patients, as confirmed by the fact that in both groups of patients, there was only one patient who presented with acute illness. Six patients of our study group, three months after the acute infection, received pegylated interferon (Peg-IFNa2a) 135 mug for a six-month period. Four of them responded very well to therapy and at the first determination HCV RNA was below the cutoff point. One of our patients with very high HCV levels (HCV RNA > 50,000,000 IU/mL), despite receiving the same therapy, did not respond well and developed cirrhosis. In conclusion, it is clear from our experience that better information is needed about the current incidence, prevalence, and risk factors for HCV infection in dialysis patients. Algorithms for the diagnosis and management of hepatitis C should be developed by academic societies. Routine screening for hepatitis C also would allow for better definition of the natural history of hepatitis C in patients with end stage renal disease. [image omitted]Figure 1. NS 5B gene phylogenetic tree analysis of the acute hepatitis C epidemic.

Predicting response to peginterferon alpha-2a, lamivudine and the two combined for HBeAg-negative chronic hepatitis B.

OBJECTIVE: In a trial of patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B, 24 week post-treatment biochemical and virological response rates with peginterferon alpha-2a with or without lamivudine were significantly higher than with lamivudine alone. The effect of pre-treatment factors on post-treatment responses was investigated. METHODS: Multivariate analyses were performed using available data from 518 patients treated with peginterferon alpha-2a with or without lamivudine, or with lamivudine alone. A post-treatment response was defined as alanine aminotransferase (ALT) normalisation and hepatitis B virus (HBV) DNA level of <20,000 copies/ml. RESULTS: In logistic regression analyses across all treatment arms, peginterferon alpha-2a (with or without lamivudine) therapy, younger age, female gender, high baseline ALT, low baseline HBV DNA and HBV genotype were identified as significant predictors of combined response at 24 weeks post-treatment. In the peginterferon alpha-2a and lamivudine monotherapy arms, patients with genotypes B or C had a higher chance of response than genotype D infected patients (p<0.001), the latter responding better to the combination than to peginterferon alpha-2a monotherapy (p = 0.015). At 1 year post-treatment, response rates by intention-to-treat analysis were 19.2% for the peginterferon alpha-2a, 19.0% for the combination, and 10.0% for the lamivudine groups, with genotypes B or C associated with a sustained combined response to peginterferon alpha-2a with or without lamivudine therapy. CONCLUSIONS: Baseline ALT and HBV DNA levels, patient age, gender, and infecting HBV genotype significantly influenced combined response at 24 weeks post-treatment, in patients treated with peginterferon alpha-2a and/or lamivudine. At 1 year post-treatment HBV genotype was significantly predictive of efficacy for patients treated with peginterferon alpha-2a with or without lamivudine.

Low conjunction of the cystic duct with the common bile duct: does it correlate with the formation of common bile duct stones?

BACKGROUND: This study aimed to evaluate the accuracy of magnetic resonance cholangiography (MRC) in detecting variants of low cystic duct conjunction, which can be a source of confusion during surgery when unrecognized. METHODS: All cases with both MRC and endoscopic retrograde cholangiography (ERC) indicating suspected common bile duct stones between January 1999 and January 2004 were retrospectively reviewed by investigators blinded to the final diagnosis. Assessment with ERC was regarded as the gold standard. The aim was to find a low conjunction of the cystic duct with the bile duct. The sensitivity and specificity of MRC were calculated in comparison with those for ERC. The cystic junction radial orientation was defined as lateral (insertion diagonally from the right), medial (insertion into the left side of the common hepatic duct), or posteroanterior (overlap of the junction with the bile duct in the posteroanterior view). A spiral cystic duct and a long parallel course were evaluated separately. RESULTS: Low insertion of the cystic duct was found on ERC in 66 of 622 patients (11%; 28 men and 38 women; mean age, 64.5 years). The sensitivity and specificity of MRC for detecting low cystic entrance were 100% (90.4% on an intention-to-diagnose basis and 100%, respectively). In 11 patients (16.6%), the radial orientation of the cysticohepatic junction could not be defined with MRC. The rate of correct MRC delineation was 95% for lateral (n = 21), 77% for medial (n = 26), and 74% for posteroanterior (n = 19) insertion of the cystic duct. CONCLUSION: The findings showed that MRC has good correlation with ERC with regard to the location and anatomic details of cystic duct insertion. Although this does not generate a separate indication for MRC before laparoscopic cholecystectomy, the anatomic information can be of additional use when MRC is clinically indicated in this setting.

Conservation of the conformation and positive charges of hepatitis C virus E2 envelope glycoprotein hypervariable region 1 points to a role in cell attachment.

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease. The HCV polyprotein contains a hypervariable region (HVR1) located at the N terminus of the second envelope glycoprotein E2. The strong variability of this 27-amino-acid region is due to its apparent tolerance of amino acid substitutions together with strong selection pressures exerted by anti-HCV immune responses. No specific function has so far been attributed to HVR1. However, its presence at the surface of the viral particle suggests that it might be involved in viral entry. This would imply that HVR1 is not randomly variable. We sequenced 460 HVR1 clones isolated at various times from six HCV-infected patients receiving alpha interferon therapy (which exerts strong pressure towards quasispecies genetic evolution) and analyzed their amino acid sequences together with those of 1,382 nonredundant HVR1 sequences collected from the EMBL database. We found that (i) despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of HVR1 were highly conserved, and (ii) HVR1 is a globally basic stretch, with the basic residues located at specific sequence positions. This conservation of positively charged residues indicates that HVR1 is involved in interactions with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). As with many other viruses, possible interaction with GAGs probably plays a role in host cell recognition and attachment.

The non-structural 5A protein of hepatitis C virus.

The non-structural (NS)5A protein of hepatitis C virus (HCV) is cleaved, after translation, by the NS3-encoded zinc-dependent serine proteinase, from the NS4B protein upstream and the NS5B protein downstream. The released, mature NS5A protein is a 56 000 MW phosphoprotein (p56), which also exists within infected cells in a hyperphosphorylated form (p58). The NS5A gene has a quasispecies distribution, meaning that various NS5A sequences co-exist, in various proportions, in infected individuals. HCV NS5A appears to be located in cytoplasmic membranes surrounding the nucleus. Its precise functions are not known. HCV non-structural proteins, including NS5A, form a large multiprotein replication complex, which probably directs the replication of the HCV genome. HCV NS5A lacking the 146 N-terminal amino acids is a potent transcriptional activator in vitro. NS5A can also bind to single-strand RNA-dependent protein kinase (PKR) and inhibit its antiviral function. An 'interferon (IFN) sensitivity-determining region' has recently been postulated in the NS5A protein central region in hepatitis C virus (HCV) genotype 1b, but strongly conflicting evidence has been published. In fact, there would seem to be no such region in the NS5A protein, even though NS5A plays an important and complex role in HCV resistance to IFN. Structure-function studies are required to identify precisely how NS5A and IFN interact.

Role of hepatitis C virus in lymphoproliferative disorders after liver transplantation.

It has been suggested that hepatitis C virus (HCV) infection could be associated with B-cell clonal expansion. The aim of this study was to analyze the relationship between lymphoproliferative disorders and HCV infection in liver transplant recipients. We studied 157 patients receiving a liver transplant between January 1989 and May 1997 with a follow-up longer than 3 months. The incidence of posttransplant lymphoproliferative disorders (PTLDs) was analyzed with reference to the indication for liver transplantation, the induction and maintenance immunosuppression, the incidence of acute rejection episodes, and Epstein-Barr virus (EBV) infection. Six PTLDs occurred after a median posttransplant follow-up of 7 months (3.8%). Four of the 6 PTLDs occurred among the 38 patients transplanted for HCV-related cirrhosis, and 2 PTLDs occurred in the 119 patients receiving a liver transplant for non-HCV liver diseases (10.5% vs. 1.7%, respectively; P =.03). The 4-year probability of PTLD was significantly higher in patients receiving a liver transplant for HCV-related cirrhosis than non-HCV liver diseases (12.3% vs. 2.2%, respectively; P =.015). Patients receiving a liver transplant for HCV-related cirrhosis were more likely to receive antithymocyte globulins (ATG). However, in patients treated with ATG, the 4-year probability of PTLD was higher among those patients receiving a liver transplant for HCV-related cirrhosis than for non-HCV liver diseases (27.1% vs. 6.4%, respectively; P =.08). EBV gene products were detected in tumor tissues in 3 of 4 patients with HCV-associated PTLD. Our data suggest that, in addition to EBV infection, 2 mutually nonexclusive factors, i.e., the use of ATG and HCV infection, could play a role in the occurrence of PTLD after a liver transplant for HCV-related cirrhosis.

Evolution of the hepatitis C virus second envelope protein hypervariable region in chronically infected patients receiving alpha interferon therapy.

Sustained hepatitis C virus (HCV) RNA clearance is achieved in 8 to 12% of patients with chronic HCV infection treated with alpha interferon (IFN-alpha) at the approved dose of 3 MU three times a week for 6 months and in about 25% of those receiving this treatment for 12 months. We used single-strand conformation polymorphism analysis combined with cloning and sequencing strategies to characterize the genetic evolution of HCV second envelope gene hypervariable region 1 (HVR1) quasispecies during and after IFN therapy in patients who failed to clear HCV RNA. Sustained HCV RNA clearance was achieved in 6% of patients. Profound changes in HVR1 quasispecies major variants were estimated to occur in 70% of the patients during and after therapy. These changes were evolutionary and were characterized by shifts in the virus population, related to selection and subsequent diversification of minor pretreatment variants. The quasispecies changes appeared to be induced by changes in the host environment likely resulting from the IFN-induced enhancement and post-IFN attenuation of neutralizing and possibly cytotoxic responses against HVR1. The remaining patients had no apparent changes in HVR1 quasispecies major variants, suggesting selection of major pretreatment variants, but some changes were observed in other genomic regions. We conclude that IFN-alpha administration and withdrawal profoundly alters the nature of circulating HCV quasispecies, owing to profound changes in virus-host interactions, in patients in whom sustained HCV RNA clearance fails to occur. These changes are associated with profound alterations of the natural outcome of HCV-related liver disease, raising the hypothesis of a causal relationship.

Genetic complexity of the hypervariable region 1 (HVR1) of hepatitis C virus (HCV): influence on the characteristics of the infection and responses to interferon alfa therapy in patients with chronic hepatitis C.

HCV exists within its host as pools of related genetic variants referred to as quasispecies. The hypervariable region 1 (HVR1) of the E2 envelope gene is subjected to strong selective pressure from neutralizing antibodies. The genetic complexity of this region is defined as the total number of genetic variants within the quasispecies population. The genetic complexity of the HVR1 region was examined in patients with chronic hepatitis C and its relationship with the epidemiology of HCV infection, and its influence on liver disease and the response to interferon treatment were determined in 114 patients with chronic hepatitis C. The genetic complexity of the HVR1 major variants was measured before treatment by using a polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) technique, and was compared with epidemiological, clinical, virological and histological features. The patients were treated with 3 megaunits of interferon (IFN) alfa for 3 to 6 months and the response to treatment was assessed at 3, 6 and 12 months. The HVR1 could be studied in 101 of the 114 patients (89%). Genetic complexity was significantly higher in patients infected through blood transfusion than intravenous drug use (mean complexity index: 5.7 +/- 2.3 vs. 4.7 +/- 1.5, respectively; P = 0.04). This relationship was independent of age and the estimated time since infection. No significant relationship was found with other parameters of infection or liver disease. In univariate analysis, the genetic complexity of HVR1 major variants did not affect the rates of ALT normalization at months 3 and 6 of IFN treatment. HVR1 genetic complexity was lower in patients with a sustained virological response than in non-responders (4.0 +/- 1.7 vs. 5.4 +/- 2.0, respectively; P = 0.07). In multivariate analysis of pretreatment parameters associated with a sustained virological response to treatment, three parameters appeared to be independent predictors of such a response: a low viral load (P < 0.04), a low anti-HCV core IgM titer (P = 0.03) and a low genetic complexity of HVR1 major variants (P < 0.04). In conclusion, the HVR1 of HCV has a quasispecies distribution in infected individuals. Its genetic complexity is significantly higher in transfusion recipients than in intravenous drug users, suggesting that the size of the initial inoculum affects the later emergence and development of viral quasispecies. The genetic complexity of HVR1, together with viral load and the anti-HCV IgM titer, are independent predictors of a sustained virological response to IFN alfa in patients with chronic hepatitis.

Interferon resistance of hepatitis C virus genotype 1b: relationship to nonstructural 5A gene quasispecies mutations.

A 40-amino-acid sequence located in the nonstructural 5A (NS5A) protein of hepatitis C virus genotype 1b (HCV-1b) was recently suggested to be the interferon sensitivity-determining region (ISDR), because HCV-1b strains with an ISDR amino acid sequence identical to that of the prototype strain HCV-J were found to be resistant to alpha interferon (IFN-alpha) whereas strains with amino acid substitutions were found to be sensitive (N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, N. Izumi, F. Marumo, and C. Sato, J. Clin. Invest. 96:224-230, 1995; N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, Y. Ogura, N. Izumi, F. Marumo, and C. Sato, N. Engl. J. Med. 334:77-81, 1996). We used single-strand conformation polymorphism (SSCP) analysis, combined with cloning and sequencing strategies, to characterize NS5A quasispecies in HCV-1b-infected patients and determine the relationships between pre- and posttreatment NS5A quasispecies mutations and the IFN-alpha sensitivity of HCV-1b. The serine residues involved in phosphorylation of NS5A protein were highly conserved both in the various patients and in quasispecies in a given patient, suggesting that phosphorylation is important in NS5A protein function. A hot spot for amino acid substitutions was found at positions 2217 to 2218; it could be the result of either strong selection pressure or tolerance to these amino acid replacements. The proportion of synonymous mutations was significantly higher than the proportion of nonsynonymous mutations, suggesting that genetic variability in the region studied was the result of high mutation rates and viral replication kinetics rather than of positive selection. Sustained HCV RNA clearance was associated with low viral load and low nucleotide sequence entropy, suggesting (i) that the replication kinetics when treatment is started plays a critical role in HCV-1b sensitivity to IFN-alpha and (ii) that HCV-1b resistance to IFN-alpha could be conferred by numerous and/or related mutations that could be patient specific and located at different positions throughout the viral genome and could allow escape variants to be selected by IFN-alpha-stimulated immune responses. No NS5A sequence appeared to be intrinsically resistant or sensitive to IFN-alpha, but the HCV-J sequence was significantly more frequent in nonresponder quasispecies than in sustained virological responder quasispecies, suggesting that the balance between NS5A quasispecies sequences in infected patients could have a subtle regulatory influence on HCV replication.

GB virus C (GBV-C) infection in patients with chronic hepatitis C. Influence on liver disease and on hepatitis virus behaviour: effect of interferon alfa therapy.

The aim of this study was to evaluate, in patients with chronic hepatitis C, 1) the prevalence and the epidemiological characteristics of GB virus C (GBV-C) infection, 2) the influence of GBV-C on hepatitis C virus (HCV) infection, 3) the pathogenicity of GBV-C in the absence of treatment and under interferon therapy, and 4) the effect of interferon alfa on GBV-C and HCV replications. One hundred fifteen patients with chronic hepatitis C were studied. Before treatment, they were tested for GBV-C RNA by PCR and GBV-C genotype was determined for positive samples. Pretreatment information was collected, including age, gender, source of HCV, estimated duration of HCV infection, alanine aminotransferase and gamma-glutamyl transpeptidase activities, cirrhosis and Knodell's score on liver biopsy, HCV genotype, HCV viral burden and anti-HCV core IgM antibodies. The genetic complexity of the hypervariable region 1 (HVR1) of HCV was studied by PCR-Single Strand Conformation Polymorphism. All patients were treated with 3 to 9 mega units of interferon alfa-2a three times per week for 3 to 6 months. The influence of GBV-C on the evolution of ALT and HCV replication during and after treatment was studied, and GBV-C and HCV RNA were monitored monthly by PCR during this period. Eighteen patients (16%) were GBV-C RNA-positive. Among 11 samples studied, GBV-C genotype 2a was present in 9 cases, 2b in one case and type 3 in one case. GBV-C RNA-positive patients were significantly younger than GBV-C RNA-negative ones (38.4 +/- 11.5 vs. 47.4 +/- 14.0, P = 0.012), a result independent of the route of transmission and the disease duration. No difference between GBV-C RNA-positive and -negative patients was found for other epidemiological parameters (e.g. gender, risk factor for parenteral viral infections, disease duration and HCV genotypes), or for the characteristics of HCV infection and related liver disease (e.g. HCV RNA level, genetic complexity of the HVR1, anti-HCV core IgM, alanine aminotransferase and gamma-glutamyl transpeptidase activities, cirrhosis and Knodell's score). GBV-C did not influence the rates of ALT normalization at months 3, 6 and 12 and of sustained hepatitis C virological response at month 12 of treatment follow-up. During treatment, GBV-C viremia became undetectable in 12 patients (67%) but relapse occurred after treatment withdrawal in all the nine patients with sufficient follow-up. In the remaining six patients (33%), GBV-C resisted interferon. Whatever the effect of interferon on GBV-C replication, the ALT levels correlated with the presence of HCV RNA. In conclusion, GBV-C infection is frequent in patients with chronic hepatitis C, who are mainly, but not exclusively, infected by GBV-C genotype 2a. GBV-C positive patients are significantly younger than GBV-C negative ones. GBV-C does not seem to affect HCV replication, liver disease and responses of HCV infection and liver disease to interferon therapy. GBV-C is sensitive to 3 mega units of interferon alfa administered three times per week in two-thirds of the patients, but relapse is constant with this dosage after treatment withdrawal.

Impaired urinary excretion of soluble IL-2 receptors in patients with systemic lupus erythematosus and rheumatoid arthritis.

Several previous studies have demonstrated increased in vivo release of soluble interleukin-2 receptors (sIL-2R) in patients with disorders associated with cellular activation. In this study attempting to understand better the role of sIL-2R released in vivo, we investigated the sIL-2R levels in paired serum and urine samples from 25 patients with systemic lupus erythematosus (SLE), 41 patients with rheumatoid arthritis (RA) and 20 healthy subjects. Using an ELISA for sIL-2R, we detected significantly increased urinary sIL-2R levels in normal individuals (868 +/- 114 units/ml) compared to the corresponding serum samples (209 +/- 25, P < 0.001, mean urine/serum sIL-2R ratio: 4.5 +/- 0.6), which suggests that the clearance of sIL-2R from circulation is largely kidney-dependent. The patients with SLE and RA exhibited significantly increased serum sIL-2R levels compared to normals (682 +/- 115 and 734 +/- 101 units/ml, respectively, P < 0.001) and these levels correlated with disease activity. However, urinary excretion of sIL-2R in these patients (SLE: 620 +/- 154 units/ml; RA: 1084 +/- 148 units/ml) was found to be significantly decreased (mean urine/serum sIL-2R ratio in SLE: 0.9 +/- 0.2; in RA: 1.9 +/- 0.2; P < 0.001) compared to normals, possibly contributing to the accumulation of these soluble receptors in the serum of autoimmune patients after their release from cells. Our findings can be attributed either to the binding of the sIL-2R to serum protein(s) or to distinct structural features of serum sIL-2R in SLE and RA patients, interfering with the urinary excretion of these molecules.

Serum-soluble interleukin-2 receptors in B-cell lymphoproliferative malignancies.

The levels of soluble interleukin-2 receptors (sIL-2R) were determined in the serum of 53 patients with B-cell lymphoproliferative malignancies, including 31 patients with non-Hodgkin lymphomas (NHL), 16 with chronic lymphocytic leukemia (CLL), and 6 with multiple myeloma. In addition, serum samples from 40 patients with various solid tumors as well as from 53 healthy individuals were used as controls. It was found that the mean serum levels of sIL-2R were significantly increased (P less than 0.001) in NHL (mean +/- standard error of the mean 2,327 +/- 320 units/ml) and CLL patients (2517 +/- 451 units/ml) as compared to normal controls (207 +/- 17 units/ml). No such difference was observed when the serum sIL-2R levels of patients with multiple myeloma or solid tumors were analyzed. Serum sIL-2R levels were closely related to the clinical stage, the presence of B-symptoms, and the disease activity of patients with NHL and CLL. In fact, response to chemotherapy was followed by marked decrease or normalization of sIL-2R levels, while in a number of patients sIL-2R values were even able to predict disease relapse. Finally, no association with histologic grade in NHL patients, could be demonstrated. We conclude that serum sIL-2R (1) are increased only in B-NHL and B-CLL but not in myeloma patients, (2) are related to the tumor burden, and (3) can serve as a valuable tumor marker for the monitoring of patients treatment.