Béla Hunyady

Colocalization of Somatostatin Receptor sst5 and Insulin in Rat Pancreatic β-Cells1

Somatostatin, also known as somatotropin release-inhibiting factor (SRIF), is secreted by pancreatic δ-cells and inhibits the secretion of both insulin and glucagon. SRIF initiates its actions by binding to a family of six G protein-coupled receptors (sst1, -2A, -2B, -3, -4, and -5) encoded by five genes. Messenger RNA for both sst2 and sst5 have been reported in the rat pancreas, and the sst2A receptor protein has been localized to rat pancreatic α and pancreatic polypeptide-secreting cells in the islets as well as to pancreatic acinar cells. In this study we have used double immunostaining to show that the sst5 protein is expressed exclusively in the β-cells of rat pancreatic islets and localizes with insulin-secreting α-cells. The sst5 receptor is not colocalized with sst2A. Thus, in the rat SRIF inhibits pancreatic insulin and glucagon secretion via different sst receptor subtypes.

Cell specific expression of the sst2A and sst5 somatostatin receptors in the rat anterior pituitary.

Somatostatin (SRIF), originally described as a hypothalamic hormone that inhibits the release of growth hormone was subsequently shown to inhibit the secretion of multiple pituitary hormones. Five genes encoding six different SRIF receptors (sst1, 2A, 2B, 3, 4 and 5) have been cloned and mRNAs for all five are expressed in the anterior pituitary. We used double immunostaining to determine which cells in the anterior pituitary bear sst2A and sst5 receptors. Our results show that these two receptors are widely distributed in the pituitary gland and are both present in a large percentage of GH cells. In addition, sst5 occurs in a small population of corticotrophs and a large percentage of lactotrophs whereas sst2A is found in only a few lactotrophs but a large number of corticotrophs. The sst2A receptor is also expressed in about a third of the gonadotrophs and thyrotrophs. Interestingly, sst2A and sst5 receptors colocalize in a small percentage of cells, most likely somatotrophs demonstrating that the same cells can contain multiple sst receptor subtypes. These results indicate that sst subtype specific analogs are likely to be useful for the selective regulation of individual pituitary hormones.

Dopamine Produced by the Stomach May Act as a Paracrine/ Autocrine Hormone in the Rat

Dopamine (DA) has been suggested to be a protective factor in the gastrointestinal tract but neither a source of DA nor its exact targets of action have been identified. In this study, we demonstrate high levels of DA (and DOPA) which persist after chemical sympathectomy in the gastric juice of rats. Immunostaining and in situ hybridization histochemistry reveal the presence of tyrosine hydroxylase (TH), DA transporter and vesicular monamine transporters in the acid-producing parietal cells. Like DA, TH enzyme activity remains after chemical sympathectomy. We also demonstrate active reuptake and storage of DA that indicates a regulated release of this neurohormone from parietal cells. DA D1b receptor mRNA is the most abundant DA receptor subtype in gastric and duodenal epithelium. Therefore, we suggest that selective DA D1b receptor agonists may be useful adjuncts in the treatment of duodenal and gastric ulcers. Gastric epithelia possess the hallmarks of functional DA neuroendocrine cells, suggesting that DA has an important role in self-protective mechanisms of the gastrointestinal tract. These findings should allow elucidation of DA role in normal and disease states in the stomach and duodenum.

Tyrosine hydroxylase assay for detection of low levels of enzyme activity in peripheral tissues

A nonisotopic assay for tyrosine hydroxylase, with optimized signal-to-noise ratios, enables determination of low levels of enzyme activity in peripheral tissues. DOPA produced by the enzyme is measured using HPLC with electrochemical detection. Increased signal-to-noise ratios are obtained by including in the reaction mixture glycerol for reduction of blank values and dihydropteridine reductase and NADPH for regeneration of the tetrahydropteridine cofactor. With this method, tyrosine hydroxylase activity can be detected in as few as 200 PC12 cells and in peripheral tissues at levels as low as 4.5 fmol/min/mg wet weight. The assay permits activity to be assessed in a variety of peripheral tissues.

Identification of endogenous peroxidase-containing cells as eosinophils in the gastrointestinal system

Endogenous peroxidase (EPX) activity in certain cells in the gastrointestinal system interferes with immunohistochemical methods based on the horseradish peroxidase-catalyzed substrate deposition. We studied the distribution and characteristics of these cells. We also report an effective and antigen-preserving EPX blocking method, to make possible the evaluation of immunoperoxidase method, to make possible the evaluation of immunoperoxidase stainings in cryostat sections. The EPX-containing cells (EPX cells) are present in every part of the gastrointestinal tract, predominantly in the tunica propria. We identified them as eosinophil cells in May-Grünwald-Giemsa stained sections. The complete match was confirmed by different fluorescence techniques. Firstly, the EPX cells were labeled by a red fluorochrome-conjugated substrate of peroxidase enzymes, rhodamine-tyramide, whereas the eosinophil cells were labeled by the green fluorochrome, 1-hydroxy-3,6,8-pyrenetrisulfonic acid, which is known to label exclusively eosinophilic granules at pH 10. Secondly, all the EPX cells reacted with a monoclonal antibody against the eosinophil peroxidase enzyme. Finally, a set of commercially available leukocyte markers was used to characterize the EPX cells colabeled by fluorochrome-tyramides. Neither macrophages nor mast cells showed EPX activity. Increased numbers and altered distribution were seen in stressed rats and in ulcerated human stomach.

13C-Urea breath test is superior in sensitivity to detect Helicobacter pylori infection than either antral histology or rapid urease test.

There is no single technique which fulfils the criterion for a reference method to detect Helicobacter pylori (Hp) infection. The aim was to compare the results of antral histology (H), rapid urease test (U) and urea breath test (UBT) from antral biopsy samples in patients having gastric or duodenal lesions during upper GI endoscopy. We used the following methods: 1) biopsy specimens for histology (Warthin-Starry staining); 2) rapid urease test; and 3) 13C-urea breath test with infrared spectrometry. The total number of patients was 166 examined by H, U, and UBT. H, U and UBT were negative (-) in 64 patients and positive (+) in 51. The true positivity and false negativity (%, number of patients in parentheses) of each method based upon the positivity of the other two tests were: H+, U+ (54): UBT+, 94.4% (51) and UBT-, 5.6% (3); H+, UBT+ (57): U+, 89.5% (51) and U-, 10.5% (6); U+, UBT+ (65): H+, 78.5% (51) and H-, 21.5% (14). If Hp infection is considered to be positive when at least two tests detect the presence of Hp, UBT shows the highest sensitivity in comparison to histology of biopsy specimens and urease test. UBT is highly recommended as a screening test for Hp infection in patients presenting upper GI endoscopic alterations.

Side-effects of pegylated interferon plus ribavirin therapy with or without protease inhibitor direct acting antiviral agents during treatment of chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection affects 2-3% of the population, approximately 170 million people worldwide, causing chronic HCV-related hepatitis with subsequent liver cirrhosis, hepatic failure, hepatocellular cancer, and liver-related mortality in a large number of patients. The gold standard therapy, pegylated interferon alpha in combination with ribavirin can eradicate hepatitis C virus infection in approx. 40% of treatment-naïve patients infected with HCV genotype G1, and only 15-20% of patients with previous treatment. Success rate is substantially improved with the development and registration of two direct acting anti-hepatitis C virus protease inhibitors (boceprevir and telaprevir) in the second decade of 21st century: combined with the standard therapy, almost three quarter of previously untreated, and more than half of previously unsuccessfully treated patients can achieve sustained viral response with protease inhibitor based triple therapies. A major barrier to successful treatment is the association of peginterferon/ribavirin therapy with frequent and sometimes serious adverse effects. In clinical trials, approximately 10-15% of treated patients discontinue peginterferon and ribavirin due to adverse events; however, in routine clinical practice, the rate of treatment discontinuation has been reported to be substantially higher. The side effects of peginterferon/ribavirin therapy affect virtually all organ systems, and addition of protease inhibitor can amplify these side effects (particularly anemia), and/or may lead to new ones (i.e., dysgeusia with boceprevir or skin rush with telaprevir). There is considerable regional and global variability in the nature and prevalence of these adverse effects as well as in the best strategies to ameliorate their impact on hepatitis C virus treatment. This article summarizes the side effects of dual and triple therapies and their management based on the labels of the drugs, on a comprehensive literature review, as well as on the recently published opinion of an international panel of experts - with the provision of providing help for the physicians treating hepatitis C virus infection to achieve the best possible success with the highest possible safety for the patients.

[IL28B CC genotype: a protective factor and predictor of the response to interferon treatment in chronic hepatitis C virus infection].

INTRODUCTION In chronic hepatitis C-virus infection the possible role of gene variants encoding cytokines has become the focus of interest. AIM The aim of the study was to investigate the effect of IL28B polymorphisms on the outcome of chronic hepatitis C-virus genotype 1 infection in the Hungarian population. In addition, the association between IL28B genotypes and the Th1/Th2 cytokine production of activated peripheral blood monocytes and lymphocytes was evaluated. METHOD Total of 748 chronic hepatitis C-virus genotype 1 positive patients (365 males and 383 females, aged between 18 and 82 years; mean age, 54±10 years) were enrolled, of which 420 patients were treated with pegylated interferon plus ribavirin for 24-72 weeks. Of the 420 patients, 195 patients (46.4%) achieved sustained virological response. The IL28B rs12979860 polymorphism was determined using Custom Taqman SNP Genotyping Assays (Applied Biosystems, Life Technologies, Foster, CA, USA). For cytokine studies, tumour necrosis factor-α, interleukin-2, interferon-γ, interleukin-2 and interleukin-4 production by LPS-stimulated monocytes and PMA-ionomycine activated lymphocytes were measured from the supernatant of the cells obtained from 40 hepatitis C-virus infected patients, using FACS-CBA Becton Dickinson test. The cytokine levels were compared in patients with different (CC, CT, TT) IL28B genotypes. RESULTS The IL28B rs12979860 CC genotype occurred in lower frequency in hepatitis C-virus infected patients than in healthy controls (26.1% vs 51.4%, OR 0.333, p<0.001). Patients carried the T allele with higher frequency than controls (73.9%, vs 48.6%, OR 3.003, p<0.001). Pegylated interferon plus ribavirin treated patients with the IL28B CC genotype achieved higher sustained virological response rate than those with the CT genotype (58.6% vs 40.8%, OR 2.057, p = 0.002), and those who carried the T allele (41.8%, OR1.976, p = 0.002). LPS-induced TLR-4 activation of monocytes resulted in higher tumour necrosis factor-α production in patients with the IL28B CC genotype compared to non-CC individuals (p<0.01). Similarly, increased tumour necrosis factor-α, interleukin-2 and interferon-γ production by lymphocytes was found in the IL28B CC carriers (p<0.01) CONCLUSIONS: The IL28B CC genotype exerts protective effect against chronic hepatitis C-virus infection and may be a pretreatment predictor of sustained virological response during interferon-based antiviral therapy. The IL28B CC polymorphism is associated with increased Th1 cytokine production of activated peripheral blood monocytes and lymphocytes, which may play a role in interferon-induced rapid immune control and sustained virological response of pegylated interferon plus ribavirin treated patients.

Az antioxidáns flavonoid silymarin szupplementációja hatásának placebokontrollos vizsgálata PEG-IFN + ribavirin antivirális kezelésben részesülo krónikus C-hepatitises betegekben

UNLABELLED Since oxidative stress may play a pathogenetic role in chronic hepatitis C, and sustained virological response to antiviral therapy is limited in HCV1 genotype infection, a double blind study was performed in HCV1 patients treated with pegylated interferon + ribavirin, to assess the efficacy of supplementation with the antioxidant flavonoid silymarin. PATIENTS AND METHODS Thirty-two naive HCV1 positive patients with biopsy proven chronic hepatitis C, to be treated with pegylated interferon + ribavirin, have been randomized: group A): 16 patients have been given the antiviral therapy for 6-12 months plus placebo for the first 3 months; group B): 16 patients have been treated with pegylated interferon + ribavirin for 6-12 months plus silymarin, 2 x 166 mg/day, was given for 3 months. Serum alanine aminotransferase and HCV-RNA levels as well as parameters of oxidative stress such as plasma or red blood cell hemolysate, malondialdehyde, superoxide dismutase, glutathione peroxidase, catalase and myeloperoxidase were determined after 0, 1, 3, 6 and 12 months during the treatment. Sustained virological response as undetectable serum HCV RNA was evaluated 24 weeks after the end of therapy. RESULTS In the silymarin group, a more rapid decrease in the malondialdehyde level as well as a marked decrease in superoxide dismutase and an increase in myeloperoxidase activity after month 12 were found, alanine aminotransferase normalized in 6/16 (vs control 9/16) cases, and sustained virological response occurred in 3/16 (vs 7/16) patients. DISCUSSION/CONCLUSION Although silymarin supportation to antiviral therapy improved oxidative stress, it was able to affect favourably neither the alanine aminotransferase nor the sustained virological response. These contradictory findings may be related to randomization bias as patients in study group B had more negative predictors of response: they were older with higher fibrosis score and even with more severe pretreatment baseline oxidative stress. Regarding the recently published in vitro experiments with silybinin on HCV replication as well as the newest convincing clinical observations, we do suggest further studies with more than three times higher doses of silymarin in controlled trials to assess the value of this supplementation in antivirally treated HCV patients.

Effects of Silymarin Supplementation in Patients with Chronic Hepatitis C Receiving PEG-IFN + Ribavirin Antiviral Therapy. A Placebo-Controlled Double Blind Study

Abstract Objectives Since oxidative stress may play a pathogenetic role in chronic hepatitis C and the sustained virological response to antiviral therapy is limited in HCV1 genotype infection, a double blind study was performed in HCV1 patients receiving peginterferon + ribavirin treatment, in order to assess the efficacy of supplementation with an antioxidant flavonoid, silymarin. Patients and Methods 32 naive HCV1-positive patients with biopsy-proven chronic hepatitis C, to be treated with peginterferon + ribavirin, have been randomised as follows: Group A: 16 patients received antiviral therapy for 6 to 12 months plus placebo for the first 3 months; Group B: 16 patients were treated with peginterferon + ribavirin for 6 to 12 months plus they received 166 mg oral silymarin twice a day for 3 months. Serum alanine aminotransferase and HCV RNA levels, as well as parameters of oxidative stress such as plasma or RBC haemolysate malondialdehyde, superoxide dismutase, glutathione peroxidase, catalase and myel...