György Szekeres

Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADP-ribose) polymerase 1 inhibitor in endotoxin-induced septic shock

The lack of efficacy of anti-inflammatory drugs, anti-coagulants, anti-oxidants, etc. in critically ill patients has shifted interest towards developing alternative treatments. Since inhibitors of the nuclear enzyme poly-(ADP-ribose) polymerase (PARP) were found to be beneficial in many pathophysiological conditions associated with oxidative stress and PARP-1 knock-out mice proved to be resistant to bacterial lipopolysaccharide (LPS)-induced septic shock, PARP inhibitors are candidates for such a role. In this study, the mechanism of the protective effect of a potent PARP-1 inhibitor, PJ34 was studied in LPS-induced (20mg/kg, i.p.) septic shock in mice. We demonstrated a significant inflammatory response by magnetic resonance imaging in the dorsal subcutaneous region, in the abdominal regions around the kidneys and in the inter-intestinal cavities. We have found necrotic and apoptotic histological changes as well as obstructed blood vessels in the liver and small intestine. Additionally, we have detected elevated tumor necrosis factor-alpha levels in the serum and nuclear factor kappa B activation in liver of LPS-treated mice. Pre-treating the animals with PJ34 (10mg/kg, i.p.), before the LPS challenge, besides rescuing the animals from LPS-induced death, attenuated all these changes presumably by activating the phosphatidylinositol 3-kinase-Akt/protein kinase B cytoprotective pathway.

Immunohistochemical distribution of vanilloid receptor, calcitonin-gene related peptide and substance P in gastrointestinal mucosa of patients with different gastrointestinal disorders.

The immunohistochemical distribution of capsaicin/vanilloid (transient receptor potential vanilloid 1, TRPV1) receptors and neuropeptides (CGRP, SP) was studied in the gastrointestinal mucosal biopsies of patients with gastritis, erosions, ulcers, polyps, adenocarcinoma, chronic inflammatory bowel diseases, polyps without and with hyperplasia, dysplasia and adenocarcinoma in colon. The studies were carried out in 127 patients and 30 people with only functional dyspepsia (without any histological alteration). The results were: (1) the positivity of TRPV1 receptor and CGRP was detected, and weak participation of SP was detected in patients with different gastric diseases; (2) the presence of TRPV1, CGRP and SP could be detected in chronic inflammation of bowel disease; (3) SP could not detected in patients with colon polyps, dysplasia and adenocarcinoma; (4) the presence of TRPV1 and CGRP was proved in colon dysplasia and adenocarcinoma. We conclude that (1) the immunohistochemical distribution of TRPV1, CGRP and SP differs in gastrointestinal diseases of the upper and lower tract, and (2) the participation of TRPV1, CGRP and SP differs significantly in these different gastrointestinal diseases.

Development and characterisation of a monoclonal antibody family against aquaporin 1 (AQP1) and aquaporin 4 (AQP4)

Recent studies have discovered the existence of water-channel molecules, the so called aquaporins (AQP) presumably involved in active, ATP dependent water transport between the intracellular and extracellular compartments. Both genetic and protein sequences and structures of the AQPs are known and crystallographic analyses of some members of the AQP family have been performed. Specific antibodies are required to examine their histological locations and analyse their roles in physiological and pathological pathways of water transportation and osmotic regulation. Until recently some polyclonal antibodies have been developed against certain members of the AQP family. However, to date highly specific monoclonal antibodies against aquaporins do not exist. We have developed a monoclonal antibody family against the aquaporin 1 (AQP1) and aquaporin 4 (AQP4) molecules. Well-conserved epitop sequences of AQP1 and AQP4 proteins were selected by computer analysis and their synthetic peptide fragments were used as the antigens of immunisation and the following screening. Antibodies were characterised by immunoserological methods (ELISA, dot-blot and immunoblot), flow cytometry and immunohistochemistry of formaldehyde-fixed and paraffin-embedded tissue samples. RT-PCR tests controlled the specificity of the immune reactions. Our monoclonal antibodies recognised AQP1 and AQP4 in all the techniques mentioned above and apparently they are useful both in various quantitative and qualitative measurements of the expressions of AQP1 and AQP4 in several species (human, rat, mouse, invertebrates, even plants). According to preliminary immunohistochemical studies our monoclonal anti-AQP1 and anti-AQP4 antibodies are appropriate tools of patho-morphological examinations on routine formol-paraffin tissue samples.

Immunohistochemical assessment and prognostic value of hepatitis B virus X protein in chronic hepatitis and primary hepatocellular carcinomas using anti-HBxAg monoclonal antibody.

Hepatitis B virus (HBV) is the most meaningful risk factor in chronic hepatitis, cirrhosis and primary hepatocellular carcinoma (PHC). The hepatitis B virus X protein (HBxAg) is a multifunctional protein with many important functions in hepatocellular carcinogenesis. A monoclonal anti-HBxAg antibody was developed in our laboratory and characterized by different methods. Using this antibody HBxAg was detected in formaldehyde fixed paraffin embedded tissue sections of 72 liver biopsies from patients with acute hepatitis, chronic hepatitis, cirrhosis and primary hepatocellular carcinoma. The co-expression of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and HBxAg was compared. The histological and cytological localization of the detected HBxAg showed a characteristic distribution in different stages of HBV infection. Strong and diffuse nuclear reaction was detected in PHC cases in contrast to the focal, cytoplasmic and nuclear labeling in the acute and chronic B hepatitis cases. Our antibody seems to be a suitable prognostic marker for routine pathohistological diagnosis and for comparative pathological and epidemiological research on the development of PHC.

Circadian clocks and tumor biology: What is to learn from human skin biopsies?

Some of the components of the circadian molecular clock have been shown to link directly to tumor suppression. Most studies on human tumorous biopsies with consistently down-regulated clock gene expression suggested a protective role for these genes against cancer formation. To highlight some limitations of this hypothesis we review these data in light of recent evidences from animal research, epidemiologic studies, and clinical data on skin tumors. We emphasize the role of circadian rhythmic orchestration in cellular metabolism with a potential in cancer development.

Participation of vanilloid/capsaicin receptors, calcitonin-gene-related peptide and substance P in gastric protection of omeprazole and omeprazole-like compounds.

The effects of omeprazole and different omeprazole-like compounds, associated with anti-ischaemic, antioxidant and poly(adenosine-diphosphate-ribose) polymerase (PARP) inhibitory properties, on the gastric acid secretion (4 h pylorus-ligated) and indomethacin-induced gastric mucosal damage connected with the specific immunohistochemical distribution of TRPV1, CRGP and SP during the effects of these compounds, were studied. The observations were carried out in CFY-strain rats (180–210 g), according to the standard methods and the above-mentioned parameters were studied in these experimental circumstances without and with application of different compounds. We found that: (1) all of the compounds dose-dependently inhibited the gastric acid secretion and mucosal damage; (2) the expression of TRPV1 receptor, CGRP and SP decreased significantly in both pylorusligated and indomethacin-treated animals and (3) the expression of TRPV1 and CGRP was reduced. Meanwhile, no change was obtained in SP expression during the gastric mucosal protection produced by omeprazole and omeprazole-like compounds. The conclusions were that (1) a functional overlap exists between the capsaicin-sensitive afferent and efferent vagal nerve during omeprazole effects; (2) chemical modification of omeprazole molecule offers a new pathway to obtain a new drug for the introduction in the clinical practice.

Hapten-mediated identification of cell membrane antigens using an anti-FITC monoclonal antibody

A monoclonal anti-FITC antibody (F4/1) was produced and demonstrated to be specific for both the free and protein-conjugated (either soluble or cell-bound) form of fluorescein, or carboxyfluorescein. When mouse thymocytes were labelled with a novel fluorescein derivative 5(6)-carboxyfluorescein succinimidyl ester (CFl-NSE), the incorporation of fluorescein was predominantly membrane-bound as demonstrated immunohistochemically. The coupling of CFl-NSE to cells displays a random distribution pattern as shown by immunoblotting of cell extracts prepared by detergent solubilization of CFl-NSE-labelled thymocytes. In addition, the Thy-1.2 antigen immunoprecipitated from a CFl-NSE-labelled thymocyte lysate with a rat monoclonal antibody (Mab) could be detected using the anti-FITC Mab. The molecular weight of the immunoprecipitated material could be estimated immediately by reference to the FITC-labelled molecular weight markers electrophoresed simultaneously.

Gastrointestinal cytoprotection: from basic science to clinical perspectives.

Abstract.An essential point of cytoprotection is that the prostaglandins are able to prevent chemical-induced gastric mucosal damage without affecting gastric acid secretion, this being originally suggested as a property specific to prostaglandins. Since then gastrointestinal cytoprotection has been shown with various agents (anticholinergic agents, H2RA, growth factors) and retinoids the latter differing from the actions of vitamin A. In examining the various components of gastrointestinal cytoprotection we have performed studies in isolated cells, stable cell lines, animal experiments, healthy human subjects, and in patients with gastrointestinal diseases. Our attention has focused on the effects of cytoprotective agents on cellular viability, mitochondrial and DNA damage, oxygen free radicals, natural antioxidant systems, mucosal biochemistry, vascular events, gastrointestinal mucosal protection as well as in their prevention of different human diseases. This paper gives a short overview on the different approaches for the exploring gastrointestinal cytoprotection.

Capsaicin-sensitive afferentation represents an indifferent defensive pathway from eradication in patients with H. pylori gastritis

AIM To study the role of capsaicin-sensitive afferent nerves in Helicobacter pylori (H. pylori) positive chronic gastritis before and after eradication. METHODS Gastric biopsy samples were obtained from corpus and antrum mucosa of 20 healthy human subjects and 18 patients with H. pylori positive chronic gastritis (n = 18) before and after eradication. Traditional gastric mucosal histology (and Warthin-Starry silver impregnation) and special histochemical examinations were carried out. Immunohistochemistry for capsaicin receptor (TRVP1), calcitonin gene-related peptide (CGRP) and substance P (SP) were carried out by the labeled polymer immunohistological method (Lab Vision Co., USA) using polyclonal rabbit and rat monoclonal antibodies (Abcam Ltd., UK). RESULTS Eradication treatment was successful in 16 patients (89%). Seven patients (7/18, 39%) remained with moderate complaints, meanwhile 11 patients (11/28, 61%) had no complaints. At histological evaluation, normal gastric mucosa was detected in 4 patients after eradication treatment (4/18, 22%), and moderate chronic gastritis could be seen in 14 (14/18, 78%) patients. Positive immuno-staining for capsaicin receptor was seen in 35% (7/20) of controls, 89% (16/18, P < 0.001) in patients before and 72% (13/18, P < 0.03) after eradication. CGRP was positive in 40% (8/20) of controls, and in 100% (18/18, P < 0.001) of patients before and in 100% (18/18, P < 0.001) after eradication. The immune-staining of gastric mucosa for substance-P was positive in 25% (5/20) of healthy controls, and in 5.5% (3/18, P > 0.05) of patients before and in 0% of patients (0/18, P > 0.05) after H. pylori eradication. CONCLUSION Distibution of TRVP1 and CGRP is altered during the development of H. pylori positive chronic gastritis. The immune-staining for TRVP1, CGRP and SP rwemained unchanged before and after H. pylori eradication treatment. The capsaicin-sensitive afferentation is an independent from the eradication treatment. The 6 wk time period might not be enough time for the restituion of chronic H. pylori positive chronic gastritis. The H. pylori infection might not represent the main pathological factor in the development of chronic gastritis.

Placental protein 17b overexpression in human uterine cervical cancer

We found overexpression of placental protein 17b (PP17b), cloned by us, in human squamous cervical carcinomas. Patients’ PP17b serum levels were elevated and declined after surgery. Healthy cervical epithelium was PP17 negative; in cervical intraepithelial neoplasia and carcinoma, basal‐type cells were negative, but squamous‐type cells were positive, for PP17. In HeLa cells, expression of PP17b correlated with cell differentiation and apoptosis.