György Illyés

Claudin-4 differentiates biliary tract cancers from hepatocellular carcinomas.

The recently identified claudins are dominant components of tight junctions, responsible for cell adhesion, polarity and paracellular permeability. Certain claudins have been shown to have relevance in tumor development, with some of them, especially claudin-4, even suggested as future therapeutic target. The aim of the present study was to analyze the expression of claudin-4 in the biliary tree, biliary tract cancers and hepatocellular carcinomas. A total of 107 cases were studied: 53 biliary tract cancers, 50 hepatocellular carcinomas, 10 normal liver and 10 normal extrahepatic biliary duct samples. Immunohistochemical analysis was performed on conventional specimens and on tissue microarrays as well. Claudin-4 was further investigated by Western blot analysis and real-time RT-PCR. Intense membranous immunolabeling was found for claudin-4 in all biliary tract cancers unrelated to the primary site of origin, namely intrahepatic, extrahepatic or gallbladder cancers. Normal biliary epithelium showed weak positivity for claudin-4. In contrast, normal hepatocytes and tumor cells of hepatocellular carcinomas did not express claudin-4. The results of Western immunoblot analysis and real-time RT-PCR were in correlation with the immunohistochemical findings. Cytokeratins, as CK7 (92%) and CK19 (83%) were mostly positive in biliary tract cancers, however, one-third of hepatocellular carcinomas also expressed CK7 (34%). HSA antibody (HepPar1) reacted with the majority of hepatocellular carcinomas (86%), while being positive in a low percentage of the biliary tract cancers (8%). In conclusion, this is the first report of a significantly increased claudin-4 expression in biliary tract cancers, which represents a novel feature of tumors of biliary tract origin. Claudin-4 expression seems to be a useful marker in differentiating biliary tract cancers from hepatocellular carcinomas and could well become a potential diagnostic tool.

Structural vascular changes in hypertension: Role of angiotensin II, dietary sodium supplementation, and sympathetic stimulation, alone and in combination in rats

Elevated circulating angiotensin (Ang) II levels, dietary sodium, and sympathetic stimulation are recurrent themes of hypertension research, but their in vivo interaction in physiologically meaningful doses has not been adequately investigated. In this study, the interaction of a subpressor dose of Ang II (50 ng · kg−1 · min−1 SC), 2% NaCl diet, and sympathetic stimulation in the form of overnight cold exposure was investigated in the development of hypertension and of structural vascular changes in male Sprague-Dawley rats. There were 8 experimental groups: sham operation and treatment (control), Ang II, 2% NaCl diet, cold exposure (5°C), Ang II plus 2% NaCl diet, Ang II plus cold exposure, cold exposure plus 2% NaCl diet, and Ang II plus 2% NaCl diet plus cold exposure (triple treatment). For each group, the duration of treatment was 12 weeks. Morphometric measurements of maximally dilated, in situ fixed, second-order (250 to 320 &mgr;m OD), intermediate-size (100 to 150 &mgr;m OD), and small (50 to 100 &mgr;m OD) mesenteric arteries were performed, and wall-to-lumen ratios (W/L) were calculated. During the 12-week study, the blood pressure (BP) load (the area under the systolic BP curve) of rats receiving the combined treatment of Ang II and 2% NaCl diet was increased (P <0.05), and that of rats receiving the combined treatment of cold exposure and 2% NaCl diet was decreased (P <0.05); there were no BP changes in the remaining groups of rats. The most pronounced changes among groups occurred in W/L of small resistance arteries. The W/L of small arteries increased in Ang II–treated (P <0.01) and in cold-stressed rats (P <0.01). The effect of Ang II was potentiated by the addition of a 2% NaCl diet. In contrast, the addition of 2% NaCl diet to cold stress reduced the W/L of small arteries (P <0.01). No other positive or negative synergism occurred among groups, including the rats receiving triple treatment. The findings confirm the potentiation of the hypertensinogenic and vascular trophic effects of Ang II by a high-sodium diet but do not provide evidence for synergism between Ang II and sympathetic stimulation. The finding of hypotension and reduced W/L of small resistance arteries in rats receiving the combined treatment of cold stress and high-sodium diet is unique because there are few known nonpharmacological vascular “hypotrophic” stimuli. The ultimate test of the hypertensinogenic potential of pressor stimuli alone or in combination is their long-term administration in physiologically meaningful doses to experimental animals.

Secretin and autism: a basic morphological study about the distribution of secretin in the nervous system.

For the first time, the relationship between secretin and autism has been demonstrated by one of us. Intravenous administration of secretin in autistic children caused a fivefold higher pancreaticobiliary fluid secretion than in healthy ones and, at least in some of the patients, better mental functions were reported after the secretin test. Because the precise localization of secretin in the brain is still not completely known, the abovementioned observation led us to map secretin immunoreactivity in the nervous system of several mammalian species. In the present work, the distribution of secretin immunoreactivity in cat and human nervous systems was compared with that of rats using an immunohistochemical approach. Secretin immunoreactivity was observed in the following brain structures of both humans and in colchicine-treated rats: (1) Purkinje cells in the cerebellar cortex; (2) central cerebellar nuclei; (3) pyramidal cells in the motor cortex; and (4) primary sensory neurons. Additionally, secretin immnoreactive cells were observed in the human hippocampus and amygdala and in third-order sensory neurons of the rat auditory system. In cats, secretin was only observed in the spinal ganglia. Our findings support the view that secretin is not only a gastrointestinal peptide but that it is also a neuropeptide. Its presence or the lack of its presence may have a role in the development of behavioral disorders.

Development of structural vascular changes in salt-fed rats*

The hypothesis that long-term administration of a physiologically relevant high salt diet to rats leads to the development of structural vascular changes that predispose to hypertension was tested. Adult male Sprague-Dawley rats were fed 2% NaCl diet for 3 or 6 months; rats fed 0.7% NaCl diet were controls. Then, the systemic circulation of the rats was perfusion-fixed at 100 mm Hg. The junction of the mesentery and small intestine, the renal cortex, and segments of left carotid artery, thoracic aorta, and second order mesenteric arteries were embedded in paraffin or epoxy for morphometric measurements. The average monthly tail systolic blood pressure (BP) of salt-fed rats at 3 and 6 months were unchanged. The following morphometric changes in salt-fed rats were observed: 1) dilatation of the carotid artery at 3 months (P <.05); 2) dilatation and reduced wall-to-lumen ratio of the second order mesenteric artery (P <.01); 3) increased wall-to-lumen ratio of small mesenteric resistance arteries (P <.01); 4) reduced wall-to-lumen ratio of renal cortical resistance arteries at 6 months (P <. 05); and 5) unchanged structure of aorta. The long-term administration of a high salt diet leads to structural vascular changes in normotensive rats. There are important regional and segmental variations in the long-term adaptation of arteries to a high salt diet.

Autoimmune pancreatitis associated with immune-mediated inflammation of the papilla of Vater: report on two cases.

Autoimmune pancreatitis (AIP) is defined histologically by periductal and interacinar lymphocytic infiltration. Immunohistochemically, the majority of these lymphocytes are identified as T cells. Epithelial HLA-DR antigen expression was also described as a marker of autoimmunity in this type of chronic pancreatitis. We report 2 cases, a 56-year-old man and a 29-year-old woman, with AIP associated with immune-mediated inflammation of the main duodenal papilla (MDP). Serologically, antinuclear antibody positivity was detected in the male patient. The female patient, treated medically for ulcerative proctitis, had no serological evidence of autoimmune disease. Macroscopic papillitis was present only in the male patient, and endoscopic biopsy samples were taken from this swollen MDP. Since we could not exclude malignancy, a pancreatic head resection was performed in both patients. The histologic and immunohistochemical studies of the resected specimens showed periductal T-lymphocytic infiltration in the pancreatic and papillary tissues. Furthermore, HLA-DR-antigen expression was also demonstrated in epithelial cells of the pancreas and MDP. The immunohistological features of endoscopic biopsy samples from the swollen MDP were identical as in the surgically resected specimens. Immune-mediated inflammation of the MDP may be associated with AIP.

Altered structure and distensibility of arteries in salt-fed rats.

Background and objectives Habitual high-sodium diet may cause stiffening of arteries. The aim of this study was to investigate the long-term effects of physiologically relevant high-sodium diet on the structure and distensibility of arteries in rats. Methods Adult male Sprague–Dawley rats were fed 2% NaCl diet for 3 or 6 months; rats fed 0.7% NaCl diet were controls. Pressure–volume (distensibility) relationships were measured in the presence and absence of calcium in excised, in-vitro perfused segments of right carotid artery and of second order mesenteric arteries. The left carotid artery and the remaining mesenteric arteries of rats were perfused in situ with papaverine followed by fixative at 100 mmHg, and then embedded in epoxy for morphometric measurements. Results The tail systolic blood pressure (SBP), and in subgroups of rats, the directly measured mean arterial pressure (MAP), of salt-fed rats at 3 and 6 months were unchanged. At 3 months, there was dilatation (increased lumen area) of both carotid and mesenteric arteries of salt-fed rats, without a change in distensibility. At 6 months, the lumen area of carotid arteries of salt-fed rats returned to control value (inward remodeling), and carotid artery distensibility remained unchanged. At 6 months, there was further dilatation (P < 0.01) and reduced distensibility (P = 0.01) of mesenteric arteries in salt-fed rats. Conclusions A three-fold increase in dietary sodium intake leads to dilatation of arteries in normotensive rats. When there is compensatory remodeling, the distensibility of arteries remains unchanged; when compensation is lacking, unopposed dilatation is associated with reduced distensibility.

Different structural vascular changes in angiotensin II-treated and cold-stressed rats

The role of the renin-angiotensin and sympathetic nervous system in the pathogenesis of structural vascular changes in experimental hypertension was investigated by comparing the effect of angiotensin II (ANG II) administration and of sympathetic stimulation by cold stress on the structure and composition of mesenteric arteries of rats. Adult male Sprague-Dawley rats were administered subcutaneously 100 ng/kg per min ANG II or exposed to 5 degrees C cold overnight for 12 weeks. Sham-operated rats were controls. At the end of treatment, the mesenteric circulation of rats was perfusion-fixed for morphometric measurements by light microscopy and volume density measurements by point counting on electron micrographs. Tail systolic blood pressure of ANG II-treated rats increased by 37 mm Hg at week 2 and remained elevated for the rest of the experiment. The systolic BP of cold-stressed rats measured at room temperature did not change. These findings were confirmed by direct measurement of mean arterial pressure in free-moving rats. Compared to control rats, medial thickening of large and small arteries in ANG II-treated and cold-stressed rats was the main finding of this study. Thickening of the media in the two treated groups of rats appeared to be due to hypertrophy of vascular muscle, as indicated by the increased width of smooth muscle cells. Loose matrix (interstitial fluid compartment) was increased in media of large arteries of ANG II-treated rats, and collagen was increased in the outer media of arteries of cold-stressed rats. Measurements of compositional changes in addition to morphometric changes are needed to detect differences in the pathogenesis of structural vascular changes in the various forms of hypertension.

Effect of inhibitors of myeloperoxidase on the development of aortic atherosclerosis in an animal model

Our earlier studies have shown that some steroids increase myeloperoxidase enzyme (MPO) release from human granulocytes, and that MPO plasma levels are significantly lower in postclimacteric people. Moreover, we have proven that MPO inhibits production of atherogenic free radical superoxide anion and MPO-inhibitors increase superoxide release. The aim of the present study was to investigate the effect of MPO-inhibitors on the early phase of aortic atherosclerosis, namely the extent of intimal plaques and the thickening of the medial layer. Adult male rabbits were fed with lipid rich food (cholesterol: 1.3%, peanut oil: 8%) for 8 weeks. During this period MPO-inhibitors were also given (4-aminobenzoicacid-hydrazide/ABAH/-13.3 mg/kg/day or indometacin-5 mg/kg/day). All animals developed intimal lipid plaques (raised fatty streaks). The relative plaque-covered areas of the aortas were compared and the media thickness of the aorta was measured on plaque-free as well as plaque-containing areas. The medial smooth muscle density and peroxidase activity of the aortic media were also determined. The media thickness increased (p<0.05) in the cholesterol+ABAH as well as in the cholesterol+indometacin groups up to 375.7 (+/-60.5) and 442.5 (+/-123.4) microm, respectively, compared to the control group (cholesterol feeding alone) where it measured only 308.4 (+/-51.67) microm. The medial peroxidase activity decreased significantly in the indometacin treated group and showed a decreasing tendency using ABAH. In parallel to this there was a tendency of increase in the relative plaque covered areas. The smooth muscle density showed no significant modifications, while inhibitors of the MPO seemed to enhance aortic medial thickness, i.e. the grade of a pre-atherosclerotic lesion, in our animal model. Collectively, the anti-atherogenic effect of certain steroid hormones might be realized through the impact on MPO activity.

Chlamydia pneumoniae in different coronary artery segments in the young.

UNLABELLED Chlamydia pneumoniae has emerged as the most likely pathogen to have a causative role in the development and/or for progression of atherosclerosis. Evidence for this is based on epidemiological and pathological studies. In an effort to better understand the significance of finding C. pneumoniae in atheromata, we examined coronary artery segments of young adults (15-34 years) with and without atherosclerosis. Left anterior descending coronary arteries (LAD) of 74 young adults who died suddenly were examined histologically and for the presence of C. pneumoniae by immunohistochemistry. C. pneumoniae was identified in advanced lesions (Stary types III to VI) in 17 of 32 cases (53%), and in early lesions (Stary type I-II) in 8 of 37 cases (21%), mainly at the proximal segments of the LAD. C. pneumoniae was not found in the intimal and medial layer of normal-appearing coronary arteries. C. pneumoniae was detected in the adventitia in 51 (67%) coronary arteries: in 27 of normal arteries and early lesions (64%), and in 24 of atherosclerotic lesions (75%). C. pneumoniae was found most often in macrophages, less offen in smooth muscle cells. We also observed a correlation between C. pneumoniae positivity and cigarette smoking. IN CONCLUSION C. pneumoniae may relate to the severity of atherosclerosis in young people, and it may thus initiate atherosclerotic injury or facilitate its progression with other risk factors.

Increased total scavenger capacity and decreased liver fat content in rats fed dehydroepiandrosterone and its sulphate on a high-fat diet

Background: Weak androgens have an antioxidant effect in vitro which is represented as a beneficial change in the antioxidant status. Objective: Our aim was to clarify whether dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulphate (DHEAS) oral administration results in beneficial antioxidant changes in Sprague-Dawley adult male rats in vivo. Methods: Groups of experimental animals were fed a high-fat or a normal-fat diet and treated with DHEA or DHEAS in the drinking fluid. The control group was fed a high-fat diet together with untreated drinking fluid. Total scavenger capacity (TSC) was measured before and after 4 weeks of treatment in blood samples using a chemiluminometric assay. Fat content, superoxide dismutase (SOD), catalase and glutathione S-transferase (GST) activity in the liver were determined by Sudan staining and spectrophotometric assessments, respectively, from the fresh frozen tissue. Results: DHEA and the DHEAS treatment showed significantly increased TSC in the groups fed a high-fat diet. The control group and the DHEA- or DHEAS-treated groups on normal diets showed no significant changes in TSC. The total score of liver fat content in the high-fat diet groups showed a marked positivity with Sudan staining, and the groups treated with DHEA or DHEAS had a markedly decreased amount of fat in the liver slides compared to the untreated group on the high-fat diet. Liver SOD activity was decreased in all high-fat diet groups and elevated only in the groups on a normal diet with DHEA or DHEAS treatment. Liver catalase and GST activities were decreased in the groups where TSC was significantly increased. Conclusion: Our results support the hypothesis that DHEA and DHEAS supplementation can improve the antioxidant status in lipid-rich dietary habits.