Sections Of Stomach Research Articles

The Effect of Trifolium, Raphanus, and Cistus Pollen Grains on Some Blood Parameters and Mesentery Mast Cells

Three kinds of pollen taxa belonging to 3 families (Fabaceae--Trifolium spp., Brassicaceae--Raphanus spp. and Cistaceae--Cistus spp.) and commonly collected by honeybees were fed to mature male rats separately, in the form of 60 mg/animal/day for a 30-day period. The objective of this study was to investigate any positive effects or possible side effects of the use of pollen on the immune system. This was achieved through blood analysis and cell count on blood, hemoglobin, erythrocyte and immune system cells. The cell concentration of mast cells, degranulization and cell localization were investigated in prepared mesentery tissue samples. Histological investigations of the stomach and duedenum sections of pollen-fed rats were carried out to learn the reason for eosinophil gastroenteritis in the alimentary canal. The eosinophil and lymphocyte levels of rats fed with pollen of Trifolium spp., Raphanus spp., and Cistus spp. were observed to have increased blood cell counts, while neutrophil and monocyte levels decreased; different values were found in basophil leucocytes between the pollen groups. Differing reductions in mesentery mast cell concentration, degranulization and cell localization were found. Within the three separate pollens, the rats having been fed with Cistus spp. pollen were observed to have higher blood lymphocyte, eosinophil, hemoglobin and hematocrit values than those fed with the others, as well as low mesentery mast cell concentration. Hemoglobin values were determined to increase at a proportion of between 10.0-11.3%. No difference was found in other blood parameters. The fat proportion of the male rats fed with the three taxa was between 4.03-8.75%, while that for protein proportion was between 16.11-24.25%. Male rats receiving these taxa did not experience allergic reactions and it is possible to argue that the low protein and fat content of these pollens have a strengthening effect on the immune systems by the increase in lymphocyte content and the amount of hemoglobin leads to an increase of oxygen transport capacity in the tissues.

Chitosan microparticles containing plasmid DNA as potential oral gene delivery system

The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. Chitosan has been shown to effectively bind DNA in saline or acetic acid solution and protect DNA from nuclease degradation. In this study, pDNA (plasmid DNA) was encapsulated in chitosan microparticles. Chitosan–DNA microparticles were prepared using a complex coacervation process and stability of plasmid DNA was investigated in this complex. The chitosan–DNA microparticles could protect the encapsulated plasmid DNA from nuclease degradation. Release of pDNA from microparticles was studied in simulated gastric, simulated intestinal medium and acidic PBS (phosphate buffer saline) (pH 4.5) buffer at 37 °C, and released pDNA was assayed spectrophotometrically. In vitro release of pDNA from chitosan microparticles was dependent on pH, as the pH of the release medium increased release profile decreased. In in vivo-animal studies blue color was observed with X-gal (4-chloro-5-bromo-3-indolyl-β-galactosidase) staining of histological stomach and small intestine sections after oral administration of pDNA–chitosan microparticles as an indicator of exogeneous gene expression.

Trypanosoma cruzi surface molecule gp90 downregulates invasion of gastric mucosal epithelium in orally infected mice

Experiments were performed to elucidate why Trypanosoma cruzi isolates 573 and 587 differ widely in their efficiency to infect gastric mucosal epithelium when administered orally to mice. These isolates have the same surface profile and a similar capacity to enter host cells in vitro. Metacyclic forms of isolates 573 and 587 and the control CL isolate expressed similar levels of gp82, which is a cell invasion-promoting molecule. Expression of gp90, a molecule that downregulates cell invasion, was lower in the CL isolate. Consistent with this profile, ~threefold fewer parasites of isolates 573 and 587 entered epithelial HeLa cells, as compared to the CL isolate. No difference in the rate of intracellular parasite replication was observed between isolates. When given orally to mice, metacyclic forms of isolate 573, like the CL isolate, produced high parasitemia (> 10 6 parasites per ml at the peak), killing ~40% of animals, whereas infection with isolate 587 resulted in low parasitemia (< 10 5 parasites per ml), with zero mortality. On the fourth day post-inoculation, tissue sections of the mouse stomach stained with hematoxylin and eosin showed a four to sixfold higher number of epithelial cells infected with isolate 573 or CL than with isolate 587. The rate of intracellular parasite development was similar in all isolates. Mimicking in vivo infection, parasites were treated with pepsin at acidic pH and then assayed for their ability to enter HeLa cells or explanted gastric epithelial cells. Pepsin extensively digested gp90 from isolate 573 and significantly increased invasion of both cells, but had minor effect on gp90 or infectivity of isolates 587 and CL. The profile of g82 digestion was similar in isolates 573 and 587, with partial degradation to a ~70 kDa fragment, which preserved the target cell binding domain as well as the region involved in gastric mucin adhesion. Gp82 from CL isolate was resistant to pepsin. Assays with parasites recovered from the mouse stomach 2 h after oral infection showed an extensive digestion of gp90 and increased infectivity of isolate 573, but not of isolate 587 or CL. Our data indicate that T. cruzi infection in vitro does not always correlate with in vivo infection because host factors may act on parasites, modulating their infectivity, as is the case of pepsin digestion of isolate 573 gp90.

Tissue transglutaminase antibodies in celiac disease, comparison of an enzyme linked immunosorbent assay and a dot blot assay

Aims. – The purpose of our study is to determine the sensitivity, specificity and predictive values of an enzyme linked immunosorbent assay (ELISA) and a dot blot assay for the detection of IgA class anti-tissue transglutaminase antibodies (IgA-AtTGA) and to compare these results with those of IgA class anti-endomysium antibodies (IgA-AEA), IgA class anti-reticulin antibodies (IgA-ARA) and IgA class anti-gliadin antibodies (IgA-AGA). Patients. – Serum samples from 143 patients (97 children, 46 adults) with untreated celiac disease (CD) confirmed by intestinal biopsy and 74 disease controls (64 children, 10 adults) were studied. Methods. – The anti-tissue transglutaminase antibodies were detected by dot blot assay and an ELISA using guinea pig tissue transglutaminase (gp-tTG) as antigen. The anti-endomysium antibodies were detected by an indirect immunofluorescence technique on cryostat sections of human umbilical cord. The anti-reticulin antibodies were also investigated by indirect immunofluorescence on cryostat sections of kidney, liver and stomach of rat. The anti-gliadin antibodies were determined by an ELISA. Results. – The sensitivity of an ELISA for the detection of anti-tissue transglutaminase antibodies was 86% in children and 87% in adults and the sensitivity of dot blot assay was 57% in children and 54% in adults. The specificity of an ELISA and dot blot for the detection for anti-tissue transglutaminase antibodies was, respectively, 96% and 88% lower than that of anti-endomysium antibodies (100%). The sensitivity of anti-gliadin antibodies was 97% in children and 91% in adults and their specificity was 85%. The sensitivity of anti-reticulin antibodies was 94% in children and 87% in adults. Their specificity was 100%. Conclusions. – The sensitivity and specificity of an ELISA for the detection of anti-tissue transglutaminase antibodies were better than that of dot blot assay. However, this dot blot assay could screen four celiac patients who have not had anti-tissue transglutaminase antibodies by an ELISA. The sensitivity of anti-endomysium antibodies was better than that of anti-tissue transglutaminase antibodies, anti-reticulin antibodies and anti-gliadin antibodies but in children aged less than 2 years, the sensitivity of anti-gliadin antibodies was better than that of anti-tissue transglutaminase antibodies.

Relationship between cell proliferation and tenascin-C expression in canine gastrointestinal tumours and normal mucosa

Tenascin-C is an extracellular matrix glycoprotein that has been implicated in cell proliferation and adhesion by in vitro experiments. Its expression is known to be increased in canine and human gastrointestinal tumours. The aim of this study was to investigate the possible relationship between cell proliferation and tenascin expression in these tumours. In tissue sections of normal stomach, small intestine and colon, and gastrointestinal epithelial tumours, the monoclonal antibody Ki-67, which is directed against a proliferation-associated nuclear antigen, was used to identify proliferating cells. Serial sections were also stained for tenascin. Serial sections stained for tenascin and Ki-67 were compared to determine whether there is a correlation between tenascin expression and tumour cell proliferation. In the normal gastric mucosa, Ki-67 positive cells were confined to the neck region and in the normal small intestinal mucosa positive cells were confined to the lower parts of the crypts. In adenomas and carcinomas, the frequency of positive cells was increased at the edges of adenomas and invasive tumour margins of carcinomas and there was inter- and intra-tumoural heterogeneity. Carcinomas with lymphatic invasion showed a high Ki-67-index. There was no relation between cell proliferation and tenascin expression in both normal tissues and tumours studied. The absence of a correlation between tenascin and Ki-67 expression suggests that the main function of tenascin in both normal tissues and tumours of the canine gastrointestinal tract is antiadhesion rather than proliferation.

Protective immunity against Helicobacter is characterized by a unique transcriptional signature

Immunization with a whole-cell sonicate vaccine of Helicobacter felis in conjunction with cholera toxin as a mucosal adjuvant induces long-term protective immunity in a majority of laboratory mice. We have combined gene expression profiling and immunohistochemical analysis on a set of immunized animals to better understand the mechanism of protection. The stomachs of protected animals exhibited a strikingly different transcriptional profile compared with those of nonprotected or control mice, indicating that vaccination targets the appropriate site and leaves a molecular signature. Among the genes whose up-regulation is significantly correlated with protection are a number of adipocyte-specific factors. These include the fat-cell-specific cytokines adipsin, resistin, and adiponectin and the adipocyte surface marker CD36. Interestingly, potentially protective T and B lymphocytes can be found embedded in the adipose tissue surrounding protected stomachs but never in control or unprotected stomachs. Adipsin-specific immunohistochemical staining of protected stomach sections further revealed molecular cross-talk between adjacent lymphoid and adipose cell populations. We propose a mechanism of protection that involves the effector responses of either or both lymphocyte subclasses as well as the previously unappreciated paracrine functions of adipose tissue surrounding the resident lymphocytes.

Role of the Helicobacter pylori outer-membrane proteins AlpA and AlpB in colonization of the guinea pig stomach.

The human gastric pathogen Helicobacter pylori expresses several putative outer-membrane proteins (OMPs), but the role of individual OMPs in colonization of the stomach by H. pylori is still poorly understood. The role of four such OMPs (AlpA, AlpB, OipA and HopZ) in a guinea pig model of H. pylori infection has been investigated. Single alpA, alpB, hopZ and oipA isogenic mutants were constructed in the guinea pig-adapted, wild-type H. pylori strain GP15. Guinea pigs were inoculated intragastrically with the wild-type strain, single mutants or a mixture of the wild-type and a single mutant in a 1: 1 ratio. Three weeks after infection, H. pylori could be isolated from stomach sections of all animals that were infected with the wild-type, the hopZ mutant or the oipA mutant, but from only five of nine (P = 0.18) and one of seven (P = 0.02) animals that were infected with the alpA or alpB mutants, respectively. The hopZ and oipA mutants colonized the majority of animals that were inoculated with the strain mixture, whereas alpA and alpB mutants could not be isolated from animals that were infected with the strain mixture (P < 0.01). Specific IgG antibody responses were observed in all animals that were infected with either the wild-type or a mutant, but IgG levels were lower in animals that were infected with either the alpA or the alpB mutants, compared to the wild-type strain (P < 0.05). In conclusion, absence of AlpA or AlpB is a serious disadvantage for colonization of the stomach by H. pylori.

Failure to detect Chlamydia pneumoniae in senile calcific aortic stenosis or calcified congenital bicuspid aortic valve by immunofluorescence, polymerase chain reaction and electron microscopy

Introduction: Chlamydia pneumoniae has been identified in arterial atherosclerosis. Aortic valves affected by senile calcific aortic stenosis (SCAS) or calcification of a congenital bicuspid valve (CCBAV) may have interior environments similar to atherosclerosis. This study aimed to detect C. pneumoniae within either SCAS or CCBAV. Methods: 60 valves showing either SCAS ( n=36) or CCABV ( n=22) and control valves ( n=2) were studied for the presence of C. pneumoniae by the following three techniques: (1) indirect immunofluorescence (IF) was performed on 36 SCAS valves, 22 CCBAV valves and 2 control aortic valves using a HEp-2 cell line infected with C. pneumoniae as a positive control. Negative controls comprised duplicate slides of the same valves with omission of the primary antibody step. A section of human stomach was also used as a negative control. A semiquantitative scoring method was used to grade positive IF staining. (2) Polymerase chain reaction (PCR) was performed on 30 SCAS valves, 20 CCBAV valves and 1 control valve using C. pneumoniae as a positive control and negative controls comprised a Ureaplasma sp. and human DNA from peripheral blood mononuclear cells. (3) Electron microscopy (EM) was performed upon 13 SCAS, 8 CCBAV and 2 control valves. Results: All three methods failed to detect the presence of C. pneumoniae in any of the 60 aortic valves examined. False positive IF staining was encountered in 81% of test valves and in 76% of negative control valve sections (positive in calcified material due to nonspecific binding of FITC-conjugated secondary antibody). No staining was observed in the negative control stomach sections. Conclusions: This study failed to detect C. pneumoniae within aortic valves showing SCAS or CCBAV. Studies using IF alone to detect C. pneumoniae in calcified tissues should be interpreted with caution, since nonspecific binding of FITC-conjugated secondary antibody by calcium in the cusps may be misinterpreted as evidence of Chlamydia. The use of appropriate controls and ancillary methods for the identification of C. pneumoniae are important in this regard.

High-fat, high-cholesterol diet increases the incidence of gastritis in LDL receptor-negative mice.

Transgenic and knockout mice are widely used as models for atherogenesis studies. While developing a Helicobacter infection model in LDL receptor-negative (LDLR(-/-)) mice, we noticed that mice fed a high-fat, high-cholesterol diet often contracted gastritis independent of infection. To further investigate this finding, we studied 27 male and 18 female LDLR(-/-) mice fed high-fat, 1% or 1.25% cholesterol diets for 3 to 4 months. The extent of atherosclerosis was morphometrically analyzed in the whole aorta, and the degree of gastric inflammation was scored histologically in hematoxylin-eosin-stained stomach sections. The autoantibody titers to epitopes of oxidized LDL were also measured. Mice fed high-fat, high-cholesterol diets had a significantly higher incidence of gastritis than mice fed normal chow, 62% versus 5%, respectively (P<0.0001). This effect was specific for LDLR(-/-) mice, because no difference in gastritis was found in wild-type mice fed either diet. Animals with gastritis showed slightly more atherosclerosis than animals without gastritis: 16.3+/-6.4% versus 12.8+/-3.4% in males and 9.4+/-3.5% versus 6.5+/-3.3% in females. Cholesterol-fed mice also had significantly higher IgG autoantibody titers against modified LDL than normal chow-fed animals, but no difference was seen between the gastritis and nongastritis groups. We conclude that the standard high-fat, high-cholesterol diet commonly used in many murine models to induce atherosclerosis increased the incidence of gastritis significantly in LDLR(-/-) mice.

Cytokeratin Immunoreactivity in Mouse Tissues: Study of Different Antibodies With a New Detection System

The cross-reactivity of a group of monoclonal antibodies (MABs) generated against human cytokeratins (CKs) was investigated in mouse tissues. Formalin-fixed and paraffin-embedded sections of lung, stomach, small and large intestine, liver, and kidney were immunostained with MABs after epitope retrieval with enzyme digestion. AE1/AE3, a "cocktail" of two MABs that recognizes basic and acidic CKs, 5D3 MAB to low molecular weight CKs (8, 18, and 19), and monospecific MABs to CK 7 and 20 were tested. Additionally, CK 17 and 34betaE12 MABs to high molecular weight CKs were evaluated in the same organs and in sections from skin and preputial glands. We employed the new universal animal system (ARK) as the detection system. The results showed intense reactivity for the first group of antibodies used, with topographic distribution similar to that in human tissues, with the exception of CK 7 in lung parenchyma, which displayed reactivity only in type II pneumocytes, with negativity of adjacent bronchial epithelium. Also of note was the lack of reaction of liver hepatocytes and renal tubular cells to AE1/AE3 and 5D3 MABs. Regarding the second group of antibodies, no reaction was obtained for CK 17 in the tissues tested. On the contrary, 34betaE12 MAB yielded intense reactivity in cells of epidermis and hair follicles. Compared to other detection systems used previously in this animal, ARK produced a well-defined reactivity at the cellular level without any background. We conclude that a useful panel of anti-CK antibodies commonly used in human pathology can be applied successfully to mouse tissues after enzyme digestion, leading to a more accurate definition of cellular populations in this laboratory animal.

Early weaning and prolonged nursing induce changes in cell proliferation in the gastric epithelium of developing rats.

Food deprivation stimulates cell proliferation in the gastric epithelium of suckling, but not weanling rats. This study was designed to investigate the role of diet on proliferation in developing animals, using early weaning and prolonged nursing models. Rat pups were subjected to these dietary conditions at d 15 and were killed 3 or 7 d afterwards. One day before killing, half of pups were deprived of food. Body weights were recorded. After mitosis blockade, the histologic sections of the stomach were used for the evaluation of cell proliferation and methapasic cell distribution along the gland, and for the measurement of mucosa thickness. Body weight was impaired at 18 d by early weaning and at 22 d by prolonged nursing. Food restriction promoted a 10-15% weight loss regardless of dietary conditions. At 18 d, food deprivation inhibited cell division (P: < 0.01) and reduced the thickness of the mucosa (P: < 0.05) in rats that were weaned early. At 22 d, only the thickness of the mucosa was different between the groups that were subjected to early weaning and prolonged nursing (P: < 0.05), regardless of feeding state. The frequency of dividing cells along the gland was affected by early weaning in 18- and 22-d-old rats. These results suggest the following: 1) food deprivation effects are dependent on dietary condition at 18 d because different proliferative responses were achieved after early weaning and prolonged nursing; 2) the lack of changes after dietary manipulation in 22-d-old rats indicates a nonresponsive period during postnatal development. We conclude that milk is a modulatory factor for cell proliferation in the gastric mucosa of rats.

NMDA receptors are present in primary afferent fibers in the gut

Background: Glutamate neurotransmission, and mRNA and immunoreactivity for the NMDA receptor (NMDA-R) have been found in myenteric and submucosal neurons of the rat and guinea pig. However, it is not clear whether the NMDA-R is also present in extrinsic primary afferent fibers in the gut. This is important, since the NMDA-R would then be appropriately localized to participate in nociception. Methods: We localized peripheral NMDA-R in the rat gut and in the L6 dorsal root ganglion (DRG) using subunit specific antibodies, immunofluorescence and confocal microscopy. Antibodies recognizing the NR2C and NRI (Santa Cruz Biotechnology, Santa Cruz, CA) subunits of the NMDA-R were used to label tissue sections of stomach, small and large intestine, and whole mounts of duodenum, jejunum and proximal colon. Antibodies recognizing the NRI (Santa Cruz Biotechnology), and the NR2A and NR2B subunits of the NMDA-R (Chemicon, Temecula, CAl were used to localize the receptor in L6 DRG. Results: Throughout the gut, both the NRI and NR2C antibodies stained many long nerve processes, although these were more apparent with the particulate staining of the NRI antibody. In the duodenum, jejunum and proximal colon, submucosal neurons had greater NR I immunoreactivity than myenteric neurons. In the jejunum, small submucosal blood vessels, probably arterioles, were outlined by NRI or NR2C immunoreactivity. In the distal colon, some NRI-expressing nerve fibers were evident in the submucosa and the circular muscle layer. In the L6 DRG, the NRI and the NR2A1NR2B antibodies labeled practically all cell profiles. With either antibody, staining was most intense in the cytoplasm, having a granular appearance, and was also found in the plasma membrane, but not in the nucleus. Labeling was more intense in medium or small diameter cells, but was also apparent in large cells. Labeling was specific in both gut and DRG, since pre-absorption of the NRI antibody with the receptor fragment used for immunization abolished staining. Conclusion: The NMDA-R is present on the peripheral terminals of extrinsic primary afferents innervating the gut, where they may play an important role in modulating nociceptive and non-nociceptive sensory information from the gut. Supported by NIH grants DK52388 (EFG), DK40919 (EAM) and AstraZeneca R&D Molndal, Sweden

Image analysis method to assess adhesion of Helicobacter pylori to gastric epithelium using confocal laser scanning microscopy

We have used confocal scanning microscopy of FITC-labelled bacteria to assess binding of Helicobacter pylori to stomach sections and to assess the effect of inhibitors on binding to the Lewis antigens. We have quantified the binding using an image manipulation package that is readily available on the web. Our results demonstrate heterogeneity of binding of Helicobacter pylori to tissue sections and that binding can be inhibited using synthetic Lewis B oligosaccharide.

Isolation of recombinant protective Helicobacter pylori antigens.

A total of seven clones producing both new and previously described Helicobacter pylori proteins were isolated from a library of H. pylori genomic DNA. The screening approach by which these proteins were detected relied on the use of antisera raised in mice vaccinated with Helicobacter felis sonicate plus cholera toxin, a regimen which protects mice from H. pylori challenge. This strategy was designed to maximize the possibility of obtaining antigens which might be capable of conferring protection from H. pylori infection. Two of the clones were shown to encode the urease enzyme and the heat shock protein HspB, which have already been identified as protective antigens. The other five clones were sequenced, protein coding regions were deduced, and these sequences were amplified by PCR for incorporation into Escherichia coli expression vectors. The proteins produced from these expression systems were purified to allow testing for protective efficacy in an H. pylori mouse model. All five proteins were able to facilitate the clearance of a challenge with H. pylori, as judged by an assay of gastric urease activity and light microscopy on stomach sections. These results clearly indicate that the screening strategy has successfully identified candidate vaccine antigens.

Identification of a functional Ca2+-sensing receptor in normal human gastric mucous epithelial cells

The purpose of the present study was to determine whether human gastric mucous epithelial cells express a functional Ca2+-sensing receptor (CaR). Human gastric mucous epithelial cells were isolated from surgical tissues and cultured on glass coverslips, plastic dishes, or porous membrane filters. Cell growth was assessed by the MTT assay, CaR localization was detected by immunohistochemistry and confocal microscopy, CaR protein expression was assessed by Western immunoblotting, and intracellular Ca2+ concentration ([Ca2+]i) was determined by fura 2 spectrofluorometry. In paraffin sections of whole stomach, we found strong CaR immunohistochemical staining at the basolateral membrane, with weak CaR-staining at the apical membrane in mucous epithelial cells. Confocal microscopy of human gastric mucous epithelial cell cultures showed abundant CaR immunofluorescence at the basolateral membrane and little to no CaR immunoreactivity at the apical membrane. Western immunoblot detection of CaR protein in cell culture lysates showed two significant immunoreactive bands of 140 and 120 kDa. Addition of extracellular Ca2+ to preconfluent cultures of human gastric mucous epithelial cells produced a significant proliferative response. Changes in [Ca2+]i were also observed in response to graded doses of extracellular Ca2+ and Gd3+. The phospholipase C inhibitor U-73122 specifically inhibited Gd3+-induced changes in [Ca2+]i in the gastric mucous epithelial cell cultures. In conclusion, we have identified the localization of a functional CaR in human gastric mucous epithelial cells.

Histologic sectioning produces TUNEL reactivity. A potential cause of false-positive staining.

○ Objective.-To determine if the DNA strand breaks caused by tissue sectioning result in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) reactivity. Methods.-The incidence and location of TUNEL-positive nuclei were determined in 5- and 15-μm sections of human stomach. Five- and 15-μm sections of tonsil were stained as a positive control. Results.-In 5-μm gastric sections, 69% of nuclei were labeled; in 15-μm sections, only 30% were labeled. In the latter sections, almost all labeled nuclei were located at the cut surface of sections. Labeled nuclei did not have apoptotic morphology. Apototic bodies and tingible body macrophages were labeled throughout 15-μm sections of tonsil. Conclusions.-Tissue sectioning creates TUNEL reactivity. The morphologic findings on routine stains should be considered the gold standard for the detection of apoptosis on tissue sections.

Histologic sectioning produces TUNEL reactivity. A potential cause of false-positive staining.

To determine if the DNA strand breaks caused by tissue sectioning result in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) reactivity. The incidence and location of TUNEL-positive nuclei were determined in 5- and 15-micron sections of human stomach. Five- and 15-micron sections of tonsil were stained as a positive control. In 5-micron gastric sections, 69% of nuclei were labeled; in 15-micron sections, only 30% were labeled. In the latter sections, almost all labeled nuclei were located at the cut surface of sections. Labeled nuclei did not have apoptotic morphology. Apototic bodies and tingible body macrophages were labeled throughout 15-micron sections of tonsil. Tissue sectioning creates TUNEL reactivity. The morphologic findings on routine stains should be considered the gold standard for the detection of apoptosis on tissue sections.

Multiple Cryptosporidium serpentis Oocyst Isolates from Captive Snakes Are Not Transmissible to Amphibians

Viable Cryptosporidium serpentis oocysts originating from 6 captive snakes were gastrically delivered to 12 Cryptosporidium-free African clawed frogs and 9 tadpoles and 3 recently metamorphosed adults of Cryptosporidium-free wood frogs. On days 7 and 14 postinoculation, no life-cycle stage of Cryptosporidium was observed in any of the histological sections of stomach, jejunum, ileum, cloaca, and cecum. However, viable inoculum-derived C. serpentis oocysts were recovered from the water in which the amphibians were kept. Amphibians may disseminate C. serpentis oocysts in the natural habitat.

Interferon therapy in LKM-1 positive patients with chronic hepatitis C: follow-up by a quantitative radioligand assay for CYP2D6 antibody detection

Background/Aims: Liver/kidney microsomal type 1 (LKM-1) antibodies described by indirect immunofluorescence using frozen sections of kidney, stomach and rat liver define a group of patients with type 2 autoimmune hepatitis. Sera react with a non-glycosylated 50-kD protein of the endoplasmic reticulum, which was recently identified as cytochrome P4502D6 (CYP2D6). LKM-1 antibodies may also be associated with hepatitis C virus infection (HCV+/LKM-1+). For this subset of patients, the choice of steroids or interferon alpha therapy may be difficult because of the association of hepatitis C virus infection and autoimmune manifestations. Recently we developed a quantitative immunoprecipitation radioligand assay using 35S-methionine-labeled CYP2D6 protein produced by in vitro transcription and translation reaction. This method detects antibodies against linear and conformational epitopes in both AIH-2 and HCV+/LKM-1+ patients. The aim of this study was to analyze the time-course of HCV+/LKM-1+ patients, applying our radioligand assay over a long follow-up. Methods: We studied five patients who were positive for CYP2D6 antibodies from among 235 chronic hepatitis C virus hepatitis patients (2.1%) treated with interferon alpha for a minimal follow-up of 2 years. We analyzed LKM-1 antibody titer sequentially by radioligand assay, HCV RNA titer and alanine aminotransferase activity in these patients. Results: We found no aggravation of liver disease in this group of patients. Three of these patients showed a sustained biochemical and virological response after interferon. Two others responded partially to interferon therapy. Alanine aminotransferase level sand HCV-RNA decreased during interferon therapy in responder patients. CYP2D6 antibodies did not change in three responder patients during follow-up. One responder patient decreased CYP2D6 antibody level by radioligand assay, but indirect immunofluorescence titers showed a similar pattern. One partial responder patient decreased CYP2D6 antibody level but was negative by indirect immunofluorescence. Conclusions: Our results show that patients with hepatitis C virus who are positive for CYP2D6 antibodies may be treated with interferon, and respond in the same way as CYP2D6 antibody negative patients. Radioligand assay could be helpful for monitoring HCV+/LKM-1+ patients receiving interferon therapy.

Localization of the beta(beta)3-adrenoceptor in the human gastrointestinal tract: an immunohistochemical study.

Activation of human and non-human colonic beta(beta)3-adrenoceptors causes smooth muscle relaxation. beta3-Adrenoceptor agonists protect against experimental indomethacin-induced jejunal ulceration. The mechanism of protection may involve spasmolytic/vasodilatory agonist activity. The precise localization of beta3-adrenoceptors in the human gut is not known. To localize the beta3-adrenoceptor within the human gastrointestinal tract using the immunohistochemical technique. Human beta3-adrenoceptors were immuno-localized in paraffin sections of human oesophagus (OS), stomach (ST), duodenum (DU), ileum (IL), sigmoid colon (SC), rectum (R) and gall-bladder (GB) using the rabbit polyclonal antibody anti-P12. Staining was graded , ++, + and 0. Immunostaining of SC was also done with pre-incubation of anti-P12 with P12 peptide. Western blotting of anti-P12 on human and murine IL and SC isolated membrane proteins was performed. All epithelia, vascular endothelial cells and ganglia scored 0. Smooth muscle of the vasculature, muscularis propria, muscularis mucosae and mucosa was graded, respectively, as follows; OS ( , , ,-), ST (++, , ++, ++), DU (++, , , +), IL (++, ++, ++, +), SC ( , ++, ++, ++), R (++, ++, +, +), GB ( , , -, 0). Pre-incubation of anti-P12 with P12 peptide almost abolished SC smooth muscle positivity. Western blot analysis using anti-P12 on human, but not murine, IL and SC membrane proteins revealed a single 5 5 kDa band, a size consistent with the predicted size of a partially glycosylated form of the human beta3-adrenoceptor. This immunohistochemical study has localized the beta3-adrenoceptor to vascular and nonvascular smooth muscle in the human gastrointestinal tract. These findings support a role for the beta3-adrenoceptor in the control of blood flow and motility in the human gastrointestinal tract.