Attenuation of Clostridium difficile toxin-induced damage to epithelial barrier by ecto-5'-nucleotidase (CD73) and adenosine receptor signaling.

BACKGROUND: Clostridium difficile (Cdf) releases toxins (TcdA and TcdB) that damage the intestinal epithelial barrier. Ecto-5'-nucleotidase (CD73) is expressed on intestinal epithelial cells, and it is hypothesized to protect against toxin-induced epithelial damage through the cleavage of 5'-AMP to adenosine (Ado) and subsequent activation of adenosine receptors (AdoRs). Herein, we sought to assess the potential protective effects of CD73 and AdoR signaling on the injurious effects of Cdf toxins. METHODS: Barrier function was assessed with T84 colonocytes. Transepithelial electrical resistance (TEER), paracellular fluorescein isothiocyanate (FITC)-dextran flux, and tight junction protein (ZO-1) integrity were monitored. Intrarectal installation of Cdf toxin was used to assess epithelial damage in vivo. KEY RESULTS: TcdA/B caused reduced TEER and increased paracellular flux in vitro. Concurrent treatment with 5'-AMP attenuated these responses to Cdf toxin; an effect that was blocked with ZM241385 (AdoRA2 antagonist). APCP, a CD73 inhibitor, also suppressed the protective effects of 5'-AMP on paracellular flux. 5'-AMP reduced toxin-induced disruption of ZO-1, an effect that was abolished by APCP and ZM241385. Inhibition of CD73 with APCP during Cdf toxin exposure led to increased intestinal barrier permeability and epithelial damage in vivo. Intrarectal instillation of 5'-AMP had no effect on toxin-induced intestinal injury. CONCLUSIONS & INFERENCES: Our data suggest that CD73 has a protective role against TcdA/B-induced damage. 5'-AMP treatment attenuated the damaging effects of Cdf toxin in vitro, and inhibitors of CD73 (APCP) and AdoRs (ZM241385) revealed that the cleavage of 5'-AMP to Ado was necessary for the protective effects. Inhibition of CD73 in vivo increases colonic tissue damage and epithelial permeability during Cdf toxin exposure.

Differential effects of CB(1) neutral antagonists and inverse agonists on gastrointestinal motility in mice.

BACKGROUND: Cannabinoid type 1 (CB(1)) receptors are involved in the regulation of gastrointestinal (GI) motility and secretion. Our aim was to characterize the roles of the CB(1) receptor on GI motility and secretion in vitro and in vivo by using different classes of CB(1) receptor antagonists. METHODS: Immunohistochemistry was used to examine the localization of CB(1) receptor in the mouse ileum and colon. Organ bath experiments on mouse ileum and in vivo motility testing comprising upper GI transit, colonic expulsion, and whole gut transit were performed to characterize the effects of the inverse agonist/antagonist AM251 and the neutral antagonist AM4113. As a marker of secretory function we measured short circuit current in vitro using Ussing chambers and stool fluid content in vivo in mouse colon. We also assessed colonic epithelial permeability in vitro using FITC-labeled inulin. KEY RESULTS: In vivo, the inverse agonist AM251 increased upper GI transit and whole gut transit, but it had no effect on colonic expulsion. By contrast, the neutral antagonist AM4113 increased upper GI transit, but unexpectedly reduced both colonic expulsion and whole gut transit at high, but not lower doses. CONCLUSIONS & INFERENCES: Cannabinoid type 1 receptors regulate small intestinal and colonic motility, but not GI secretion under physiological conditions. Cannabinoid type 1 inverse agonists and CB(1) neutral antagonists have different effects on intestinal motility. The ability of the neutral antagonist not to affect whole gut transit may be important for the future development of CB(1) receptor antagonists as therapeutic agents.

Cholinergic regulation of epithelial ion transport in the mammalian intestine.

Acetylcholine (ACh) is critical in controlling epithelial ion transport and hence water movements for gut hydration. Here we review the mechanism of cholinergic control of epithelial ion transport across the mammalian intestine. The cholinergic nervous system affects basal ion flux and can evoke increased active ion transport events. Most studies rely on measuring increases in short-circuit current (ISC = active ion transport) evoked by adding ACh or cholinomimetics to intestinal tissue mounted in Ussing chambers. Despite subtle species and gut regional differences, most data indicate that, under normal circumstances, the effect of ACh on intestinal ion transport is mainly an increase in Cl- secretion due to interaction with epithelial M3 muscarinic ACh receptors (mAChRs) and, to a lesser extent, neuronal M1 mAChRs; however, AChR pharmacology has been plagued by a lack of good receptor subtype-selective compounds. Mice lacking M3 mAChRs display intact cholinergically-mediated intestinal ion transport, suggesting a possible compensatory mechanism. Inflamed tissues often display perturbations in the enteric cholinergic system and reduced intestinal ion transport responses to cholinomimetics. The mechanism(s) underlying this hyporesponsiveness are not fully defined. Inflammation-evoked loss of mAChR-mediated control of epithelial ion transport in the mouse reveals a role for neuronal nicotinic AChRs, representing a hitherto unappreciated braking system to limit ACh-evoked Cl- secretion. We suggest that: i) pharmacological analyses should be supported by the use of more selective compounds and supplemented with molecular biology techniques targeting specific ACh receptors and signalling molecules, and ii) assessment of ion transport in normal tissue must be complemented with investigations of tissues from patients or animals with intestinal disease to reveal control mechanisms that may go undetected by focusing on healthy tissue only.

[Quantification of T CD4+ lymphocytes and viral RNA in patients with HIV/AIDS]. / Cuantificación de linfocitos T CD4+ y de RNA viral en pacientes con VIH/SIDA.

Quantification of CD4+ T lymphocytes and viral RNA in patients with HIV/AIDS. Levels of progression markers (viral load and CD4+ T lymphocytes) in 410 patients with HIV/AIDS that were in different clinical stages of the disease and under different therapeutic schemes were quantified. The objective was to determine the correlation between values of progression markers and clinical stage of the patients. Commercial methodologies for the quantification of lymphocytes, subpopulations and circulating viral RNA were used. Results indicate that there was a correlation between low CD4+ values and high viral load in patients with antiretroviral treatment but not in patients without treatment. Furthermore, analysis of 1,208 samples processed during 1999 showed that 46% of the patients had less than 200 CD4+ T lymphocytes/mL blood and more than 500 copies of circulating viral RNA. Implications of these results in public health in Mexico are discussed.

Characterization of GP39-42 and GP24 antigens from Taenia solium cysticerci and of their antigenic GP10 subunit.

Diagnosis of neurocysticercosis is performed by Western blotting with an enriched fraction of glycoproteins (LLGP). GP39-42 and GP24 are immunodominant antigens. These antigens were electroeluted and characterized by biochemical methods. When GP39-42 or GP24 were reduced, a band of 10 kDa (named GP10) appeared; this band was also analyzed. The most abundant amino acids in the three GPs were lysine, phenylalanine, asparagine, glycine, and leucine. The amino terminal portion was sequenced, and the following order was obtained for the three GPs: EKNKPKNVAXSTKKGYEYVXEF. The glycan portion was 8.4%, 18.2%, and 18.3% in GP39-42, GP24, and GP10, respectively. The three GPs contained mannose, N-acetyl-D-glucosamine, and galactose. GP39-42 and GP24 seem to be oligomeric forms of GP10. When reduced LLGP was reacted with samples obtained from patients with neurocysticercosis or pigs with cysticercosis, a band corresponding to GP10 was always observed. Furthermore, hyperimmune serum from rabbits immunized with GP39-42 or with GP24 recognized GP10 as well as GP39-42 and GP24. The data obtained in this paper suggest that GP10 might be a useful tool for diagnosis.

Contamination of Salmonella blockley in the environment of a poultry farm.

Cecal and environmental samples were collected and examined for the presence of Salmonella on a farm where a high prevalence of Salmonella blockley in chickens was observed. Of 895 cecal and 525 environmental samples examined, 242 (27.0%) and 202 (38.5%) samples, respectively, yielded S. blockley. From the analysis of plasmid profile patterns, 11 different plasmid profile patterns were found among 444 isolates, with plasmid patterns c and d the most frequent among the isolates from ceca and the environment. Salmonella blockley was isolated from environmental samples such as floor litter, walls, drinking water, waste water, dust, and soil collected when barns were occupied and was positive in drinking water, waste water, and soil when samples were collected from empty barns with occupied neighboring barns, but it was negative in all environmental samples with the exception of soil when the environmental samples were collected from empty barns with empty neighboring barns.

Prevalence and persistence of Salmonella in broiler chicken flocks.

Cecal contents of 2,345 broiler chickens consisting of 28 flocks originated from 12 farms were examined for the prevalence of Salmonella to know the actual status of infection with Salmonella in the chicken flocks. Salmonella was isolated from 336 (14.3%) samples. From these isolates, eight serovars were identified. Of the 336 Salmonella isolates, 242 (72.0%) were serotyped as S. Blockley, 60 (17.9%) S. Hadar, 15 (4.5%) S. Bredeney, nine (2.7%) S. Schwarzengrund, four (1.2%) S. Anatum, three (0.9%) S. Enteritidis, two (0.6%) S. Ohio, and one (0.3%) S. Livingstone. The same serovars of Salmonella were repeatedly found in the chickens from the same farms. S. Typhimurium and S. Enteritidis were detected in pooled broken eggshell samples collected from the hatchery. Analysis of plasmid profiles revealed 11 patterns of S. Blockley and seven patterns of S. Hadar. Strains of the same plasmid profiles of S. Blockley were isolated repeatedly from the same farm over one year after the first isolation.

Effect of indomethacin suppositories on rectal polyposis in patients with familial adenomatous polyposis.

BACKGROUND: Oral sulindac is known to reduce polyps in patients with familial adenomatous polyposis (FAP). The authors speculated that rectal administration of indomethacin would be effective therapy for adenomas in the rectal remnant of FAP. METHODS: Eight patients with FAP who had been treated by total colectomy with ileorectal anastomosis were administered an indomethacin suppository (50 mg) once or twice daily during a period of 4 or 8 weeks. The number of polyps at the same site within the rectum was counted under proctoscopy prior to, at the end of, and after the treatment. In four patients, proliferative activity of the rectal mucosa was assessed by immunohistochemical staining for MIB-1. RESULTS: In six of the eight patients who initially had ten or more polyps, the number of polyps decreased to fewer than five, whereas such a decrease could not be observed in the remaining two patients. In the six patients, the number of polyps increased after indomethacin was discontinued. The proliferative activity of the rectal mucosa was higher at the end of treatment than it was prior to indomethacin administration. CONCLUSIONS: Indomethacin suppositories may be effective in the management of rectal adenomatosis in patients with FAP.

Phosphorylation of 558T of moesin detected by site-specific antibodies in RAW264.7 macrophages.

To determine, whether 558Thr in the carboxyl-terminal domain of moesin is phosphorylated in cells other than platelets, rabbit phosphorylation state-specific antibodies were made to the chemically phosphorylated synthetic hexapeptide KYKpTLR of the moesin sequence, as well as to the unphosphorylated form. The affinity-purified antibody populations were specific for either the phosphorylated or the unmodified peptide conjugated to BSA. Site-specific phosphorylation of moesin is detected in RAW macrophages by Western blot analysis, and immunofluorescence studies demonstrate that phosphorylated moesin is localized in filopodial protrusions. After pretreatment with the phosphatase inhibitor calyculin A, a similar effect to that seen in platelets in found, namely a substantial increase in moesin phosphorylation at 558Thr and redistribution of phospho-moesin together with F-actin into one or more ring-like structures in the cytoplasm, presumably due to binding of phosphorylated moesin to F-actin.

Posttraumatic intestinal stenosis: clinical and radiographic features in four patients.

PURPOSE: To characterize posttraumatic intestinal stenosis clinically and radiographically. MATERIALS AND METHODS: The clinical records and radiographic and pathologic findings were reviewed in four patients with posttraumatic stenosis. RESULTS: The patients experienced abdominal symptoms from 1 to 18 weeks after the trauma. While the small intestine was affected in two patients with ileus, the colon was involved in the other two patients with rectal bleeding or diarrhea. Barium studies showed an irregular contour within the severely narrowed intestine in three patients, even 25 weeks after the trauma. In these three patients, pathologic examinations of the resected specimens revealed a circumferential, open ulcer, whereas a scarred ulcer was present in the other patient. CONCLUSION: Posttraumatic intestinal stenosis is clinically characterized by a delayed onset of symptoms that differ according to the site of involvement. This condition should be included in the differential diagnosis of intestinal stenosis.

Gastrointestinal involvement in tuberous sclerosis. Two case reports.

Two patients with tuberous sclerosis had multiple semispherical polyps in the large intestine, predominantly in the rectosigmoid colon. These were hamartomas with an excess of smooth muscle fibers in the stroma. In addition, the 21-year-old man had multiple tiny protrusions in the esophagus and oral fibroepithelial polyps. In the 31-year-old woman, who had pulmonary lymphangiomyomatosis with positive estrogen and progesterone receptors, hamartomatous gastric polyps, colonic leiomyoma, and adenomatous rectal polyps were also identified. Our findings suggest that gastrointestinal polyposis may be one of the phenotypes of tuberous sclerosis complex, possibly linking this disease with other hereditary polyposis syndromes.

Immuno-electron-microscopic localization of enkephalin in the secretory granules of C cells in the chicken ultimobranchial glands.

In the chicken, enkephalin-immunoreactive cells and nerve fibers are distributed in the ultimobranchial glands, which consist of C-cell groups and cyst structures. Ultrastructural features of the enkephalin cells and nerve fibers were examined by immuno-electron microscopy using both the streptavidin-biotin-peroxidase method and the protein A-colloidal gold method. Immunoreactivity for enkephalin was located on the secretory granules of C cells. In 1-day-old chickens, three types of C cells were distinguished on the basis of their granule size. Type-I cells were filled with large secretory granules (200-600 nm in diameter). These elements represented a majority of the C-cell population. Type-II cells contained medium-sized granules (100-280 nm in diameter). Type-III cells displayed small secretory granules (60-200 nm in diameter). The latter cells were elongate or irregular in shape and frequently extended cytoplasmic processes into the connective tissue stroma or contacted other C cells. Enkephalin-immunoactivity was revealed by dense deposits of immunogold particles on the secretory granules of type-II and type-III cells. There were only a few type-I cells showing immunoreactivity for enkephalin. A double immunogold labeling procedure demonstrated that calcitonin and enkephalin were colocalized in the same secretory granules of type-I and type-II cells. Type-III cells were devoid of immunoreactivity for calcitonin. Enkephalin-immunoreactive nerve fibers were characterized by the presence of granular vesicles, 60-160 nm in diameter, and frequently established direct contact with the surface of C cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Staining of pancreatic centroacinar cells, liver bile canaliculi and testicular Leydig cells with a monoclonal antibody against adrenocortical cells.

The immunoreactivity of a monoclonal antibody against cell suspensions from guinea pig adrenal glands was examined at light- and electron-microscopic levels. In addition to the cell surface membrane of adrenocortical cells, the antibody labeled specific sites in the pancreas, liver and testis, but did not label any of the other tissues examined. In the pancreas, microvilli-like processes and the cell surface membrane of centroacinar cells were immunoreactive to the antibody. The microvilli of interlobular duct cells and pancreatic duct cells were also immunoreactive. In the liver, bile canalicular microvilli of hepatocytes were exclusively labeled. Membrane structures of cell organelles, mainly mitochondria, in testicular Leydig cells were also labeled. Immunoblot analysis showed that the monoclonal antibody bound to two common bands at molecular weights of approximately 62 kDa and 110 kDa in the pancreas, liver, testis, and adrenal gland. The two bands reacted with the digoxigenin-conjugated lectin, Sambucus nigra agglutinin (SNA), which recognizes sialic acid linked alpha (2-6) to galactose. Reaction patterns of SNA in the pancreas, liver and testis were similar to those of the monoclonal antibody; pancreatic centroacinar cells and interlobular duct cells, hepatocyte bile canaliculi and testicular Leydig cells were densely stained with SNA. Thus, the monoclonal antibody recognizes two common membrane glycoproteins containing sialic acids in the pancreas, liver, testis and adrenal cortex.

Production of a monoclonal antibody against cell-surface glycoprotein of guinea pig adrenocortical cells.

A monoclonal antibody (MAb) that reacted with the cell-surface antigens of adrenocortical cells was generated against cell suspensions from guinea pig adrenal glands. Cell-surface membranes of the adrenocortical cells in all zones, i.e., zona glomerulosa, zona fasciculata, and zona reticularis, were labeled with the antibody. Adrenal medulla remained unlabeled. Immunoelectron microscopy showed that entire plasma membranes, i.e., plasma membranes between adjacent cells and free cell-surface membranes, including sinusoidal microvilli, were immunoreactive to the antibody. Immunoblot analysis demonstrated that the antibody bound to two prominent bands at molecular weights of approximately 62,000 and 110,000. Two bands were stained with lectin-digoxigenin conjugates. The 110 KD band reacted with Datura stramonium (DSA) and Maackia amurensis (MAA) agglutinins, indicating the presence of N-acetyl-glucosamine and sialic acid-linked alpha (2-3) to galactose; the 62 KD band reacted with SNA, indicating the presence of sialic acid-linked alpha (2-6) to galactose. In adrenocortical cells, the reaction pattern of Sambucus nigra (SNA) agglutinin was similar to that of the (MAb), whereas reaction patterns of DSA and MAA were different. Both neuraminidase digestion and prior absorption of the antibody with N-acetyl-neuraminic acid completely prevented the immunolabeling of adrenocortical cells. These results indicate that the MAb mainly recognizes the 2-6 sialylated cell-surface antigen of adrenocortical cells.

[Toxicological studies on a new cephamycin, MT-141. XI. Immunological properties].

Immunogenicity, eliciting antigenicity of MT-141 and its cross-reactivity with other beta-lactam antibiotics were studied in mice, guinea pigs and rabbits. The results were as follows. Injections of MT-141 failed to produce IgE-type antibody in mice but injections of the MT-141 conjugated to rabbit serum albumin produced a trace of IgE-type antibody. No antibody was produced in the guinea pigs immunized with the MT-141 conjugated to rabbit serum albumin in alum or Freund's complete adjuvant. The conjugated MT-141 also failed to elicit anaphylactic shock in the immunized guinea pigs. The subcutaneous treatments with MT-141 in Freund's complete adjuvant produced an amount of hemagglutination antibody in rabbits. The intravenous treatments with MT-141 produced no antibody in rabbits. When rabbits were subcutaneously immunized with the MT-141 conjugated to rabbit serum albumin in Freund's complete adjuvant, production of specific antibody in the rabbits was demonstrated by observations of passive cutaneous anaphylaxis and hemagglutination. The results of hapten-induced inhibition of passive hemagglutination, passive cutaneous anaphylaxis, anaphylactic shock and hemagglutination by using conjugates of antibiotics and rabbit serum albumin as immunogens and conjugates of antibiotics and bovine gamma-globulin as eliciting antigen showed that MT-141 did not cross-react with other antibiotics. MT-141 did not cause the in vitro direct Coombs' reaction in the human blood even at a high concentration of 160 mg/ml. It is concluded from these results that immunological activity of MT-141 preparation is weak.

[Toxicological studies on a new cephamycin, MT-141. VIII. Its fertility test in rats].

A fertility study of MT-141 was performed in SD rats with the intramuscular (i.m.) injections at the dose levels of 400, 800 and 1,600 mg/kg/day. The male rats were injected with MT-141 for 63 days before mating and during the mating period, while the female rats were injected with MT-141 from the 14th day before mating up to the day 7 of gestation. All pregnant rats were sacrificed on day 20 of gestation followed by external, visceral and skeletal observations of their fetuses. The results are summarized as follows. The suppression of body weight gain was observed in males given above 800 mg/kg/day i.m. and in females of all treated groups during early period of gestation. However, no significant differences were found between treated groups and the control with regard to copulation rate and conception rate. Though no defects were observed for visceral and skeletal specimens in the fetuses of treated groups, MT-141 produced a delayed ossification of forelimbs in the fetuses at the doses above 800 mg/kg/day and of sternebrae at the dose of 1,600 mg/kg/day. It is concluded from the above-mentioned results that the maximal "no 'effective" dose of MT-141 on the fertility is above 1,600 mg/kg/day i.m. in parental rats and less than 800 mg/kg/day i.m. for the fetuses.

Regeneration of 2,3-bisphosphoglycerate and ATP of stored erythrocytes by phosphoenolpyruvate; a new preservative for blood storage.

In ACD solution which contains PEP and sucrose was used as a preservative solution as well as a medium for rejuvenating depleted erythrocytes. 2,3-DPG and ATP in whole blood or red cell concentrates were increased effectively by incubating the cells with the solution at 37 degrees C for 30-60 min. The transport of PEP through the erythrocyte membrane was essential to the increase of 2,3-DPG and ATP. During storage of the cells at 4 degrees C in the presence of PEP, no increase of 2,3-DPG and ATP was observed because no transport of PEP into the cells occurred. By incubation at 37 degrees C for 30 min at the end of various storage periods, however, the levels of ATP and 2,3-DPG in the cells were raised. Addition of ascorbic acid, purine or purine nucleosides to the ACD-sucrose-PEP solution improved the PEP effect of maintaining ATP and/or 2,3-DPG in erythrocytes during storage at 4 degrees C.