Studies on a factor in intestinal strangulation fluid from germfree and conventional rats inhibiting serum bactericidal activity on Escherichia coli.
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Animals↗
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On the chemical nature of the blood borne cardiotoxic material released from the feline small bowel in regional shock.
The cardiotoxic material released into blood from the feline small intestine during a 2 or 3 h regional shock period (inflow pressure to the small intestine 30--35 mmHg during a continuous activation of the regional sympathetic vasoconstrictor fibres at 6 Hz) has been analyzed with regard to three properties: 1. molecular mass determined by molecular filtrations; 2. solubility in a nonpolar solvent (ether); 3. heat stability by heating plasma to 80 degrees C for 30 min. The results obtained suggest that the cardiotoxic material consists of at least two heat stable fractions. One is water soluble with a molecular mass between 500 and 1 000 d and the other is lipid soluble with an unknown molecular mass.
Adrenergic Fibers↗
Contribution of presystemic hepatic extraction to the low oral bioavailability of green tea catechins in rats.
Green tea and green tea catechins have been shown to possess potent cancer-preventive activities in rodent cancer models. At present, epidemiological evidence of the protective effect of green tea consumption against the development of human cancers is not conclusive. Oral bioavailability of green tea catechins has been shown to be low in animals and possibly in humans. This study is designed to determine the contribution of first-pass hepatic elimination to the low oral bioavailability of green tea catechins. Green tea catechin mixture was dosed to rats by intravenous or intraportal infusion. Blood samples were collected after dosing and analyzed using high-performance liquid chromatography with the coulometric electrode array detection system. The systemic clearance of epigallocatechin gallate (EGCG), epigallocatechin (EGC), and epicatechin (EC) was 8.9, 6.3, and 9.4 ml/min, respectively. The steady state volume of distribution (V(ss)) of EGCG, EGC, and EC was 432, 220, and 187 ml, respectively. We found that high percentage of green tea catechins escaped first-pass hepatic elimination, with 87.0, 108.3, and 94.9% of EGCG, EGC, and EC, respectively, available in the systemic blood following intraportal infusion. Our results suggest that factors within the gastrointestinal tract such as limited membrane permeability, transporter mediated intestinal secretion, or gut wall metabolism may contribute more significantly to the low oral bioavailability of green tea catechins.
Animals↗
Neuropeptide Y Y4 receptor homodimers dissociate upon agonist stimulation.
The pancreatic polypeptide-fold family of peptides consists of three 36-amino acid peptides, namely neuropeptide Y (NPY), peptide YY, and pancreatic polypeptide (PP). These peptides regulate important functions, including food intake, circadian rhythms, mood, blood pressure, intestinal secretion, and gut motility, through four receptors: Y1, Y2, Y4, and Y5. Additional receptor subtypes have been proposed based on pharmacology observed in native tissues. Recent studies with other G-protein-coupled receptors have shown that homo- and heterodimerization may be important in determining receptor function and pharmacology. In the present study, the recently cloned rhesus (rh) Y4 receptor was evaluated using radioligand binding, and the pharmacological profile was found to be very similar to the human Y4 receptor. To study homo- and heterodimerization involving the Y4 receptor using bioluminescence resonance energy transfer 2 (BRET(2)), the carboxy termini of the rhesus Y1, Y2, Y4, and Y5 receptors were fused to Renilla luciferase, and rhY4 was also fused to green fluorescent protein. Dimerization was also studied using Western blot analysis. Using both BRET(2) and Western analysis, we found that the rhY4 receptor is present at the cell surface as a homodimer. Furthermore, agonist stimulation using the Y4-selective agonists PP and 1229U91 can dissociate these dimers in a concentration-dependent manner. In contrast, rhY4 did not heterodimerize with other members of the NPY receptor family or with human opioid delta and mu receptors. Therefore, homodimerization is an important component in the regulation of the Y4 receptor.
Animals↗
Increased bioavailability of the food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in MRP2-deficient rats.
MRP2 is an apical transporter expressed in hepatocytes and the epithelial cells of the small intestine and kidney proximal tubule. It extrudes organic anions, conjugated compounds, and some uncharged amphipaths. We studied the transport of an abundant food-derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in vitro, using an MRP2 transfected epithelial cell line (MDCK II) and intestinal explants from Wistar and MRP2-deficient TR(-) rats in Ussing chambers. In the experiments with the transfected cell line, we could demonstrate more than 3-fold higher transport from basolateral to apical than vice versa, whereas the transport in the parent cell line was equal in both directions. These results were confirmed in studies using isolated pieces of small intestine from Wistar and TR(-) rats in the Ussing chamber. Subsequent in vivo experiments demonstrated that after oral administration, absorption of PhIP was 2-fold higher in the TR(-) rat than in the Wistar rat. Consequently, PhIP tissue levels in several organs (liver, kidney, lung, and colon) were 1.7- to 4-fold higher 48 h after oral administration. MRP2 mediated transport of unchanged PhIP probably involves intracellular GSH, because GSH depletion by BSO-treatment in Wistar rats reduced intestinal secretion in the Ussing chamber to the same level as in TR(-) rats. In accordance, BSO treatment increased oral bioavailability in intact Wistar rats. This study shows for the first time that MRP2-mediated extrusion reduces oral bioavailability of a xenobiotic and protects against an abundant food-derived carcinogen.
ATP Binding Cassette Transporter, Subfamily B↗
Influence of renal failure on ciprofloxacin pharmacokinetics in rats.
Ciprofloxacin pharmacokinetics have been shown to be modified in patients with renal failure (e.g., the intestinal secretion of ciprofloxacin is increased). This study investigated the influence of renal failure on the pharmacokinetics of ciprofloxacin following oral and parenteral administration to rats of a dose of 50 mg/kg of body weight. After parenteral administration, only renal clearance (CLR) was reduced in nephrectomized rats (5.3+/-1.4 versus 17.8+/-4.7 ml/min/kg, P < 0.01, nephrectomized versus control rats). However, nonrenal clearance was increased in nephrectomized rats (32+/-4 versus 15+/-5 ml/min/kg, P < 0.01, nephrectomized versus control rats), suggesting compensatory mechanisms for reduced renal function. After oral administration, apparent total clearance and CLR were reduced (P < 0.01) in nephrectomized rats (117+/-25 and 6.8+/-4.4 ml/min/kg, respectively) compared with the values for control rats (185+/-9 and 22.6+/-5.3 ml/min/kg, respectively) and the area under the concentration-time curve was higher (P < 0.01) for nephrectomized rats (436.3+/-90.5 mg. min/liter) than for control rats (271.3+/-14.3 mg.min/liter). Terminal elimination half lives in the two groups remained constant after oral and parenteral administration. These results suggest an increased bioavailability of ciprofloxacin in nephrectomized rats, which was confirmed by a nonlinear mixed-effect model.
Administration, Oral↗
Fluid accumulation in mouse ligated intestine inoculated with Clostridium perfringens enterotoxin.
Clostridium perfringens enterotoxin, when inoculated into the ligated intestinal loop of mice, caused marked distension due to fluid accumulation. The increase in weight of the intestinal loop was proportional to the log dose of enterotoxin within a range from 1 to 16 micrograms. The fluid accumulation was arrested by washing the loop with saline or by injection of the specific anti-enterotoxin serum into the loop 5 or even 30 min after inoculation of the enterotoxin. A significant increase in weight of the loop was found as early as 10 min after inoculation of the toxin. These results may suggest that entergotoxin is neither bound firmly to the mucosal membrane nor permeates into the cells of the intestinal wall. The mouse intestinal loop test is economical, simple to perform, and applicable for quantitative determination of the enteropathogenic activity of C. perfringens enterotoxin.
Animals↗
Permeability and enterotoxic factors of nonagglutinable vibrios Vibrio alcaligenes and Vibrio parahaemolyticus.
Enterotoxic factor (EF) was present and could be developed in nonagglutinable vibrios, but not the permeability factor (PF). Presence of PF without EF in Vibrio parahaemolyticus is significant. Both EF and PF were absent in V. alcaligenes.
Animals↗
Response of ligated intestinal loops in chickens to the enterotoxin of Clostridium perfringens.
Approximately 45-cm length of jejunoileum of 7-week-old chickens was found to be responsive and suitable for testing the enterotoxin of Clostridium perfringens by the ligated intestinal loop technique. Injections of 20 to 30 mug of enterotoxin per loop caused positive response of fluid accumulation. Chickens were found to be more convenient and economical for this purpose than other laboratory and domestic animals.
Animals↗
Immunological control mechanism against cholera toxin: interference with toxin binding to intestinal receptors.
The immunological control mechanism against cholera toxin (CT) in the small intestine of rats was studied in vivo. CT binding to intestinal receptors was determined by injected radiolabeled CT into the loops of rat small intestine and subsequently separating purified microvillus membranes from mucosal scrapings of those loops. substantial radioactivity (10(5) cpm/mg of microvillus protein) was present in microvillus fractions of small intestine exposed to 125I-labeled CT compared to radioactivity (10(2) cpm/mg) in fractions from intestine exposed to radiolabeled bovine serum albumin (BSA) used as a control. CT binding to intestinal receptors was significantly inhibited (P less than 0.02) in rats immunized with crude toxin by a combined intraperitoneal and oral method compared to CT binding in animals immunized with BSA or controls, suggesting a specific relationship between intestinal antitoxin and inhibition of binding. Furthermore, ligated ileal loops from CT-immunized animals showed a significant decrease in fluid accumulation when exposed to CT compared to loops from control or BSA-immunized animals, suggesting that antitoxins also interfered with the biological action of CT under conditions of immunization. These studies provide direct evidence that intestinal antitoxins protect against CT-induced diarrhea by interfering with the attachment of the toxin to the intestinal microvillus surface.
Animals↗
Enzyme-linked immunosorbent assay for immunoglobulin G and immunoglobulin A antibodies to Shigella flexneri antigens.
An enzyme-linked immunosorbent assay has been developed to detect class-specific antibodies to Shigella flexneri lipopolysaccharide antigens. This enzyme-linked immunosorbent assay system has been used to measure antibodies present in serum or intestinal secretions without further purification. It is considerably more sensitive than passive hemagglutination, allowing detection of as little as 1.3 ng of specific immunoglobulin G antibody per ml in immune sera. Optimal conditions for this assay are outlined in this report.
Antibodies, Bacterial↗
Synergistic protection against experimental cholera by immunization with cholera toxoid and vaccine.
Rabbits were immunized with two parenteral injections of Wellcome toxoid PX389A, Wyeth toxoid 20101, or Merck bivalent vaccine. Other groups of rabbits were immunized with combinations of the Merck vaccine and each of the two toxoids. Antitoxin responses were monitored in each group of rabbits before livecell challenge of each animal by the ligated intestinal loop assay. Inaba and Ogawa strains of Vibrio cholerae were used for challenge experiments. Basically, the data indicate that the toxoids were equivalent in antigenic potency and antitoxin responses were unaffected by combination of the toxoids with the whole-cell vaccine. The 50 microgram doses of each toxoid as well as the 4 X 10(9) cells of the bivalent vaccine provided the same magnitude of protection against live-cell challenge with either Inaba or Ogawa vibrios. Immunization with either toxoid in combination with the bivalent vaccine resulted in a synergistic protective response against live-cell challenge of intestinal loops with V. cholerae. Synergistic protection was observed when toxoid and vaccine were administered together by the oral and parenteral routes. Maximum protection was obtained when rabbits were immunized with the combined toxoid-whole-cell vaccine administered by both oral and parenteral routes.
Animals↗
Production of neutralizing monoclonal antibodies to Escherichia coli heat-stable enterotoxin.
In an effort to develop new approaches to the study and control of infectious diarrhea, we prepared murine monoclonal antibodies to the Escherichia coli heat-stable enterotoxin (STa). The toxin was purified from E. coli culture media and conjugated to bovine serum albumin. The STa-bovine serum albumin conjugate was used to immunize BALB/c mice, and the immune spleen cells from these mice were fused with SP2/0 myeloma cells. Resultant hybridomas were screened in an enzyme-linked immunosorbent assay protocol against 500 ng of STa-bovine serum albumin bound to microtiter wells as the solid-phase antigen. Five stable clones were selected and grown further in ascites fluid, which demonstrated anti-STa activity at dilutions of up to 1:500,000 in the enzyme-linked immunosorbent assay for heat-stable enterotoxin. In a competitive enzyme-linked immunosorbent assay format, the antibodies recognized several human and porcine strains of STa to various extents, but did not recognize E. coli heat-labile toxin, cholera toxin, or staphylococcal enterotoxin B. The antibodies were all able to bind lactoperoxidase-labeled [125I]STa, and antibody 20B3 was also able to dissociate [125I]STa bound to toxin receptors on rat jejunal villous cells. Preincubation of STa with antibodies 20B3 or 20F5 led to a concentration-dependent neutralization of toxin activity in a suckling mouse intestinal secretion assay. These antibodies are likely to provide new tools for the continued study of STa structure-function relationships and may lead to improved diagnosis and treatment of E. coli-induced infectious diarrhea.
Animals↗
Intranasal immunization with SAG1 protein of Toxoplasma gondii in association with cholera toxin dramatically reduces development of cerebral cysts after oral infection.
SAG1 protein of Toxoplasma gondii was evaluated as a protective antigen in mucosal immunization with cholera toxin as an adjuvant. CBA/J mice intranasally immunized with a combination of SAG1 and cholera toxin exhibited significantly fewer cysts in the brain after oral infection with the 76K strain of T. gondii than control mice. This acquired protection lasted at least 5 months. Protected mice developed high levels of serum anti-SAG1 immunoglobulin G antibodies as well as an enhanced systemic cellular response, as assessed by the proliferation of splenocytes in response to SAG1 restimulation in vitro. This cellular proliferation was associated with an increase of interleukin-2 and interleukin-5 synthesis and with barely detectable gamma interferon production. Splenic immune T cells were shown to convey modest protection to recipients against development of brain cysts following oral infection with T. gondii. Significant production of anti-SAG1 immunoglobulin A was induced in intestinal secretions of protected mice. These results indicate that intranasal immunization with SAG1 and cholera toxin can induce mucosal and systemic immune responses and affords partial and long-lasting resistance against the establishment of chronic toxoplasmosis.
Administration, Intranasal↗
Secretory phospholipase A2 is the principal bactericide for staphylococci and other gram-positive bacteria in human tears.
We examined human tears for molecules that killed gram-positive bacteria. The principal mediator of bactericidal activity against staphylococci proved to be a calcium-dependent enzyme, secretory phospholipase A2. Whereas the concentration of secretory phospholipase A2 in the normal tear film exceeded 30 microg/ml, only 1.1 ng (<0.1 nM) of the enzyme per ml sufficed to kill Listeria monocytogenes and 15 to 80 ng/ml killed Staphylococcus aureus. Despite its efficacy against gram-positive bacteria, secretory phospholipase A2 lacked bactericidal activity against gram-negative organisms (Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa) when tested in the ionic environment of tears. Given the presence of secretory phospholipase A2 in tears, intestinal secretions, and leukocytes, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against gram-positive bacteria.
Adult↗
Cholera toxin B subunit activates arachidonic acid metabolism.
Cholera toxin (CT) increases intestinal secretion of water and electrolytes and modulates the mucosal immune response by stimulating cellular synthesis of arachidonic acid (AA) metabolites (e.g., prostaglandin E2), as well as the intracellular second messenger cyclic AMP (cAMP). While much is known about the mechanism of CT stimulation of adenylate cyclase, the toxin's activation of phospholipase A2, which results in increased hydrolysis of AA from membrane phospholipids, is not well understood. To determine whether CT activation of AA metabolism requires CT's known enzymatic activity (i.e., ADP-ribosylation of GSalpha), we used native CT and a mutant CT protein (CT-2*) lacking ADP-ribose transferase activity in combination with S49 wild-type (WT) and S49 cyc- murine Theta (Th)1.2-positive lymphoma cells deficient in GSalpha. The experimental results showed that native CT stimulated the release of [3H[AA from S49 cyc- cells at a level similar to that for S49 WT cells, indicating that GSalpha is not essential for this process. Further, levels of cAMP in the CT-treated cyc- cells remained the same as those in the untreated control cells. The ADP-ribosyltransferase-deficient CT-2* protein, which was incapable of increasing synthesis of cAMP, displayed about the same capacity as CT to evoke the release of [3H]AA metabolites from both S49 WT and cyc- cells. We concluded that stimulation of arachidonate metabolism in S49 murine lymphoma cells by native CT does not require enzymatically functional CT, capable of catalyzing the ADP-ribosylation reaction. These results demonstrated for the first time that stimulation of adenylate cyclase by CT and stimulation of AA metabolism by CT are not necessarily coregulated. In addition, the B subunits purified from native CT and CT-2* both simulated the release of [3H]AA from S49 cyc- cells and murine monocyte/macrophage cells (RAW 264.7), suggesting a receptor-mediated cell activation process of potential importance in enhancing immune responses to vaccine components.
Animals↗
Zonula occludens toxin is a powerful mucosal adjuvant for intranasally delivered antigens.
Zonula occludens toxin (Zot) is produced by toxigenic strains of Vibrio cholerae and has the ability to reversibly alter intestinal epithelial tight junctions, allowing the passage of macromolecules through the mucosal barrier. In the present study, we investigated whether Zot could be exploited to deliver soluble antigens through the nasal mucosa for the induction of antigen-specific systemic and mucosal immune responses. Intranasal immunization of mice with ovalbumin (Ova) and recombinant Zot, either fused to the maltose-binding protein (MBP-Zot) or with a hexahistidine tag (His-Zot), induced anti-Ova serum immunoglobulin G (IgG) titers that were approximately 40-fold higher than those induced by immunization with antigen alone. Interestingly, Zot also stimulated high anti-Ova IgA titers in serum, as well as in vaginal and intestinal secretions. A comparison with Escherichia coli heat-labile enterotoxin (LT) revealed that the adjuvant activity of Zot was only sevenfold lower than that of LT. Moreover, Zot and LT induced similar patterns of Ova-specific IgG subclasses. The subtypes IgG1, IgG2a, and IgG2b were all stimulated, with a predominance of IgG1 and IgG2b. In conclusion, our results highlight Zot as a novel potent mucosal adjuvant of microbial origin.
Adjuvants, Immunologic↗
Differences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of Escherichia coli.
The response of adult rabbit small intestine to the heat-stable (ST) and heat-labile (LT) enterotoxins of Escherichia coli has been investigated by employing the ligated loop technique. Fluid accumulation was determined in relation to enterotoxin dose and duration of gut exposure. The individual responses to ST and LT differed in a characteristic manner. Onset of net fluid accumulation in response to ST appeared to be immediate even at the lowest dose tested. Onset of net fluid accumulation in response to LT was rapid at high doses but delayed at low doses. Maximum volume per length ratios elicited by ST occurred between 4 and 6 h after injection of loops over the entire range of doses tested. However, maximum ratios elicited by LT occurred no less than 10 h after injection even at low doses. Fluid accumulation elicited by LT increased in duration with increasing dose; high doses of LT producing a response which was sustained for at least 18 h. The net effect of these differences in reaction characteristics is a sharp increase in the proportion of the cumulative net secretory response attributable to LT with time. Therefore, a 6-h assay time is appropriate for the titration of ST, whereas an 18-h assay is not. The 18-h assay was found more appropriate for toxin-antitoxin neutralization studies since only LT was neutralized by anti-enterotoxin serum. LT of E. coli (swine) strain P-263 and (human) strain 334 was neutralized by antibody stimulated by enterotoxin from E. coli (human) strain H-10407. LT was labile to mild acid conditions, whereas ST was not.
Animals↗