Rabbit Ileal Mucosa Research Articles

Investigating the effects of Modified Sijunzi Decoction on the diversity of intestinal microflora of severe scald rabbits based on 16S ribosomal RNA high-throughput sequencing

Investigating the effects of Modified Sijunzi Decoction on the diversity of intestinal microflora of severe scald rabbits based on 16S ribosomal RNA high-throughput sequencing

EX VIVO MODEL OF RABBIT INTESTINAL EPITHELIUM APPLIED TO THE STUDY OF COLONIZATION BY ENTEROAGGREGATIVE ESCHERICHIA COLI

The diarrheal syndrome is considered a serious public health problem all over the world and is considered a major cause of morbidity and mortality in developing countries. The high incidence of enteroaggregative Escherichia coli in diarrheal syndromes classified as an emerging pathogen of gastrointestinal infections. After decades of study, your pathogenesis remains uncertain and has been investigated mainly using in vitro models of adhesion in cellular lines. The present study investigated the interaction of enteroaggregative Escherichia coli strains isolated from childhood diarrhea with rabbit ileal and colonic mucosa ex vivo, using the in vitro organ culture model. The in vitro adhesion assays using cultured tissue were performed with the strains co-incubated with intestinal fragments of ileum and colon over a period of 6 hours. Each strain was tested with three intestinal fragments for each region. The fragments were analysed by scanning electron microscopy. Through scanning electron microscopy we observed that all strains adhered to rabbit ileal and colonic mucosa, with the typical aggregative adherence pattern of "stacked bricks" on the epithelium. However, the highest degree of adherence was observed on colonic mucosa. Threadlike structures were found in greater numbers in the ileum compared to the colon. These data showed that enteroaggregative Escherichia coli may have a high tropism for the human colon, which was ratified by the higher degree of adherence on the rabbit colonic mucosa. Finally, data indicated that in vitro organ culture of intestinal mucosa from rabbit may be used to elucidate the enteroaggregative Escherichia coli pathogenesis.

Adhesion and cytotoxicity of Aeromonas caviae to rabbit intestinal epithelium ex vivo.

Aeromonads are considered potential pathogens for humans and animals and are responsible for the etiology of intestinal and extraintestinal diseases. The presence of Aeromonas spp. in food and water shows that it is an important vehicle of infection in humans. The pathology caused by these bacteria involves several virulence factors, such as the ability to produce toxins, adhesion and invasion. The present study investigated the interaction of five Aeromonas caviae strains isolated from human diarrheic faeces with rabbit ileal and colonic mucosa ex vivo, using in vitro organ culture model. The in vitro adhesion assays using cultured tissue were performed with A. caviae strains co-incubated with intestinal fragments of ileum and colon over a period of 6h. The fragments were analyzed by light and electron microscopy. All strains adhered to rabbit ileal and colonic mucosa ex vivo, with higher degree of adherence presented on colonic mucosa. The typical aggregative adherence pattern was observed among strains studied. Through electron and light microscopy, we observed extensive colonization of ileal and colonic mucosa, large mucus production, biofilm formation and morphological alterations such as intense vacuolization, structural disorganization, cell extrusion and destruction of the villi. These results demonstrate that in vitro organ culture of intestinal mucosa from rabbit may be used to investigate Aeromonas spp. Finally, our results support the pathogenic potential of Aeromonas emphasising their importance in public health.

The CFTR Associated Protein CAP70 Interacts with the Apical Cl-/HCO3-Exchanger DRA in Rabbit Small Intestinal Mucosa

DRA (down regulated in adenoma) is an intestinal anion exchanger, acting in parallel with NHE3 to facilitate ileal and colonic NaCl absorption. Furthermore it is involved in small intestinal bicarbonate secretion. Because DRA has a PDZ interaction motif, which may influence its properties, we searched for DRA-interacting PDZ adapter proteins in the small intestine. Using an overlay assay with the recombinant DRA C-terminus as a ligand, a 70 kDa protein was labeled, which was restricted to the brush border membrane in rabbit duodenal and ileal mucosa and was not detected in the colon. Destruction of the C-terminal PDZ interaction motif abolished this band, suggesting a specific protein-protein interaction. The 70 kDa protein was identified as CAP70 (CFTR associated protein of 70 kDa) by an anti-CAP70 antibody and by two in vitro binding assays after cloning CAP70 from rabbit duodenum and ileum. The interaction was recapitulated in HEK cells transfected with DRA and PDZK1, the human orthologue of CAP70. Corresponding to the overlay assay, no CAP70 mRNA or protein was detected in the colon. In vitro protein-protein interaction studies revealed specific binding of DRA to the 2nd and 3rd PDZ domain, while CFTR is known to interact with PDZ1, PDZ3, and PDZ4. The composition of macromolecular complexes assembled by CAP70 in the distal small bowel is unknown. Its restricted expression shows that it cannot be involved in NaCl absorption in the proximal colon. We suggest that CAP70 mediates regulatory functions specific to the small intestine.

Absence of intestinal secretion on supernatants from macrophages stimulated with Clostridium difficile toxin B on rabbit ileum

Several studies have documented the involvement of both Clostridium difficile, toxins, A and B in the pathogenesis of antibiotic-associated diarrhea. Recently, we demonstrated that IL-1β is the intestinal secretory factor released by macrophages stimulated with toxin A. The aim of this study was to evaluate the importance of macrophages stimulated with toxin B on rabbit ileal ion transport. The changes in ion transport were analyzed by studying the short-circuit current of the rabbit ileal mucosa mounted in Üssing chambers. The supernatants of macrophages treated with toxin B (3.6×10 −7 M) had no effect on the ion transport (change in short-circuit current ΔI sc =28.0±9.2 vs. control=26.8±3.6 μA cm −2). Supernatants of macrophages stimulated with toxin A (3.2×10 −7 M), our positive control, induced a significant change in ileal ion transport (Δ I sc=55.2±5.7 mA cm −2). It was also observed that, like toxin A, toxin B stimulated macrophages to produce TNF-α (555.0±37.9 pg/ml vs. control=182.0±39.8 pg/ml; p<0.05). Nevertheless, in contrast to toxin A, toxin B did not stimulate IL-1β synthesis (28.0±7.5 pg/ml vs. control=40.0±14.4 pg/ml; p>0.05). We conclude that the supernatants of macrophages stimulated with toxin B are not able to stimulate ion transport and that both toxins stimulate the genesis of TNF-α, but only toxin A induces the synthesis of IL-1β, which, we have earlier reported, causes an electrogenic intestinal response in rabbit ileum.

Supernatants from Macrophages Stimulated with Microcystin‐LR Induce Electrogenic Intestinal Response in Rabbit Ileum

Microcystin-LR is a cyclic heptapeptide hepatotoxin produced by the cyanobacterium Microcystis aeruginosa. This microorganism often forms toxic blooms in freshwater lakes and reservoirs for drinking water supply, producing serious disorders in humans and animals. Some have suggested that certain biological activities of microcystin may depend upon the stimulation of immune cells. Therefore, the aims of this research were to examine electrogenic intestinal secretion, in vitro, caused by the supernatants from macrophages stimulated with microcystin-LR, as well as to investigate the presence of interleukin-1beta and tumour necrosis factor-alpha in these supernatants. We found that the supernatants of macrophages stimulated with microcystin-LR (0.1, 0.3 and 1.0 microg/ml) caused electrogenic intestinal effects (change in short circuit currents (delta SCC)=57.6, 50.8 and 73.3, respectively, versus control=19.6 microA.cm(-2)) in a time-dependent way (microcystin-LR (1.0 microg/ml)=63.2, 108.8, 120.4 and 132.3 microA.cm(-2) at time 0, 40, 50 and 60 min., respectively). In addition, the intestinal secretory activity present in these supernatants was blocked (57%) by the prior treatment of macrophages with dexamethasone. We also demonstrated that microcystin-LR (0.1, 0.3 and 1.0 ,microg/ml) is capable of stimulating the synthesis of tumour necrosis factor-alpha (375.4, 369.0 and 610.8 pg/ml, respectively, versus control=165.0 pg/ml) and interleukin-1beta (198.9, 189.3 and 522.1 pg/ml, respectively, versus control=39.7 pg/ml). These findings demonstrate that microcystin-LR induces the release of interleukin-1beta and tumour necrosis factor-alpha by peritoneal macrophages in vitro, and that the supernatants from these macrophages induce electrogenic secretion in rabbit ileal mucosa.

Characterization of non-membrane-damaging cytotoxin of non-toxigenic Vibrio cholerae O1 and its relevance to disease

The non-membrane-damaging cytotoxin which causes dramatic cell rounding of cultured HeLa cells was purified to homogeneity from a clinical strain (WO5) of non-toxigenic Vibrio cholerae O1 Inaba belonging to the El Tor biotype. The purified protein has a denatured molecular weight of 35 kDa and a native molecular weight of approximately 37 kDa indicating the monomeric nature of the protein. The 15 N-terminal amino acid sequence of non-membrane-damaging cytotoxin showed complete homology to the hemagglutinin protease previously purified and characterized from V. cholerae O1. Purified non-membrane-damaging cytotoxin from V. cholerae O1 was immunologically and biochemically identical to that previously purified from V. cholerae O26. Non-membrane-damaging cytotoxin was found to be enterotoxic in rabbit ileal loop assay inducing accumulation of non-hemorrhagic fluid at 100 μg and elicited a concentration dependent increase in short circuit current and tissue conductance of rabbit ileal mucosa mounted on Ussing chambers. A significant serum immunoglobulin G response against non-membrane-damaging cytotoxin was elicited by patients infected with V. cholerae O139 but not with V. cholerae O1. These properties make non-membrane-damaging cytotoxin a potential virulence factor of V. cholerae which should be taken into consideration while making live, attenuated recombinant vaccine strains against cholera.

Intestinal secretory factor released by macrophages stimulated with Clostridium difficile toxin A: role of interleukin 1beta.

Clostridium difficile toxin A is associated with enterocolitis in animals and humans. However, the mechanisms of its secretory and damaging effects are not totally understood. In this work, we examined the intestinal secretion of electrolytes and water caused by supernatants from macrophages stimulated with toxin A in rabbit ileal mucosa mounted in Ussing chambers. We also investigated the mechanism by which the intestinal secretory factor (ISF) is released from stimulated macrophages. Supernatants from macrophages stimulated with toxin A caused potent intestinal secretion (change in short-circuit current [DeltaIsc], 76 microA x cm-2; P < 0.01). The release of the ISF was pertussis toxin sensitive (reduction, 61%; P < 0.01) and was also reduced (P < 0.05) by a protein synthesis inhibitor (67%), protease inhibitors (57%), a phospholipase A2 inhibitor (54%), a cyclo-oxygenase inhibitor (62%), a dual cyclo- and lipoxygenase inhibitor (48%), a platelet-activating factor (PAF) receptor antagonist (55%), and tumor necrosis factor alpha (TNF-alpha) synthesis inhibitors (48%). However, this release was not inhibited by a lipo-oxygenase inhibitor. Monoclonal anti-interleukin 1beta (IL-1beta) but not anti-IL-1alpha antibody blocked (72%; P < 0.01) the secretory action of the ISF, as did recombinant human IL-1 receptor antagonist (80%; P < 0.01). High levels of IL-1beta (3,476 pg/ml) were detected by an enzyme-linked immunosorbent assay in the above supernatants. Furthermore, the addition of IL-1beta to the serosal side caused a potent secretory effect (DeltaIsc, 80 microA x cm-2; P < 0.01). These results show that macrophages stimulated with toxin A release an ISF capable of provoking intestinal secretion. The regulation of this factor is dependent upon the activation of the G protein. In addition, prostaglandins, PAF, and TNF-alpha are involved in the release of the ISF. We conclude that IL-1beta is probably the ISF released by macrophages in response to toxin A.

137 ATTACHING AND EFFACING BACTERIA INDUCE AN INCREASE IN TISSUE PERMEABILITY THAT IS SPECIES-SPECIFIC

Introduction. A three-steps of Enteropathogenie Escherichia coli (EPEC) pathogenicity include a fimbrial-mediated loose adherence to the enterocyte apical membrane (step. 1), followed by intimate adherence with formation of characteristic attaching and affacing (A/E) lesions (step 2). The genetic elements necessary for step 2 are clustered in a 35kb region called Locus for Enterocyte Effacement (LEE). Finally, step 3 involves an intracellular signalling cascade that seems to mediate ion secretion. Other data shows that EPEC decrease tissue resistance and increase transepithelial flux of mannitol, which may be a complementary mechanism of diarrhea. Aim. We investigated whether the decrease in tissue resistance is species-specific and whether intimate adherence is necessary for the resistance drop to occur. Methods. Stripped rabbit ileal mucosa was mounted in Ussing chambers, batheds with Ringer's solution, gassed with 95%O2-5%CO2 and potential difference (PD) and short-cirtcuit current (Isc) were monitored after challenging the tissue with PBS-resuspended cultures of wild type A/E forming bacteria and genetically derived strains. Tissue resistance (Rt) was calculated from PD and Isc according to Ohm's law and its variation (ΔRt) was expressed as percentage of the basal Rt value obtained ca 10 min after mounting the tissue. Results and conclusions. Rabbit specific A/E strain RDEC-1 induced a ΔRt of -17% (±3) while negative control (i.e. no fimbria, no A/E formation) strain DH5α induced a decrease of -7% (±2). Wild type, human specific EPEC strain E2348/69 did not induce a significant Δrt while transforming it with the plasmid (pM5) containing RDEC-1 fimbria gene evokes a ΔRt of -27% (±4). No significant ΔRt were noted transforming pM5 into a negative control strain. Finally, cloning RDEC-1 LEE and pM5, but not the LEE alone, into DH5α induced a ΔRt comparable to wild type RDEC-1. In summary, our data show that the ability of decreasing Rt is dependent on species-specific step 1 adherence. However, fimbrial adherence per se is not sufficient to give this effect, since elements contained in the LEE, most likely involved in A/E lesion formation, are required. Using optical and electron microscopy we are currently looking for a morphologic correlation between A/E lesions and decreased Rt. We are also engineering more selective genetic constructs to find out which gene(s) in the LEE is (are) involved in the process.

Octreotide Inhibition of Serotonin-Induced Ileal Chloride Secretion

Octreotide (SMS, synthetic miniature somatostatin) effectively alleviates the secretory diarrhea of the malignant carcinoid syndrome. Although SMS inhibits tumor release of serotonin (5HT) and other bioactive agents, it also inhibits the diarrhea in patients who continue to exhibit elevated serum levels of 5HT. This observation suggests that SMS may directly inhibit mediator-stimulated intestinal ion secretion at the mucosal level. To test this hypothesis, intestinal ion secretion was studied in rabbit ileal mucosa mounted in Ussing chambers. Maximal changes in short circuit current (ΔI sc) were observed as an indicator of mucosal ion secretion. The application of pathophysiologic concentrations of 5HT (10 -5 M ) to the mucosal preps resulted in a ΔI sc of 52 ± 1995 ± 6 μA/cm 2. This 5HT-stimulated ΔI sc was significantly inhibited by serosal furosemide (10 -3 M) or use of a chloride-depleted medium, indicating that 5HT stimulates electrogenic chloride secretion in the rabbit ileum. Pretreatment with a therapeutic concentration of SMS (10 -8 M) resulted in a significant inhibition of 5HT-stimulated electrogenic CI - secretion (9 ± 1 μA/cm 2) ( P < 0.005). This inhibitory effect of SMS was not seen in tissue pretreated with pertussis toxin. The results of these experiments demonstrate that octreotide inhibits 5HT-stimulated electrogenic chloride secretion at the mucosal level. Additionally this inhibitory effect of octreotide is likely mediated by activation of the inhibitory subunit of membrane-bound GTP-binding regulatory proteins. These results thus provide experimental evidence in support of the ability of SMS to ameliorate the carcinoid diarrhea by a direct effect on stimulated mucosal ion secretion.

Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.

The intracellular signaling involved in the mechanism of action of zonula occludens toxin (ZOT) was studied using several in vitro and ex vivo models. ZOT showed a selective effect among various cell lines tested, suggesting that it may interact with a specific receptor, whose surface expression on various cells differs. When tested in IEC6 cell monolayers, ZOT-containing supernatants induced a redistribution of the F-actin cytoskeleton. Similar results were obtained with rabbit ileal mucosa, where the reorganization of F-actin paralleled the increase in tissue permeability. In endothelial cells, the cytoskeletal rearrangement involved a decrease of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This actin polymerization was time- and dose-dependent, and was reversible. Pretreatment with a specific protein kinase C inhibitor, CGP41251, completely abolished the ZOT effects on both tissue permeability and actin polymerization. In IEC6 cells ZOT induced a peak increment of the PKC-alpha isoform after 3 min incubation. Taken together, these results suggest that ZOT activates a complex intracellular cascade of events that regulate tight junction permeability, probably mimicking the effect of physiologic modulator(s) of epithelial barrier function.

Effects of Deconjugated Bile Acids on Electrolyte and Nutrient Transport in the Rabbit Small Intestine In Vitro

To obtain new information on the poorly investigated mechanisms of the diarrheogenic effects of bile acids, we investigated the effects of the unconjugated bile acids chenodeoxycholate and ursodeoxycholate on the small intestine transport of electrolytes and nutrients in the rabbit jejunal and ileal mucosa mounted in vitro in Ussing or influx chambers. When added to the ileal mucosa at a concentration of 1 m M, both bile acids induced a secretory shift in ion transport; absorption of Na and Cl was abolished and secretion of bicarbonate was enhanced. No changes in short circuit current or electrolyte transport were observed when the bile acids were added to the jejunal mucosa. The addition of 1 m M chenodeoxycholate to the luminal side of the ileum induced a marked inhibition in the uptake of glucose, phenylala‐nine, and glutamic acid. A similar inhibition, though of a lesser magnitude, was also induced by both bile acids on jejunal nutrient transport. Further studies on the inhibitory effect of chenodeoxycholate on glucose influx showed that the inhibition was dose‐dependent; at 1 m M it was maximal and resulted in the complete abolition of the active component, with only the diffusional pathway still operative. We conclude that unconjugated bile acids act as secretagogues in the ileum and significantly blunt the active transport of nutrients in the small intestine. Both effects may well play a role in the diarrheogenic action of these bile acids.

Effects of Deconjugated Bile Acids on Electrolyte and Nutrient Transport in the Rabbit Small Intestine In Vitro

To obtain new information on the poorly investigated mechanisms of the diarrheogenic effects of bile acids, we investigated the effects of the unconjugated bile acids chenodeoxycholate and ursodeoxycholate on the small intestine transport of electrolytes and nutrients in the rabbit jejunal and ileal mucosa mounted in vitro in Ussing or influx chambers. When added to the ileal mucosa at a concentration of 1 mM, both bile acids induced a secretory shift in ion transport; absorption of Na and Cl was abolished and secretion of bicarbonate was enhanced. No changes in short circuit current or electrolyte transport were observed when the bile acids were added to the jejunal mucosa. The addition of 1 mM chenodeoxycholate to the luminal side of the ileum induced a marked inhibition in the uptake of glucose, phenylalanine, and glutamic acid. A similar inhibition, though of a lesser magnitude, was also induced by both bile acids on jejunal nutrient transport. Further studies on the inhibitory effect of chenodeoxycholate on glucose influx showed that the inhibition was dose-dependent; at 1 mM it was maximal and resulted in the complete abolition of the active component, with only the diffusional pathway still operative. We conclude that unconjugated bile acids act as secretagogues in the ileum and significantly blunt the active transport of nutrients in the small intestine. Both effects may well play a role in the diarrheogenic action of these bile acids.

Quantitative studies of invasion of rabbit ileal mucosa by Salmonella typhimurium strains which differ in virulence in a model of gastroenteritis

An asymmetric organ culture system in which ileal tissues, freshly removed from rabbits, can be maintained structurally and functionally for up to 4 h has been developed. The composition of the solutions used to maintain ileal tissue in vitro were as follows. The serosal surface was bathed in the World Health Organization (WHO) rehydration formulation: NaCl, 60 mM; NaHCO3, 30 mM; KCl, 20 mM; and glucose, 111 mM. The mucosal surface was bathed in the same solution with two important changes: all the sodium was replaced by choline, which is not absorbed, and tissue culture medium (consisting of commercial minimal essential medium to which was added fetal calf serum and glutamine to final concentrations of 10% [vol/vol] and 2.0 mM, respectively) was added to the choline-containing medium to a final concentration of 10% (vol/vol). The initial invasiveness (first 2 h) of seven strains of Salmonella typhimurium differing in virulence (defined in terms of clinical origin or the ability to induce fluid loss in monkeys or rabbit ileal loops) was assessed quantitatively in an in vitro invasion assay with the organ culture system. The virulent strains (TML, W118, and WAKE) were found to be about 25- to 100-fold more invasive than the avirulent strains (SL1027, M206, LT7, and Thax-1). Thus, a clear correlation between initial mucosal invasion and virulence of S. typhimurium in a model which is relevant to human gastroenteritis was established. This is the first time, to our knowledge, that quantitative studies of invasiveness have been carried out in vitro on freshly isolated functioning gut.

Bicarbonate secretion in rabbit ileum: Electrogenicity, ion dependence, and effects of cyclic nucleotides

Background: Ileal HCO3− secretion is not well understood. The aim of this study was to examine its Na+ and Cl− dependencies, electrogenicity, and responses to amiloride, 4-acetamido-4′-isothiocyano-stilbene-2,2′-disulfonate (SITS), and cyclic nucleotides. Methods: The serosa to mucosa HCO3− flux (Jsm) across rabbit ileal mucosa mounted between HCO3−-free mucosal solution and HCO3−-containing serosal solutions was determined by titration. Results: In SO42−-containing Ringer's solution, Jsm varied with [Na+] in two phases, one with a high and one with a low affinity for Na+; amiloride inhibited the high- and SITS inhibited the low-affinity phase. Switching from SO42−-to Cl−-containing Ringer's solution caused a SITS-inhibitable 42% increase in Jsm. Changes in Jsm were coupled 3:2 with changes in short-circuit current. Cyclic nucleotide effects on Jsm were as follows. In SO42−-containing Ringer's solution at 141 (but not 80) mmol/L Na+, theophylline caused equal increases in Jsm and short-circuit current that equaled the combined effects of 8-Br-5′-cyclic guanosine monophosphate (cGMP) and 8-Br-5′-cyclic adenosine monophosphate (cAMP). Serosal SITS blocked these effects, but amiloride did not. In Cl−-containing Ringer's solution, theophylline and bumetanide together (but not separately) increased Jsm. Conclusions: (1) Basolateral HCO3− entry occurs via Na+/H exchange and a SITS-inhibitable process (Na+-HCO3− cotransport?). (2) Most HCO3− exit across the brush border occurs by a Cl−-independent process and some by Cl−/HCO3− exchange. (3) At low cellular [Cl−], HCO3− can be secreted via anion channels activated by cAMP and cGMP. (4) Ileal HCO3− secretion is electrogenic.

PH-dependent transepithelial transport of cephalexin in rabbit intestinal mucosa

Previous studies have demonstrated carrier-mediated cephalexin (CPX) transport in rat jejunum under basal conditions. The purpose of this study was to compare fluxes of cephalexin (CPX) and benzylpenicillin (BZP) across rabbit intestine to those reported in rat intestine, to determine whether these fluxes exhibit regioselectivity and to investigate the pH dependence of these fluxes. Fluxes of CPX were examined in rabbit jejunal, ileal and distal colonie mucosa in Ussing chambers. Concurrently, transepithelial (TE) potential difference, TE electrical conductance and mannitol fluxes were determined to evaluate tissue viability and integrity. Mucosal (m)-to-serosal(s) fluxes of CPX were ~ 3-times the s-to-m fluxes (0.66 vs 0.18% h −1 3 cm −2, respectively) in both jejunum and ileum under short-circuit conditions. Distal colonic m-to-s and s-to-m fluxes were ~ 0.18% h −1 3 cm −2. Inhibition of Na +/K +-ATPase activity with ouabain or incubation with the Na +H + exchange inhibitor, amiloride, reduced by 70 and 40%, respectively, the m-to-s fluxes of CPX in the ileum. Effects of changes in luminal pH on TE transport of CPX and mannitol were studied in ileal and colonic mucosa. At a luminal pH of 5.5, ileal m-to-s transport of CPX was increased to 2.85 ± 0.12% h −1 3 cm −2 while mannitol flux and electrical properties were unchanged. In distal colon, pH 5.5 in the luminal bathing solution did not alter CPX or mannitol fluxes. pH dependent carrier-mediated transport was not exhibited by BZP. Thus CPX appears to be absorbed by a pH dependent, carrier-mediated mechanism confined to the small intestine whereas BZP absorption appears to occur by an entirely passive process.

Cloning of a gene (zot) encoding a new toxin produced by Vibrio cholerae

Live oral candidate cholera vaccines have previously been constructed by deletion of Vibrio cholerae sequences encoding the enzymatically active A subunit of the cholera toxin. However, volunteer studies have shown that these non-cholera toxin-producing strains still provoke mild to moderate diarrhea in some individuals. We recently reported the identification of a second toxin produced by V. cholerae which may be responsible for this residual diarrhea (A. Fasano, B. Baudry, D. W. Pumplin, S. S. Wasserman, B. D. Tall, J. M. Ketley, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 88:5242-5246, 1991). This new toxigenic factor increases the permeability of rabbit ileal mucosa by affecting the structure of the intercellular tight junctions (zonula occludens). We now report the identification and cloning of the gene encoding this new toxin. This gene, named zot (for zonula occludens toxin), consists of a 1.3-kb open reading frame which could potentially encode a 44.8-kDa polypeptide. The location of the zot gene encoding the new toxin is immediately upstream of the ctx operon encoding cholera toxin.

Endothelin: a potent stimulator of intestinal ion secretion in vitro

Effects of endothelin (ET) on electrical properties and Na + and Cl − fluxes in stripped rabbit ileal mucosa were investigated in vitro in Ussing chambers. Results demonstrate that serosal addition of ET-1, ET-2, ET-3 or the precursor 38 amino acid ‘big endothelin’ produce dose-dependent increases in short-circuit current ( I sc ) with maximal effects at approx. 100 nM, 100 nM, 10 nM and 100 nM, respectively and half-maximal effects at 1.4 nM, 5 nM, 1.4 nM and 20 nM, respectively. Mucosal addition of ET-3 failed to elicit a response. Changes in I sc elicited by ET-3 are accompanied by decreases in net fluxes of both Na + and Cl −. The cyclooxygenase inhibitors, indomethacin and piroxicam, inhibited the increase in I sc produced by ET-3 and indomethacin also abolished the changes in Na + and Cl − fluxes produced by ET-3. However, no changes in the release of PGE 2, thromboxane B 2 or 6-keto-prostaglandin F 1α could be detected up to 20 min after the addition of ET-3. Preincubation of tissues with neuronal agonists or antagonists, antihistamines or an LTD 4/LTE 4 receptor antagonist, SKF 104353, failed to alter the response to ET-3. Furthermore, removal of serosal Ca 2+ also failed to inhibit the change in I sc produced by ET-3. These results indicate that endothelin is a potent intestinal secretagogue which does not appear to elicit its response through stimulation of PGE 2, thromboxane A 2 or prostacyclin.

Roles of motility and flagellar structure in pathogenicity of Vibrio cholerae: analysis of motility mutants in three animal models

Wild-type Vibrio cholerae of both El Tor and classical biotypes (strains N16961 and 395, respectively) and nonmotile mutant derivatives with and without flagellar structures were characterized in three different animal models: (i) the rabbit ileal loop, (ii) the removable intestinal tie adult rabbit diarrhea (RITARD) model, and (iii) the suckling mouse model. Both the wild-type strains and nonmotile mutants were toxinogenic in the rabbit ileal loop and the suckling mouse models. However, all of the nonmotile mutants produced significantly less fluid accumulation than did the wild-type parental strains. The two nonmotile mutants of strain N16961 did not adhere to rabbit ileal mucosa, but both nonmotile mutants derived from strain 395 exhibited adherence. In the RITARD model, the motile El Tor strains were more virulent than both the flagellate and aflagellate nonmotile mutants (all infected rabbits died within 18 h), while the nonmotile mutants, when fatalities occurred, required 78 to 105 h to produce a fatal outcome. Likewise, the motile classical parent 395 produced a fatal outcome within ca. 25 h, while nonmotile mutants required 69 to 96 h. The nonmotile flagellate strain KR31 was not significantly more virulent than the nonmotile aflagellate strain KR26. Of the two classical nonmotile mutants, KR1, which produces a coreless sheathlike structure, was clearly more virulent (5 of 10 rabbits died within 96 h), while KR3 (nonmotile, aflagellate) did not produce fatalities in any of the 10 rabbits tested. Similarly, no significant difference in diarrheagenicity or colonizing ability was detected between the two nonmotile mutants derived from the El Tor strain, but the classical nonmotile mutant with the coreless sheath caused significantly greater diarrhea and colonized for a longer time than did the isogenic nonmotile aflagellate strain, KR3. No significant differences between the nonmotile mutants were detected in competition studies done with suckling mice. Analysis of the wild-type and mutant strains in these three animal models clearly demonstrated a role for motility in V. cholerae pathogenicity, while analysis of only the nonmotile mutants derived from the classical parent suggested a role for flagellar structures.

Enteroaggregative Escherichia coli elaborate a heat-stable enterotoxin demonstrable in an in vitro rabbit intestinal model.

Enteroaggregative Escherichia coli (EAggEC) have been associated with persistent diarrhea in young children, but little is known about its pathogenesis. We assayed for enterotoxic activity in culture filtrates (CF) of EAggEC strains in Ussing chambers mounted with rabbit ileal mucosa. CF from strain 17-2, a prototype Chilean EAggEC strain, caused a greater rise in potential difference and short circuit current (SCC) than that seen in HB101 control, and this effect was abolished by protease pretreatment and partially stable after heat treatment. Ultrafiltration of 17-2 CF preparations localized the active moiety to the 2-5 kD Mr size range. CF from HB101 transformed with the 17-2 plasmid showed Ussing chamber activity. less than 10-kD CF fractions from five of six other EAggEC strains screened in Ussing chambers gave SCC responses of similar magnitude to 17-2. The 17-2 CF activity was not neutralized after pretreatment with polyclonal anti-STa antibody. Additionally, all of the seven EAggEC strains studied were nonreactive by heat-stable enterotoxin variant STa ELISA, were negative in the suckling mouse assay, and failed to hybridize with heat-stable enterotoxin variant STh and STp DNA probes. In summary, our data indicate that 17-2 produces a low molecular weight, partially heat-stable, protease-sensitive enterotoxin which appears to be plasmid associated, and genetically and immunologically distinct from E. coli STa. Preliminary screening suggests that this tox+ phenotype may be common among EAggEC.