Suckling Mouse Intestine Research Articles

Intestinal Kinetics And Dynamics Of Escherichia Coli Heat-Stabile Enterotoxin In Suckling Mice

The heat-stabile enterotoxin produced by Escherichia coli (ST) induces diarrhea by binding to receptors on intestinal cells, activating guanylyl cyclase, and increasing cyclic GMP. High- and low-affinity receptors for this toxin have been identified previously. ST induces intestinal secretion in suckling mice in picomole doses, suggesting a role for high-affinity receptors in this process. The present studies examine the relative roles of high- and low-affinity receptors in this process. The time course of changes in free ST concentrations in suckling mouse intestine was determined after intragastric inoculation. Also, binding characteristics of high- and low-affinity receptors and their coupling to guanylyl cyclase were defined in intestinal membranes from suckling mice. Intestinal concentrations of toxin and receptor binding characteristics empirically determined were used in a dynamic model correlating fractional occupancy of high- and low-affinity receptors with intestinal secretion to estimate their relative contributions to ST-induced diarrhea.

Ultrastructural study on experimental infection of rotavirus in a murine heterologous model.

Viral replication, histopathological and ultrastructural changes were observed for a period of nine days in the small intestine of suckling mice infected with a simian rotavirus (SA11). Samples taken from duodenum, jejunum and ileum were prepared for light microscopy, transmission and scanning electron microscopy analysis. Histopathologic effect could be detected within 8 hr post-infection, when only a few altered cells were observed. Damage was extensive after 16 hr post-infection, showing swollen enterocytes and reduced and irregularly oriented microvilli at intestinal villi tips. Virus particles were detected at 16 and 48 hr post-infection, budding from the viroplasm into the rough endoplasmic reticulum cisternae in ileum enterocytes. Clear evidence of viral replication, observed by electron microscopy was not described before in heterologous murine models. Regeneration of the intestinal villi began at the third day post-infection. Despite some differences observed in clinical symptoms and microscopic analysis of homologous and heterologous rotavirus infections, we concluded that mechanisms of heterologous rotavirus infection in mice follow similar patterns to those observed in the homologous models.

Oral passive immunization effect of anti-human rotavirus IgY and its behavior against proteolytic enzymes.

The neutralization titer of anti-human rotavirus (HRV) IgY was completely inactivated by pepsin at pH 2.0. However, it was not significantly affected by trypsin or chymotrypsin under certain conditions. The immunological activity of the IgY was observed in the intestine of suckling mice for 2 h after oral administration and the activity rapidly decreased thereafter. The effects of oral supply of IgY were thus estimated for HRV-induced diarrhea in suckling mice and it was found that a previous supply of the IgY (1 h before HRV infection) completely prevented the HRV-induced diarrhea. The preventive effect was decreased as the time gap between IgY administration and HRV infection was longer. However, the oral supply of the IgY within 24 h after HRV infection was still effective and decreased the incidence of HRV diarrhea in suckling mice.

Disaccharidase Activities in Small Intestine of Rotavirus-Infected Suckling Mice

A histochemical study of the time course of the appearance and location of lactase and alpha-glucosidase (used to detect sucrase and maltase) activities was carried out on control and rotavirus-infected mice from 7 to 14 days old. The overall pattern of enzyme activity was in agreement with previous quantitative studies on the activities of these enzymes. No evidence was obtained to support the idea that lactase deficiency was the result of repopulation of villi (denuded of lactase-producing villus cells) with immature lactase-negative cells. Low lactase activity was more likely to reflect profound changes in metabolically crippled cells, and recovery of lactase activity with recovery of normal metabolic functions. The location of enzyme activity to brush border regions rather than the cytoplasm of villus enterocytes enhances the significance of previous quantitative studies on these enzymes. The timing and duration of diminished lactase activities were such that they were unlikely to cause the induction or perpetuation of diarrhea in murine rotavirus diarrhea. The appearance in infected animals of alpha-glucosidase 3 days earlier than normal indicates that, in addition to reversible changes seen with lactase, developmental changes were accelerated that affected both crypt and villus cells.

Histochemical detection of α-D-galactosidase with 5-Br-4-Cl-3-indoxyl α-D-galactoside

5-Br-4-Cl-3-indoxyl alpha-D-galactoside was used as a new substrate in azoindoxyl, indigogenic, and tetrazolium procedures for the detection of alpha-D-galactosidase in the light microscope. Compared with the simultaneous azo-dye methods using 1-naphthyl or 6-Br-2-naphthyl alpha-D-galactoside as substrates and hexazotized pararosaniline for simultaneous coupling, primarily the azoindoxyl method with hexazotized pararosaniline and the indigogenic technique proved to be superior. The azoindoxyl reaction is recommended for the localization of alpha-D-galactosidase in lysosomes when freeze-dried celloidin-coated cryostat sections are used: the indigogenic procedure should be employed for the detection of the total activity of the enzyme in combination with the technique of semipermeable membranes. The tetrazolium reaction delivered high amounts of formazan as the final reaction product; however, its localization was less precise than with the azoindoxyl and indigogenic methods. Conspicious species differences were found; except for the small intestine of suckling mice the highest activities of alpha-D-galactosidase were present in different immature and mature rat organs when compared with mouse, hamster, guinea-pig, marmoset monkey, and human tissues.

Characterization of the Vibrio cholerae ToxR regulon: identification of novel genes involved in intestinal colonization.

A gene fusion library of Vibrio cholerae classical strain O395 was generated by using a broad host range vector for delivery of the transposon TnphoA. The insertion library was screened for colonies expressing alkaline phosphatase-positive (PhoA+) fusion proteins on LB agar at 30 degrees C in the presence of 0.2% glucose. Over 600 PhoA+ strains were isolated and then tested for regulation of their gene fusions in broth media that permitted high or low expression of cholera toxin. This strategy resulted in the isolation of 60 TnphoA (Tn5 IS50L::phoA) fusions to genes encoding secreted proteins that are apparently coordinately regulated with cholera toxin. Introduction of a toxR null mutation into 10 of these fusion strains confirmed that these TnphoA gene fusions are controlled either directly or indirectly by the cholera toxin transcriptional activator encoded by toxR. A combination of Southern and immunoblot analysis identified 17 distinct ToxR-regulated genes in V. cholerae O395. Many of these insertions were located in one of the two cholera toxin operon copies of strain O395, as well as a large gene cluster involved in the biogenesis of the toxin-coregulated pilus colonization factor. In addition, insertions were identified in genes that had no effect on either cholera toxin or toxin-coregulated pilus expression. Several of these insertions were localized to a cluster of four genes, the disruption of any of which by TnphoA reduced the ability of strain O395 to colonize the intestines of suckling mice. The product encoded by this second gene cluster was named accessory colonization factor to describe its possible role in cholera pathogenesis. These studies reinforce the contribution of ToxR-regulated genes to the virulence properties of V. cholerae. This report also demonstrates a new approach for the identification of bacterial virulence factors, based on the characterization of genes that are regulated by the same environmental signals that control the expression of a known virulence factor.

Structural features of the apical and tubulovesicular membranes of rodent small intestinal tuft cells.

Tuft cells are present in most columnar epithelia derived from endoderm including the small intestine. They are characterized by long, wide apical microvilli and an extensively developed cytoplasmic tubulovesicular system. We examined in detail the structural features of the apical plasma membrane of small intestinal tuft cells from adult guinea pigs, rats, and adult and suckling mice with freeze-fracture and conventional transmission electron microscopy methods and utilized cationized ferritin and horseradish peroxidase as tracers to determine whether tuft cells endocytose macromolecules. The microvillus membrane of intestinal tuft cells has few P-face intramembrane particles, displays little alkaline phosphatase activity, and is highly enriched in cholesterol. Tuft cell tight junctions resemble those of absorptive cells in strand count and strand-to-strand crosslinks but, unlike those of absorptive cells, they display many abluminal free-ending strands. Tuft cells of adult and suckling mouse intestine show no evidence of internalization of cationized ferritin or, in suckling mice, uptake of horseradish peroxidase. We conclude that the microvillus membrane of small intestinal tuft cells is protein-poor but cholesterol-rich and that small intestinal tuft cells do not endocytose macromolecules in bulk from the intestinal lumen.

Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin.

The transposon TnphoA was used to generate fusions between phoA, the gene for alkaline phosphatase (PhoA), and genes encoding proteins that are secreted by Vibrio cholerae. One of the PhoA+ mutants isolated showed a dramatic reduction in its ability to colonize the intestines of suckling mice. This mutant no longer produced a 20.5-kDa protein (TcpA) that we show is the major subunit of a V. cholerae pilus. Amino-terminal sequence analysis of the TcpA pilus subunit showed that it shares amino acid homology with the pilins produced by several other pathogenic bacteria. The TcpA pilus was coordinately expressed with cholera toxin under various culture conditions, and this effect appeared to be dependent on the transcriptional activator encoded by the toxR gene. We conclude that the toxR gene plays a central role in the transcriptional regulation of multiple virulence genes of V. cholerae.

Stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the gastrointestinal tract of the suckling mouse

1. 1. The effects of epidermal growth factor (EGF), cortisone and thyroxine on deoxyribonucleic acid (DNA) synthesis in the esophagus, stomach, small intestine and colon have been studied in suckling mouse. 2. 2. Daily administration of EGF [4 μg/g body weight (bw)/day] during 3 days to 8-day-old mice induced a significant increase of the incorporation of [ 3H]thymidine into DNA in the stomach, the small intestine, and the two halves of the colon. 3. 3. The DNA synthesis in the esophagus remained unaffected by the EGF treatment. 4. 4. The maximal increase of [ 3H]thymidine incorporation into DNA was observed in the colon, and represented 112%. 5. 5. Daily administration of cortisone acetate (25 μg/g bw/day) or thyroxine (1 μg/g bw/day) during 3 days to 8-day-old mice had no significant influence of the DNA synthesis of any part of the gastrointestinal tract. 6. 6. These results show that EGF is able to affect the DNA synthesis in the stomach, small intestine and colon of suckling mice.

Histopathological studies on experimental marine toxin poisoning. I. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin-1 and pectenotoxin-1

K. Terao, E. Ito, T. Yanagi and T. Yasumoto. Histopathological studies on experimental marine toxin poisoning. I. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin-1 and pectenotoxin-1. Toxicon 24, 1141 – 1151, 1986 — Sequential ultrastructural changes were studied in mouse digestive organs after i.p. injections of dinophysistoxin-1 and pectenotoxin-1, causative agents of diarrhetic shellfish poisoning. Dinophysistoxin-1, a diarrheagenic substance, produced severe mucosal injuries in the small intestine within 1 hr after the administration of the toxin. The injuries were divided into 3 consecutive stages: extravasation of villi vessels, degeneration of absorptive epithelium and desquamation of the degenerated epithelium from the lamina propria. In contrast to dinophysistoxin-1, pectenotoxin-1, a non-diarrheagenic toxin from diarrhetic shellfish poisoning causative mussels, resulted in no abnormalities in the small intestine, but did cause characteristic liver injuries. Within 1 hr after the injection of pectenotoxin-1 numerous non-fatty vacuoles appeared in the hepatocytes around the periportal regions of the hepatic lobules. Electron microscopic observations with colloidal iron demonstrated that these vacuoles originated from invaginated plasma membranes of the hepatocytes.

Isolation, primary structure and synthesis of heat-stable enterotoxin produced by Yersinia enterocolitica

Five heat-stable enterotoxins were isolated from the culture supernatant of Yersinia enterocolitica and purified to homogeneity by DEAE-Sephacel and high-performance liquid chromatographies. They caused acute fluid accumulation in the intestine of suckling mice. The amino acid sequence of one of the enterotoxins was determined to be Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys, by Edman degradation of its pyridylethylated derivative and a combination of fast atom bombardment mass spectrometry and carboxypeptidase B digestion. This structure was unambiguously confirmed by chemical synthesis. The other enterotoxins had longer or shorter amino acid sequences at their N termini, but the same sequence at their C termini. The six half-cystine residues formed intramolecular disulfide linkages, as shown by measurement of the molecular masses of the enterotoxins by fast atom bombardment mass spectrometry. The sequence of 13 amino acid residues at the C terminus showed similarity to those of heat-stable enterotoxins isolated from enterotoxigenic Escherichia coli [Aimoto, S. et al. (1982) Eur. J. Biochem. 129, 257-263; Takao, T. et al. (1983) FEBS Lett. 152, 1-5] suggesting that these similar sequences are related to the common biological and immunological properties of enterotoxins produced by Y. enterocolitica and enterotoxigenic E. coli.

Heat-stable Enterotoxin (STh) of Human Enterotoxigenic Escherichia coli (Strain SK-1). Structure-activity Relationship

Abstract For examination of the structure-activity relationship of STh, five shorter analogs of STh lacking one to five N-terminal amino acid residues of STh were synthesized. The synthetic peptides were confirmed to have the same intramolecular disulfide linkages as those in native STh. These peptides caused fluid accumulation in the intestine of suckling mice at almost the same molar levels as that of native STh. The toxicities of these peptides were also neutralized by antisera raised against native STh. These results indicated that the toxic and antigenic sites of STh are both located in the amino acid sequence between the Cys residue at the 5th position from the N-terminus and the C-terminal Tyr residue.

IMMUNOLOGIC STUDIES OF ENTERIC CORONAVIRUS-LIKE PARTICLES(CVLP)

CVLP have been implicated in gastrointestinal illness sporadically among children and in an intensive care nursery outbreak at our hospital. Fecal examination by electron microscopy(EM) is the only means of diagnosis. We developed an enzyme-linked immunosorbent assay(ELISA) using ammonium sulfate precipitated CVLP from feces. The purified CVLP was immunogenic in rabbits. Sera containing antibodies against bovine enteric, canine, OC43, and 229E coronaviruses(CV) did not block binding of rabbit anti-CVLP to microtiter wells coated with purified CVLP. Immune EM using these antisera and nonaggregated CVLP also suggested that CVLP are antigenically distinct from other CV. False positives occurred when stools negative for CVLP by EM were tested. This nonspecific reaction was not blocked by sonicates of E.coli, Strep. fecalis, B.fragilis, or C.difficile, or by C.difficile antitoxin. Absorption of rabbit anti-CVLP serum with suckling mouse intestine did not improve specificity. More sophisticated purification of fecal CVLP is needed to improve ELISA sensitivity and specificity, and to characterize the antigen.

Organ culture of the small intestine of the suckling mouse in a serum-free medium.

Proximal and distal parts of the small intestine of 8-day-old suckling mice can best be maintained for 48 h in a serum-free organ culture system, Leibovitz L-15, at room air and room temperature. As determined by light and electron microscopy, the villous architecture was preserved as well as the classical ultrastructure of the enterocytes. Incorporation of 3H-thymidine and 3H-leucine continued during the culture period, reflecting a sustained synthesis of DNA and proteins for at least 48 h. The hydrolytic activities of the brushborder membrane, namely of lactase (L), trehalase (T), glucoamylase (GA) and alkaline phosphatase (AlPase) were measured in the explants as well as the culture medium. The overall enzymatic activities were increased as compared to the controls. In the tissue, L, GA and T activities remained stable or even increased during culture while in the medium an accumulation of enzymatic activities was noted especially for GA an AlPase. These results show that the morphological as well as the functional integrity of the mucosa is preserved for at least 48 h when small intestine of suckling mice is cultured in a serum-free medium.

Medium-dependent production of extracellular enterotoxins by non-O-1 Vibrio cholerae, Vibrio mimicus, and Vibrio fluvialis.

Fluid accumulation at 4 h in the intestines of suckling mice enabled us to distinguish non-O-1 Vibrio cholerae, V. mimicus, and V. fluvialis clinical isolates from environmental isolates. Enterotoxin production was culture medium dependent. Filtrates of cultures grown in tryptic soy broth without glucose but with added 0.5% NaCl did not exhibit marked enterotoxin activity in the assay. Culture filtrates of all clinical strains grown in brain heart infusion broth supplemented with 0.5% NaCl induced large amounts of fluid accumulation in mouse intestines. However, most environmental strains grown in brain heart infusion broth amended as described above were unable to induce fluid accumulation. The enterotoxin present in culture filtrates lost activity at 56 degrees C and appeared to be distinct from previously described virulence factors, including the well-described cholera toxin. The new enterotoxin could represent an important virulence mechanism common to all three species.

Enteropathogenicity of Aeromonas hydrophila and Plesiomonas shigelloides: prevalence among individuals with and without diarrhea in Thailand

To evaluate the enteropathogenicity of Aeromonas hydrophila and Plesiomonas shigelloides, the rate of isolation of these organisms was compared among individuals with and without diarrhea in Thailand. In two groups of American travelers, A. hydrophila, but not P. shigelloides, was associated with episodes of travelers diarrhea more often than when individuals did not have diarrhea (P less than 0.025). Among three populations of Thais, A. hydrophila and P. shigelloides were isolated with similar frequencies from individuals with and without diarrhea. The biochemical characteristics, production of cytotoxin, and ability to distend suckling mouse intestine were similar among A. hydrophila isolates from individuals with and without diarrhea. However, cytotoxic A. hydrophila strains distended rabbit and suckling mouse intestine and produced destructive lesions in intestinal mucosa of both species of animal. P. shigelloides strains produced neither cytotoxin nor distended intestine. Oral administration of whole cultures (10(9)) of cytotoxic A. hydrophila or P. shigelloides failed to cause diarrhea in rhesus monkeys. Volunteer studies or intestinal biopsies of patients with diarrhea may be required to establish whether A. hydrophila is a gastrointestinal pathogen in humans.

Pathology of Rotavirus Infection in Suckling Mice: A Study by Conventional Histology, Immunofluorescence, Ultrathin Sections, and Scanning Electron Microscopy

Pathologic changes induced in the small intestine of suckling mice by rotavirus infection were studied by conventional histology, immunofluorescence, scanning electron microscopy, and electron microscopy of ultrathin sections. Infection could be detected within 24 hours in a few mice, but after 2 days it was well established. Swollen, often vacuolated infected cells were found on the sides and tips of villi from which they rapidly became detached; microvilli showed variable irregularity. Immature enterocytes from crypts replaced lost infected cells. By the tenth day very few infected cells could still be found. Both tubular structures and spherical particles occurred in the infected cells. Only tubular structures were found in nuclei.

Simplified purification and biophysicochemical characteristics of Kanagawa phenomenon-associated hemolysin of Vibrio parahaemolyticus.

Kanagawa phenomenon-associated hemolysin (K-hemolysin) was purified by Sephadex gel and ion-exchange column chromatography, after the culture supernatant had been adsorbed on and eluted from diethylaminoethyl-Sepharose CL-6B, and acid precipitated. K-hemolysin was a heat-stable and trypsin-susceptible protein with an apparent molecular weight of 44,000, the subunit of which was 22,000. The isoelectric point was 4.9. The minimum hemolytic dose was 0.1 mug/ml. The fifty percent lethal dose by intravenous injection was 1.4 mug. Electron microscopy of the small intestine of suckling mice orally challenged with the highest dose (50 mug) not only showed disappearance of epithelial cell microvilli, but also structural disturbances of the endoplasmic reticulum and mitochondrial swelling. One blueing dose representing permeability factor activity was 0.3 mug, and positive reaction in the rabbit ileal loop appeared at above 125 mug. Besides these data in experimental models, we discovered the appearance of an antibody in patients which neutralizes K-hemolysin during the course of the disease. This finding reinforces our view that K-hemolysin plays a most significant role in the pathogenesis of this enteric human disease.

The effect of Reo Virus Type 3 on the small intestine of suckling mice

The effect of Reo Virus Type 3 on the small intestine of suckling mice

Selective transfer of plasma proteins across mammary gland in lactating mouse.

Specific plasma proteins were labeled with 131I, and their half-lives in lactating and nonlactating mice were determined. The proteins included mouse IgF and IgM, mouse, bovine, rabbit, and human IgG, human serum and salivary IgA, human transferrin and albumin, and mouse and human immunoglobin light chains. The rates at which these proteins are transferred across the mammary gland in mice suckling their young were calculated: transmammary protein transfer was found to be highly selective. The G-class immunoglobulins readily traversed the mammary gland, the rate being dependent on the IgG load presented to the transport system. Immunoglobulin lambda-chains and human serum IgA crossed the mammary gland even more rapidly than did the G immunoglobulins. Human IgA without secretory piece was transported into milk more rapidly than IgA with secretory piece attached. Protein transport across the mouse mammary gland was found to be similar to and yet different from protein transport across either the suckling mouse intestine or the mouse placenta as to which proteins crossed and at what relative rates.