RESUMO
The RDEC-1 strain Escherichia coli is an enteroadherent bacterium that produces diarrhea in the rabbit. A histopathologically similar disease has been described in humans. The RDEC-1 bacterium adheres to the epithelium of lymphoid follicles in rabbit ileal Peyer's patches by 4 h postinoculation, 3-4 d before its adherence to absorptive epithelium. The purpose of this study was to determine whether the RDEC-1 bacterium adheres to a specific cell type in the lymphoid follicle epithelium. RDEC-1 bacteria were given in a dose of 2 X 10(6) by the orogastric route to postweanling rabbits. The distal ileal Peyer's patch, taken from 5 control rabbits and 43 rabbits at intervals in the first 24 h postinoculation, was examined by routine and high-voltage electron microscopy. The RDEC-1 bacterium adhered specifically to M (membranous) rather than absorptive epithelial cells of the lymphoid follicle epithelium. Further understanding of how the bacterium attaches to M cells, which transport antigens to intraepithelial lymphocytes, could be useful in designing vaccines to protect mucosal surfaces.
Assuntos
Diarreia/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/fisiologia , Tecido Linfoide/microbiologia , Nódulos Linfáticos Agregados/microbiologia , Animais , Adesão Celular , Epitélio/ultraestrutura , Fímbrias Bacterianas/ultraestrutura , Microscopia Eletrônica , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/ultraestrutura , CoelhosRESUMO
Escherichia coli (strain RDEC-1) adheres to M cells of rabbit Peyer's patch lymphoid follicle epithelium. The RDEC-1 strain contains an 85 X 10(6) D plasmid that codes for pili, which, when purified, adhere to gut absorptive epithelium. This study compared the in vivo lymphoid follicle adherence of the RDEC-1 strain with that of a Shigella flexneri (ShD15) that contained the 85 X 10(6) D plasmid and expressed the RDEC-1 pili, a control E. coli, and a control S. flexneri (ShD12). The bacteria were given in a dose of 10(10) to 0.7-1.1 kg rabbits. The rabbits were sacrificed at 2, 4, 6, and 12 h postinoculation. Peyer's patch tissue was examined by electronmicroscopy and direct fluorescence microscopy. The piliated ShD15 and RDEC-1 bacteria adhered in large numbers at 2 and 4 h postinoculation, but only the RDEC-1 strain persisted and increased in numbers past that time. Control strains did not adhere. The ShD15 strain adhered to and was rapidly taken into M cells, precipitating an acute inflammatory reaction within the follicle and adjacent lumen. Initial lymphoid follicle M cell adherence of the ShD15 strain was associated with the possession of the adherence pilus plasmid. The failure of the ShD15 strain to survive and colonize the lymph follicle epithelium contrasts with the success of the RDEC-1 strain and indicates that the RDEC-1 strain possesses virulence factors in addition to pili.
Assuntos
Escherichia coli/genética , Fímbrias Bacterianas/fisiologia , Tecido Linfoide/microbiologia , Nódulos Linfáticos Agregados/microbiologia , Plasmídeos , Animais , Epitélio/microbiologia , Epitélio/ultraestrutura , Escherichia coli/patogenicidade , Escherichia coli/fisiologia , Escherichia coli/ultraestrutura , Infecções por Escherichia coli/fisiopatologia , Imunofluorescência , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Nódulos Linfáticos Agregados/ultraestrutura , CoelhosRESUMO
Spontaneous and dexamethasone-induced noninsulin-dependent diabetes mellitus (NIDDM) in rats is associated with loss of glucose-stimulated insulin secretion (GSIS) and a reduction in both GLUT-2-positive beta cells and high Km glucose transport. To determine if the chronology and correlation of these abnormalities is consistent with a causal relationship, Zucker (fa/fa) rats were studied longitudinally before and during 10 d of dexamethasone-induced (0.4 mg/kg per d i.p.) NIDDM. Within 24 h of dexamethasone treatment blood glucose rose and GSIS declined, becoming paradoxically negative (-87 +/- 12 microU/ml per min) on day 10. Blood glucose was negatively correlated with GSIS (r = -0.92; P < 0.001). 3-0-methyl-D-glucose (3MG) transport was unchanged at 12 h, 23% below normal on day 1, and declined further to a nadir 59% below normal. The GLUT-2-positive beta cell area did not decline until 48 h, reaching a nadir of 35% of normal at 10 d. The area of GLUT-2-positive beta cells was correlated with GSIS (r = 0.77; P < 0.005). We conclude that the chronology and correlation between GSIS loss and hyperglycemia is consistent with a cause-effect relationship, but that the subtotal impairment in glucose transport by itself cannot explain the total loss of GSIS if one assumes that normal beta cells are functionally homogenous.
Assuntos
Dexametasona/toxicidade , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Ilhotas Pancreáticas/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , 3-O-Metilglucose , Animais , Arginina/farmacologia , Transporte Biológico/efeitos dos fármacos , Glicemia/metabolismo , Feminino , Transportador de Glucose Tipo 2 , Técnicas In Vitro , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Metilglucosídeos/metabolismo , Mifepristona/farmacologia , Proteínas de Transporte de Monossacarídeos/efeitos dos fármacos , Ratos , Ratos ZuckerRESUMO
GLUT2 underexpression has been reported in the beta-cells of Zucker diabetic fatty rats and db/db mice, models of spontaneously occurring NIDDM with antecedent obesity. To determine whether the beta-cells of a nonobese rodent model of NIDDM exhibit the same abnormalities in GLUT2, we studied Goto-Kakizaki rats. In these mildly diabetic animals glucose-stimulated insulin secretion was reduced at all ages examined from 8 to 48 wk. In normal control Wistar rats, immunostainable GLUT2 was present on all insulin-positive cells in the pancreatic islets. Only 85% of beta-cells were GLUT2-positive in GK rats at 12 wk of age, and only 34% were positive at 48 wk of age. GLUT2 mRNA was 50% of normal in 12-wk-old GK rats. In the latter age-group, glucose-stimulated insulin secretion was only 28% of normal at a time when 85% of beta-cells were GLUT2-positive and initial 3-O-methyl-D-glucose transport rate was 77% of the control value. We conclude that although GLUT2 is underexpressed, neither the magnitude of the underexpression of GLUT2 nor of the reduction in GLUT2 transport function in islets of GK rats is sufficient by itself to explain the profound reduction in glucose-stimulated insulin secretion.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Expressão Gênica , Glucose/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Transporte de Monossacarídeos/biossíntese , Envelhecimento/metabolismo , Animais , Arginina/farmacologia , Transporte Biológico , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos , Ratos Wistar , Especificidade da EspécieRESUMO
To determine if alterations in endothelial prostaglandin production occur after long-term cocaine use, 26 New Zealand White rabbits were randomized to a low fat diet with (n = 12) or without (n = 14) daily intravenous cocaine (2 mg/kg body weight). Rabbits were killed at 6 or 12 weeks. Segments of aorta were examined in blinded manner for histologic changes. Additional slices were incubated in oxygenated Krebs buffer and release of 6-keto-prostaglandin F1 alpha, thromboxane B2 and prostaglandin E2 was assayed by radioimmunoassay. Minimal intimal histologic changes were seen in the aorta of three cocaine-treated rabbits. At 12 weeks 6-keto-prostaglandin F1 alpha was increased in the cocaine group (p = 0.063) as compared with levels in the control group. When rabbits killed at 6 and 12 weeks were considered together, increases in thromboxane B2 (p = 0.044) and a trend to increased prostaglandin E2 (p = 0.083) were seen in the cocaine group. The ratio of thromboxane B2 to 6-keto-prostaglandin F1 alpha was increased in the cocaine group compared with that in the control group (p less than 0.02). These data suggest that an increase in prostaglandin production occurs in the vascular endothelium of rabbits ingesting cocaine before gross histologic changes are evident. In addition, thromboxane B2 increases disproportionately with respect to 6-keto-prostaglandin F1 alpha, suggesting that a milieu for thrombosis may exist in users of cocaine.
Assuntos
Aorta/efeitos dos fármacos , Cocaína/farmacologia , Endotélio Vascular/efeitos dos fármacos , Prostaglandinas/biossíntese , Animais , Aorta/metabolismo , Aorta/patologia , Endotélio Vascular/metabolismo , CoelhosRESUMO
An asymptomatic adult female rabbit had intestinal cryptosporidiosis. The ileum had blunted villi, a decrease in villus-crypt ratio and a mild edema in the lamina propria. Transmission electron microscopy showed the parasite to be a Cryptosporidium similar to those reported in mouse, guinea pig, lamb, calf, horse, monkey and man. This organism is referred to as Cryptosporidium cuniculus. Scanning electron microscopy on ileal mucosa showed altered intestinal microvilli in the attachment of the cryptosporidia. It is postulated that the organism was enveloped by the microvilli of the ileal epithelial cells which then fused and formed a continuous double membrane around the parasite.
Assuntos
Coccidiose/veterinária , Coelhos , Animais , Coccídios/ultraestrutura , Coccidiose/parasitologia , Coccidiose/patologia , Feminino , Íleo/ultraestrutura , Microscopia Eletrônica , Microscopia Eletrônica de VarreduraRESUMO
A strain of Escherichia coli (RDEC-1) has been described that in rabbits colonizes the intestine; adheres to mucosal epithelial cells of the ileum, cecum, and colon; and causes diarrhea by an unknown mechanism. This study attempted to determine the location of the bacteria in the rabbit intestine during the unexplained six- to seven-day interval between bacterial inoculation and onset of diarrhea. Specimens of ileum, cecum, colon, ileal Peyer's patches, sacculus rotundus, and appendix from control rabbits and from rabbits killed at intervals after inoculation with RDEC-1 bacteria were examined by light and direct fluorescence microscopy. Bacteria in large numbers attach to the tips of the Peyer's patch lymphoid follicles by 24 hr, but they did not attached to ileal, cecal, or colonic mucosa until three days after inoculation. The lag time between inoculation and onset of diarrhea was probably due to the need for the bacteria first to attach to and then colonize the Peyer's patch lymphoid follicles. The intestinal mucosa was probably colonized by bacteria shed from the Peyer's patches.
Assuntos
Diarreia/etiologia , Infecções por Escherichia coli/complicações , Escherichia coli/crescimento & desenvolvimento , Tecido Linfoide/microbiologia , Nódulos Linfáticos Agregados/microbiologia , Animais , Apêndice/microbiologia , Ceco/microbiologia , Colo/microbiologia , Íleo/microbiologia , Microscopia de Fluorescência , CoelhosRESUMO
The group factor 3,4 somatic antigen was transferred by recombination from a donor strain of Shigella flexneri type 2a to the Escherichia coli O15 rabbit pathogen strain RDEC-1. A hybrid clone (7482-1-1) that expressed only the S. flexneri 3,4 somatic antigen and a second clone (7482-1-7) from the same mating mixture that expressed only the O15 somatic antigen were compared for virulence in rabbits. The 7482-1-1 strain produced diarrhea in 10% of rabbits versus 84% for the 7482-1-7 strain (P less than .001). In fluorescent antibody-stained, frozen, sectioned tissues, fewer 7482-1-1 bacteria adhered to ileum (P = .013) and cecum (P = .044). Light and electron microscopic studies demonstrated that the recombinant strain, which adhered to membranous (M) cells to the same degree as the 7482-1-7 strain, was found beneath the epithelium, where its presence resulted in a marked acute inflammatory response. Both the 7482-1-1 and 7482-1-7 strains exhibited characteristic close adherence to M cells and absorptive ileal and cecal mucosa.
Assuntos
Antígenos de Bactérias/biossíntese , Escherichia coli/patogenicidade , Intestinos/microbiologia , Recombinação Genética , Animais , Adesão Celular , Escherichia coli/imunologia , Escherichia coli/metabolismo , Imunofluorescência , Mucosa Intestinal/microbiologia , Lipopolissacarídeos/análise , Microscopia Eletrônica , Antígenos O , Coelhos , Shigella flexneri/imunologiaRESUMO
Escherichia coli (RDEC-1) adheres to Peyer's patch and absorptive epithelium in the rabbit in the closely adhering manner characteristic of enteropathogenic and enterohemorrhagic E. coli. An adherence pilus (AF/R1) is important for adherence to Peyer's patch M cells in vivo and ileal brush borders in vitro. A nonpiliated mutant (42-2-37-8) of the RDEC-1 strain colonized the gut lumen less readily than the parent strain. The mutant adhered infrequently to Peyer's patch lymphoid follicle epithelium (22% vs. 84%, P less than .0001). Ileal close adherence was less frequent at 3 d, but by 9 d after inoculation had increased, approaching that of the parent RDEC-1 strain. However, adherence was focal, and fewer bacteria were present at each adherence site compared with the parent RDEC-1 strain. The result was a lower frequency of diarrhea (34% vs. 65%, P = .003) and mortality (9.4% vs. 27%, P = .035) with the 42-2-37-8 strain. Loss of AF/R1 pili compromised the ability of the RDEC-1 strain to adhere to Peyer's patch and absorptive epithelium and to produce diarrhea.
Assuntos
Aderência Bacteriana , Diarreia/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/patogenicidade , Fímbrias Bacterianas , Animais , Epitélio/microbiologia , Escherichia coli/genética , Escherichia coli/ultraestrutura , Intestinos/microbiologia , Microscopia Eletrônica , Mutação , Nódulos Linfáticos Agregados/microbiologia , CoelhosRESUMO
Pre-diabetic male Zucker diabetic fatty rats (ZDF) become diabetic between 8 and 10 weeks of age. At that time their beta cells exhibit high basal insulin secretion, absent insulin response to glucose and loss of GLUT 2 glucose transporter. Beta-cell volume, which is increased at the onset of non-insulin-dependent diabetes, declines precipitously by age 18 weeks. To determine if expression of this diabetic phenotype was dependent upon the increased food intake of these rats, they were diet-matched to lean littermates for 12 weeks beginning at 6 weeks of age. Untreated control ZDF rats received an unrestricted diet for 3 months. All of the controls became hyperglycaemic by 8 weeks of age, whereas all diet-matched rats remained euglycaemic throughout the 3 months, despite the fact that at 18 weeks of age their mean body weight equaled that of obese rats on an unrestricted diet. In the former rats glucose-stimulated insulin secretion was absent at 12 weeks of age and GLUT-2-positive beta cells had fallen below 30%. The volume fraction of their beta cells was 2.6 times normal at this age but by 18 weeks of age it had declined by 75%. Diet restriction for 3 months prevented the loss of glucose-stimulated insulin secretion and the reduction of beta-cell GLUT-2 and beta-cell volume fraction. However, neither the elevated basal insulin secretion nor the exaggerated arginine-stimulated insulin secretion of the obese rats was reversed or prevented by caloric restriction.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Ingestão de Energia , Ilhotas Pancreáticas/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Obesidade/metabolismo , Estado Pré-Diabético/metabolismo , Animais , Transportador de Glucose Tipo 2 , Hiperglicemia/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Estado Pré-Diabético/dietoterapia , Ratos , Ratos ZuckerRESUMO
RDEC-1 is a strain of Escherichia coli that, in rabbits, attaches to intestinal mucosal epithelial cells bereft of microvillar borders and causes diarrhea by an unknown mechanism. The stages of attachment of RDEC-1 bacteria to mucosal epithelial cells were examined using high-voltage electron microscopy of thick (0.5-micrometers) sections of ileum and cecum of rabbits with diarrhea. The tissues were stained with ruthenium red or antisera to strain RDEC-1 OK antigens. Micrographs, including stereopairs, demonstrated several stages of bacterial attachment. Bacteria were attached to the glycocalyxes of epithelial cell microvilli and to pedestal-like extrusions of the surfaces of epithelial cells lacking microvilli. Structures consistent with bacterial pili were rarely visualized. Attachment to microvilli appeared to be a result of the interaction of polysaccharides of the microvillar glycocalyx and the K antigen of the bacterial capsule.
Assuntos
Sítios de Ligação Microbiológicos , Diarreia/imunologia , Infecções por Escherichia coli/imunologia , Mucosa Intestinal/imunologia , Lisogenia , Animais , Diarreia/patologia , Epitopos , Escherichia coli/imunologia , Escherichia coli/ultraestrutura , Infecções por Escherichia coli/patologia , Fímbrias Bacterianas , Mucosa Intestinal/ultraestrutura , Microvilosidades/imunologia , Microvilosidades/ultraestrutura , Polissacarídeos Bacterianos/imunologia , CoelhosRESUMO
The stimulus for increased gallbladder eicosanoid synthesis during cholecystitis is unknown. This study examines the hypothesis that increased intragallbladder pressure stimulates endogenous gallbladder eicosanoid release. Rabbit gallbladders were perfused in vitro at 1 ml/minute with oxygenated Krebs-Henseleit buffer and subjected to 0, 12 or 24 mm Hg of intraluminal gallbladder pressure. Release of 6-keto-PGF1 alpha infinity PGE2 and thromboxane B2 were measured in all groups after 15 and 30 and 60 minutes of perfusion by enzyme immunoassay and gallbladders were examined histologically. Increasing intraluminal gallbladder pressure concomitantly increased gallbladder 6-keto-PGF1 alpha release 2 fold or more at all time of perfusion and altered gallbladder mucosal architecture by increasing basolateral edema in the submucosal space. Infusion of indomethacin (10 micrograms/ml in the perfusion media) decreased 6-keto-PGF1 alpha release from the in vitro perfused gallbladders subjected to 24 mm Hg by 70%. Increased gallbladder eicosanoid release during early cholecystitis may in part be related to the physical force of increased gallbladder intraluminal pressure on the gallbladder mucosa.
Assuntos
6-Cetoprostaglandina F1 alfa/análise , Colecistite/metabolismo , Dinoprostona/análise , Vesícula Biliar/fisiologia , Tromboxano B2/análise , Animais , Vesícula Biliar/anatomia & histologia , Vesícula Biliar/efeitos dos fármacos , Pressão Hidrostática , Técnicas Imunoenzimáticas , Indometacina/farmacologia , Masculino , Perfusão , CoelhosRESUMO
Although obesity is associated with insulin resistance, most obese humans and rodents remain normoglycemic because of compensatory hyperinsulinemia. This has been attributed to beta-cell hyperplasia and increased low Km glucose metabolism of islets. Since free fatty acids (FFA) can induce these same beta-cell changes in normal islets of Wistar rats and since plasma FFA are increased in obesity, FFA could be the signal from adipocytes that elicits beta-cell compensation sufficient to prevent diabetes. To determine if FFA-induced compensation is impaired in islets of rats with a diabetogenic mutation, the Zucker diabetic fatty (ZDF) rat, we cultured islets from 6-week-old obese (fa/fa) rats that had compensated for obesity and apparently normal islets from lean ZDF rats (fa/+) in 0, 1, or 2 mM FFA. Low Km glucose usage rose 2.5-fold in FFA-cultured control islets from age-matched Wistar rats, but failed to rise in either the precompensated islets of ZDF rats or in islets of lean ZDF rats. Bromodeoxyuridine incorporation increased 3.2-fold in Wistar islets but not in islets from obese or lean ZDF rats. Insulin secretion doubled in normal islets cultured in 2 mM FFA (p < 0.01) but increased only slightly in islets from lean ZDF rats (not significant) and declined in islets from obese ZDF rats (p < 0.05). We conclude that, unlike the islets of age-matched Wistar rats, islets of 6-week-old heterozygous and homozygous ZDF rats lack the capacity for FFA-induced enhancement of beta-cell function.
Assuntos
Diabetes Mellitus/fisiopatologia , Ácidos Graxos não Esterificados/farmacologia , Ilhotas Pancreáticas/metabolismo , Obesidade , Animais , Bromodesoxiuridina , Sobrevivência Celular , Células Cultivadas , Diabetes Mellitus/genética , Ácidos Graxos não Esterificados/sangue , Feminino , Glucose/metabolismo , Glucose/farmacologia , Heterozigoto , Homozigoto , Hiperinsulinismo/fisiopatologia , Técnicas In Vitro , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Masculino , Ratos , Ratos Wistar , Ratos Zucker , Valores de Referência , Caracteres Sexuais , Fatores Sexuais , Especificidade da Espécie , Fatores de TempoRESUMO
RDEC-1 is a piliated strain of Escherichia coli that was isolated from and produces diarrhea in rabbits without invading the mucosa or synthesizing one of the classical enterotoxins. Previous histological and fluorescent-antibody studies of RDEC-1 diarrhea revealed an acute inflammatory response and large numbers of RDEC-1 associated with (adhering to) the mucosal surface of the ileum, cecum, and colon. The purpose of the present investigation was to further elucidate the histopathology by scanning (SEM) and transmission (TEM) electron microscopy. SEM revealed aggregates of bacteria on the surface of the gut; their distribution was patchy in the ileum and diffuse in the cecum and colon. Bacteria were in contact with each other and appeared to be closely associated with the epithelial surface. TEM showed that the brush border region of the epithelial cells was found to be in varying stages of degeneration, and the bacteria could not be seen adhering to the mucosal cells unless the brush border was absent. Bacteria were in close contact only with epithelial cells that had lost their brush border. The space between the bacteria and the epithelial cells was 11 nm, and it appeared to be filled, in most cases, with densely stained material. This E. coli rarely penetrated epithelial cells, but when it did; it was found in the supranuclear region and never reached the lamina propria. From previous and present studies, it seems probable that RDEC-1 produces diarrhea in rabbits by a mechanism that may be cytotoxic and differs from the classic mechanisms by which E. coli produces diarrhea.
Assuntos
Diarreia/patologia , Infecções por Escherichia coli/patologia , Mucosa Intestinal/ultraestrutura , Animais , Membrana Celular/microbiologia , Epitélio/microbiologia , Epitélio/ultraestrutura , Escherichia coli/ultraestrutura , Mucosa Intestinal/microbiologia , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , CoelhosRESUMO
Purified immunoglobulin G (IgG) from the serum of patients with insulin-dependent diabetes mellitus (IDDM) of recent onset inhibits high-Km uptake of 3-O-methyl-beta-D-glucose by rat pancreatic islets. To determine if the inhibition is the result of antibodies against GLUT-2, the high-Km glucose transporter of beta cells, we incubated IDDM sera with rat islet cells and with AtT-20ins cells transfected to express GLUT-2. IDDM sera inhibited glucose uptake in islet cells and in GLUT-2-expressing AtT-20ins cells but not in AtT-20ins cells transfected to express the low-Km isoform, GLUT-1. In 24 of 30 (77%) patients with newly diagnosed IDDM, IgG binding as measured by immunofluorescence and flow cytometry of the cells transfected to express GLUT-2 was > 2 standard deviations from the mean of the nondiabetic population; 29 of 31 (96%) of nondiabetic children were negative (P < 0.0001). Increased IgG binding could be removed by absorption with GLUT-2-expressing cells but not with GLUT-1-expressing cells. We conclude that most patients with IDDM of recent onset have autoantibodies to GLUT-2.