Alterations in thymocyte populations under conditions of endotoxin tolerance / 中华医学杂志(英文版)

BACKGROUND@#Endotoxin tolerance (ET) is a protective phenomenon in which pre-treatment with a tolerance dose of lipopolysaccharide (LPS) leads to dramatically elevated survival. Accumulating evidence has shown that peripheral T cells contribute to the induction of ET. However, what happens to T cell development in the thymus under ET conditions remains unclear. The purpose of this study was to analyze the alterations in thymocyte populations (double-positive [DP] and single-positive [SP] cells) under ET conditions.@*METHODS@#Mice were intraperitoneally injected with LPS at a concentration of 5 mg/kg to establish an LPS tolerance model and were divided into two groups: a group examined 72 h after LPS injection (72-h group) and a group examined 8 days after LPS injection (8-day group). Injection of phosphate-buffered saline was used as a control (control group). Changes in thymus weight, cell counts, and morphology were detected in the three groups. Moreover, surface molecules such as CD4, CD8, CD44, CD69, and CD62L were analyzed using flow cytometry. Furthermore, proliferation, apoptosis, cytokine production, and extracellular signal-regulated kinase (ERK) pathway signaling were analyzed in thymocyte populations. The polymorphism and length of the T-cell receptor (TCR) β chain complementarity-determining region 3 (CDR3) were analyzed using capillary electrophoresis DNA laser scanning analysis (ABI 3730).@*RESULTS@#Thymus weight and cell counts were decreased in the early stage but recovered by the late stage in a murine model of LPS-induced ET. Moreover, the proportions of DP cells (control: 72.130 ± 4.074, 72-h: 10.600 ± 3.517, 8-day: 84.770 ± 2.228), CD4+ SP cells (control: 15.770 ± 4.419, 72-h: 44.670 ± 3.089, 8-day: 6.367 ± 0.513), and CD8+ SP cells (control: 7.000 ± 1.916, 72-h: 34.030 ± 3.850, 8-day: 5.133 ± 0.647) were obviously different at different stages of ET. The polymorphism and length of TCR β chain CDR3 also changed obviously, indicating the occurrence of TCR rearrangement and thymocyte diversification. Further analysis showed that the expression of surface molecules, including CD44, CD69, and CD62L, on thymocyte populations (DP and SP cells) were changed to different degrees. Finally, the proliferation, apoptosis, cytokine production, and ERK pathway signaling of thymocyte populations were changed significantly.@*CONCLUSION@#These data reveal that alterations in thymocyte populations might contribute to the establishment of ET.

Expression of cry1Ab gene from a novel Bacillus thuringiensis strain SY49-1 active on pest insects

ABSTRACT In this study, the cry1Ab gene of previously characterized and Lepidoptera-, Diptera-, and Coleoptera-active Bacillus thuringiensis SY49-1 strain was cloned, expressed and individually tested on Ephestia kuehniella (Lepidoptera: Pyralidae) and Plodia interpunctella (Lepidoptera: Pyralidae) larvae. pET-cry1Ab plasmids were constructed by ligating the cry1Ab into pET28a (+) expression vector. Constructed plasmids were transferred to an Escherichia coli BL21 (DE3) strain rendered competent with CaCl2. Isopropyl β-D-1-thiogalactopyranoside was used to induce the expression of cry1Ab in E. coli BL21(DE3), and consequently, ∼130 kDa of Cry1Ab was obtained. Bioassay results indicated that recombinant Cry1Ab at a dose of 1000 µg g-1 caused 40% and 64% mortality on P. interpunctella and E. kuehniella larvae, respectively. However, the mortality rates of Bt SY49-1 strains' spore-crystal mixture at the same dose were observed to be 70% on P. interpunctella and 90% on E. kuehniella larvae. The results indicated that cry1Ab may be considered as a good candidate in transgenic crop production and as an alternative biocontrol agent in controlling stored product moths.

Effect of Trimetaphosphate and Fluoride Association on Hydroxyapatite Dissolution and Precipitation In Vitro

The present study analyzed the action of sodium trimetaphosphate (TMP) and/or fluoride on hydroxyapatite. Hydroxyapatite powder was suspended in different solutions: deionized water, 500 µg F/mL, 1,100 µg F/mL, 1%TMP, 3%TMP, 500 µg F/mL plus 1%TMP and 500 µg F/mL plus 3%TMP. The pH value of the solutions was reduced to 4.0 and after 30 min, raised to 7.0 (three times). After pH-cycling, the samples were analyzed by X-ray diffraction and infrared spectroscopy. The concentrations of calcium fluoride, fluoride, calcium and phosphorus were also determined. Adding 1% or 3% TMP to the solution containing 500 µg F/mL produced a higher quantity of calcium fluoride compared to samples prepared in a 1,100 µg F/mL solution. Regarding the calcium concentration, samples prepared in solutions of 1,100 µg F/mL and 500 µg F/mL plus TMP were statistically similar and showed higher values. Using solutions of 1,100 µg F/mL and 500 µg F/mL plus TMP resulted in a calcium/phosphorus ratio close to that of hydroxyapatite. It is concluded that the association of TMP and fluoride favored the precipitation of a more stable hydroxyapatite.

O presente estudo avaliou a ação do trimetafosfato de sódio (TMP) e/ou fluoreto sobre a hidroxiapatita. Pó de hidroxiapatita foi suspenso em diferentes soluções: água deionizada, 500 µg F/mL, 1100 µg F/mL, 1%TMP, 3%TMP, 500 µg F/mL adicionado a 1%TMP e 500 µg F/mL associado a 3%TMP. O pH das soluções foi reduzido para 4,0 e depois de 30 min, elevado para 7,0 (três vezes). Depois do processo de ciclagem de pH, as amostras foram analisadas por difração de raios-X e espectroscopia por infravermelho. As concentrações de fluoreto de cálcio, fluoreto, cálcio e fósforo também foram determinadas. A adição de 1% ou 3% TMP na solução contendo 500 µg F/mL produziu uma maior quantidade de fluoreto de cálcio comparado às amostras tratadas com uma solução de 1100 µg F/mL. A respeito da concentração de cálcio, amostras tratadas com soluções de 1100 µg F/mL e 500 µg F/mL adicionado ao TMP foram estatisticamente similares e mostraram maiores valores. Soluções de 1100 µg F/mL e 500 µg F/mL adicionado ao TMP resultaram em uma proporção molar Ca/P mais próxima à da hidroxiapatita. Conclui-se que a associação de TMP e F favoreceu a precipitação de uma hidroxiapatita mais estável.

Tendencias en la mortalidad por cáncer en México de 1980 a 2011 / Cancer mortality trends in Mexico, 1980-2011

Objetivo. Evaluar las tendencias de mortalidad por cáncer en México entre 1980 y 2011. Material y métodos. Se calcularon las tasas de mortalidad ajustadas por edad y sexo para todos los cánceres y para las 15 localizaciones más frecuentes mediante el método directo y tomando como población estándar la población mundial de 2010. Las tendencias en las tasas de mortalidad y el cambio porcentual anual para cada tipo de cáncer se estimaron a través de un modelo de regresión joinpoint. Resultados. A partir de 2004 y como consecuencia de la reducción de la mortalidad por cáncer de pulmón (-3.2% en hombres y -1.8% en mujeres), estómago (-2.1% en hombres y -2.4% en mujeres) y cérvix (-4.7%), se observó una disminución significativa (~1% anual) en la mortalidad por cáncer en general tanto en el grupo de todas las edades como en el de 35 a 64 años para ambos sexos. La mortalidad por otros cánceres como el de mama y el de ovario, en las mujeres o el de próstata, en los hombres, mostró un aumento sostenido. Conclusiones. Algunas de las reducciones en la mortalidad por cáncer pueden ser parcialmente atribuidas a la efectividad de los programas de prevención establecidos. Sin embargo, se requiere implementar registros adecuados de cáncer con base poblacional para evaluar el impacto real de estos programas, así como diseñar y evaluar intervenciones innovadoras que permitan desarrollar políticas de prevención más costo-efectivas.

Objective. To evaluate trends in cancer mortality in Mexico between 1980-2011. Material and methods. Through direct method and using World Population 2010 as standard population, mortality rates for all cancers and the 15 most frequent locations, adjusted for age and sex were calculated. Trends in mortality rates and annual percentage change for each type of cancer were estimated by joinpoint regression model. Results. As a result of the reduction in mortality from lung cancer (-3.2% -1.8% in men and in women), stomach (-2.1% -2.4% in men and in women) and cervix (-4.7%); since 2004 a significant (~1% per year) decline was observed in cancer mortality in general, in all ages, and in the group of 35-64 years of both sexes. Other cancers such as breast and ovarian cancer in women; as well as for prostate cancer in men, showed a steady increase. Conclusions. Some of the reductions in cancer mortality may be partially attributed to the effectiveness of prevention programs. However, adequate records of population-based cancer are needed to assess the real impact of these programs; as well as designing and evaluating innovative interventions to develop more cost-effective prevention policies.

Edaravone protects endotoxin-induced liver injury by inhibiting apoptosis and reducing proinflammatory cytokines

Studies have shown that edaravone may prevent liver injury. This study aimed to investigate the effects of edaravone on the liver injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS) in female BALB/c mice. Edaravone was injected into mice 30 min before and 4 h after GalN/LPS injection. The survival rate was determined within the first 24 h. Animals were killed 8 h after GalN/LPS injection, and liver injury was biochemically and histologically assessed. Hepatocyte apoptosis was measured by TUNEL staining; proinflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in the liver were assayed by ELISA; expression of caspase-8 and caspase-3 proteins was detected by Western blot assay; and caspase-3 activity was also determined. Results showed that GalN/LPS induced marked elevations in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Edaravone significantly inhibited elevation of serum AST and ALT, accompanied by an improvement in histological findings. Edaravone lowered the levels of TNF-α and IL-6 and reduced the number of TUNEL-positive cells. In addition, 24 h after edaravone treatment, caspase-3 activity and mortality were reduced. Edaravone may effectively ameliorate GalN/LPS-induced liver injury in mice by reducing proinflammatory cytokines and inhibiting apoptosis.

Histopathology and the lethal effect of Cry proteins and strains of Bacillus thuringiensis Berliner in Spodoptera frugiperda J.E. Smith Caterpillars (Lepidoptera, Noctuidae) / Histopatologia e efeito letal de cepas e proteínas Cry de Bacillus thuringiensis Berliner para lagartas de Spodoptera frugiperda J.E. Smith (Lepidoptera, Noctuidae)

Among the phytophagous insects which attack crops, the fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera, Noctuidae) is particularly harmful in the initial growth phase of rice plants. As a potential means of controlling this pest, and considering that the entomopathogen Bacillus thuringiensis Berliner demonstrates toxicity due to synthesis of the Cry protein, the present study was undertaken to evaluate this toxic effect of B. thuringiensis thuringiensis 407 (pH 408) and B. thuringiensis kurstaki HD-73 on S. frugiperda. The following method was used. Both bacterial strains were evaluated in vitro in 1st instar S. frugiperda caterpillars, by means of histopathological assays. The Cry1Ab and Cry1Ac proteins, codified by the respective strains of B. thuringiensis, were evaluated in vivo by bioassays of 1st instar S. frugiperda caterpillars in order to determine the Mean Lethal Concentration (LC50). The results of the histopathological analysis of the midget of S. frugiperda caterpillars demonstrate that treatment with the B. thuringiensis thuringiensis strain was more efficient, because the degradations of the microvilosities started 9 hours after treatment application (HAT), while in the B. thuringiensis kurstaki the same effect was noticed only after 12 HAT. Toxicity data of the Cry1Ab and Cry1Ac proteins presented for the target-species LC50 levels of 9.29 and 1.79 μg.cm-2 respectively. The strains and proteins synthesised by B. thuringiensis thuringiensis and B. thuringiensis kurstaki are effective in controlling S. frugiperda, and may be used to produce new biopesticides or the genes may be utilised in the genetic transformation of Oryza sativa L.

Entre os insetos fitófagos que atacam as culturas, Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera, Noctuidae) destaca-se como uma praga polífaga que causa prejuízos na fase inicial da cultura do arroz. No seu controle, o entomopatógeno Bacillus thuringiensis Berliner revela-se tóxico devido à síntese de proteínas Cry. Nesse contexto, o objetivo deste trabalho foi avaliar a toxicidade das cepas e proteínas Cry de B. thuringiensis thuringiensis 407 (pH 408) e B. thuringiensis kurstaki HD-73 sobre S. frugiperda. As duas cepas bacterianas foram avaliadas, in vitro, em lagartas de 1º instar de S. frugiperda, através de ensaios de histopatologia. As proteínas Cry1Ab e Cry1Ac, codificadas pelas respectivas cepas de B. thuringiensis, foram avaliadas in vivo, através de bioensaios com lagartas de 1º instar de S. frugiperda para determinação da Concentração Letal Média (CL50). Os resultados da análise histopatológica do intestino médio das lagartas S. frugiperda mostram que o tratamento com a cepa B. thuringiensis thuringiensis foi mais eficiente e a degradação das microvilosidade iniciou-se 9 horas após a aplicação dos tratamentos (HAT). Para B. thuringiensis kurstaki, o mesmo efeito foi observado, 12 HAT. Os dados de toxicidade das proteínas de Cry1Ab e Cry1Ac revelaram para a espécie-alvo uma CL50 de 9,29 e 1,79 μg.cm-2, respectivamente. As cepas e proteínas sintetizadas por B. thuringiensis thuringiensis e B. thuringiensis kurstaki são eficientes no controle de S. frugiperda, e poderão ser usadas na produção de novos biopesticidas ou a utilização dos genes na transformação genética de Oryza sativa L.

Presence and significance of Bacillus thuringiensis Cry proteins associated with the Andean weevil Premnotrypes vorax (Coleoptera: Curculionidae)

The Andean weevil Premnotrypes vorax represents an important cause of damage to Colombian potato crops. Due to the impact of this plague on the economy of the country, we searched for new alternatives for its biological control, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 300 B. thuringiensis strains obtained from potato plantations infested with P. vorax were analyzed through crystal morphology, SDS-PAGE, PCR and bioassays. We used site- directed mutagenesis to modify the Cry3Aa protein. Most of the B. thuringiensis isolates had a bipyramidal crystal morphology. SDS-PAGE analyses had seven strains groups with σ-endotoxins from 35 to 135 kDa. The genes cry 2 and cry 1 were significantly more frequent in the P. vorax habitat (PCR analyses). Three mutant toxins, 1 (D354E), 2 (R345A, ∆Y350, ∆Y351), and 3 (Q482A, S484A, R485A), were analyzed to assess their activity against P. vorax larvae. Toxicity was low, or absent, against P. vorax for isolates, wild type cry 3Aa and cry 3Aa mutants. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against other insect pests of agricultural importance, and for designing Cry proteins with improved insecticidal toxicity. Rev. Biol. Trop. 57 (4): 1235-1243. Epub 2009 December 01.

El gorgojo andino Premnotrypes vorax es una causa importante de daño en los cultivos colombianos de este tubérculo. Debido al impacto que esta plaga tiene sobre la economía del país, nos interesamos en buscar alternativas nuevas para el control biológico de P. vorax, basadas en la bacteria entomopatógena Bacillus thuringiensis. Se recolectaron un total de 300 cepas de B. thuringiensis a partir de plantaciones de papa infestadas con P. vorax, las cuales fueron analizadas por medio de la morfología del cristal, SDS-PAGE, PCR y ensayos biológicos. La mayoría de los aislamientos de B. thuringiensis presentaron cristales bipiramidales. Los análisis de SDS-PAGE indicaron la presencia de siete grupos de cepas con σ- endotoxinas que variaban entre 35 a 135 kDa. Las pruebas con PCR demostraron que los genes cry 2 y cry 1 fueron significativamente más frecuentes en el medioambiente de P. vorax. Además, se utilizó la mutagénesis sitio-dirigida para modificar la proteína Cry3Aa. Se analizaron tres toxinas mutantes, 1 (D354E), 2 (R345A, ∆Y350, ∆Y351), y 3 (Q482A, S484A, R485A), para determinar su actividad contra larvas de P. vorax. Los ensayos de toxicidad señalaron escasa, o nula, actividad hacia P. vorax tanto para las cepas, la toxina Cry3Aa de referencia y las proteínas Cry3Aa mutantes. La caracterización genética de la colección puede proveer oportunidades para la selección de cepas que pueden evaluarse por medio de bioensayos contra otros insectos-plaga de importancia agrícola, y para el diseño de proteínas Cry con actividad toxica mejorada.

Toxigenicity testing of clinical isolates of non-typhoidal salmonellae in Vero cell culture & Caenorhabditis elegans model.

The non-typhoidal salmonellae (NTS) are recognized agents of gastroenteritis worldwide. Some of the NTS do not produces cytotoxic changes in tissue culture and not much is known about the endotoxicity of the clinical isolates of NTS (mostly Salmonella enterica serotype Typhimurium and Salmonella enterica serotype Enteritidis). We examined the exotoxic (cytotoxin) and endotoxic activity of clinical isolates of NTS in two assay models namely Vero cell culture and the nematode, Caenorhabditis elegans. Bacteria-free culture supernatants of 40 isolates NTS were tested in 96 well microtitre plate containing confluent monolayers of Vero cells. For the effects on C. elegans, the worms were exposed to bacteria free culture supernatants in 24 well microtitre plate for 24 h and then transferred to OP50 Escherichia coli lawn culture. The endotoxic activity of the live bacterium was studied by feeding the worms in the lawn culture of NTS separately. No cytopathic effect was observed with NTS tested in Vero cell culture assay. Likewise, the worms exposed to the bacteria-free culture supernatants were found active up to 7 days. In the co-culture killing assay, worms were found dead with characteristic stiff and straight appearance by 16(th) day. The worms were alive up to 21 days in OP50 E. coli. Bacteria-free culture supernatants did not have any deleterious effect on worms or in Vero cell culture, suggesting that there is no soluble toxic factor (diffusible toxin) in the culture supernatants. However, live NTS were found to be lethal to the worms; indicating that direct interaction between viable NTS and C. elegans is necessary for killing.

Bacillus thuringiensis in caterpillars and associated materials collected from protected tropical forests in northwestern Costa Rica

Bacillus thuringiensis (Bt) synthesizes crystalline inclusions that are toxic to caterpillars (Lepidoptera) and other orders of invertebrates. Materials associated with 37 caterpillars from 16 species, collected while feeding on 15 different species of host plants in dry, cloud and rain forests located in the Area de Conservación Guanacaste in northwestern Costa Rica, were examined for the presence of Bt. From a total of 101 derived samples, 25 Bt isolates were cultured: 56% from host plant leaves, 8% from caterpillar guts and 36% from caterpillar fecal pellets. Bt was isolated from at least one sample in 38% of the systems constituted by the food plant, gut and fecal pellets corresponding to a single caterpillar. Four different morphologies of crystalline inclusions were observed, with bipyramidal and irregular crystal morphologies being the most prevalent.

Bacillus thuringiensis (Bt) sintetiza inclusiones cristalinas que resultan tóxicas para algunas larvas de lepidópteros y otros órdenes de invertebrados. Su presencia fue examinada en materiales asociados a 37 orugas de mariposas de 16 especies, las cuales fueron colectadas mientras se alimentaban en 15 especies diferentes de plantas hospederas en bosques secos, nubosos y húmedos localizados dentro del Área de Conservación Guanacaste (ACG) en el noroeste de Costa Rica. A partir de un total de 101 muestras se obtuvo 25 aislamientos de Bt: 56% a partir de material foliar de las plantas hospederas, 8% a partir del contenido intestinal de las larvas y 36% a partir de sus excrementos. Esta bacteria fue cultivada a partir de al menos uno de los 3 diferentes tipos de muestra asociados a una oruga particular (planta hospedera, intestino, excremento) en 38% de los casos posibles. En la colección de aislamientos obtenida se observaron cuatro morfologías de inclusiones cristalinas, siendo aquellas bipiramidales e irregulares las más prevalentes.

Effect of Pseudomonas aeruginosa (MTCC 1688) on the tissue phosphatases activity on Macrobrachium rosenbergii (De Man).

A time course study on the endotoxin toxicity of the gram negative bacteria, Pseudomonas aeruginosa MTCC 1688 on the tissue phosphatases activity on the giant freshwater prawn, Macrobrachium rosenbergii was conducted. The results revealed marked elevation of both acid and alkaline phosphatase activity in the haemolymph and body muscle. The hepatopancreas showed reduced phosphatase activity compared to control. The enzymes, being non-specific in action and particularly the acid phosphatase being of lysosomal origin, their increase in muscle and haemolymph has pathogenic significance in the inoculum treated prawns.

Effect of Salmonella typhimurium toxin on the expression of rabbit intestinal functions.

BACKGROUND & OBJECTIVES: Infection by Salmonella Typhimurium is one of the leading causes of intestinal dysfunction, however the underlying mechanism of this effect is largely unknown. Hence the effect of enterotoxin secreted by Salmonella Typhimurium-(S-LT) was studied on D-glucose absorption and brush border enzymes in rabbit ileum. mRNA levels encoding these proteins were also analysed. METHODS: Adult male New Zealand white rabbits were used. The polymyxine B extract of enterotoxin obtained from Salmonella Typhimurium was tested for the presence of enterotoxicity by rabbit ileal loop test. D-glucose uptake by ileal tissue was measured by the tissue accumulation method. Intestinal brush border membranes were isolated and the effect of S-LT on various brush border enzymes studied. RESULTS: S-LT significantly inhibited (P < 0.01) the absorption of Na+ dependent D-glucose uptake but had no effect on Na+ independent sugar uptake in rabbit ileum. The activities of brush border sucrase (72% P < 0.001) and lactase (47% P < 0.01) and alkaline phosphatase (43% P < 0.01) were also significantly reduced in infected animals as compared to the controls. Northern blot analysis revealed that mRNA levels encoding Na+ glucose co-transporter (SGLT1), brush border lactase and sucrase activities were unaffected in Salmonella infected rabbit ileal loops. INTERPRETATION & CONCLUSION: The findings suggest that the intestinal dysfunctions observed in Salmonella infection are unrelated to mRNA expression encoding Na+ glucose co-transporter and brush border enzyme proteins in rabbit ileum.

Effect of Indigofera tinctoria Linn on liver antioxidant defense system during D-galactosamine/endotoxin-induced acute hepatitis in rodents.

Effects of pre-treatment with the alcoholic extract of I. tinctoria (500 mg/kg body wt/day, p.o. for 21 days) on liver antioxidant defense system during acute hepatitis induced by D-galactosamine (D-GalN)/endotoxin (LPS extracted by phenol water method from E. coli serotype 0111.B4; 300 mg and 30 micrograms/kg body wt/day, i.p., 18 hr before the assay) were investigated on the activities of enzymic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase, and levels of total reduced glutathione in the liver of normal and experimental groups of male albino rats. Since lipid peroxidation and associated membrane damage is a key feature of D-galN/LPS-induced liver injury, the levels of lipid peroxides, was estimated and used as an index of oxidative stress. D-GalN/endotoxin-induced hepatic damage was manifested by a significant decrease in the activities of antioxidant enzymes, decreased glutathione levels and increased levels of lipid peroxides. I. tinctoria pre-treated rats showed considerable protection against D-galN/endotoxin, induced oxidative stress as evidenced by a significant increase in the activities of all the antioxidant enzymes studied and significant decrease in the levels of lipid peroxides. Results indicate that pretreatment with I. tinctoria extract in rats is very effective in reducing D-GalN/endotoxin-induced oxidative stress suggesting an antioxidant effect.

Salmonella 3, 10:r: toxicity in rabbit ileum and liver by light and electron microscopy.

Salmonella 3, 10:r:- (a monophasic variety of otherwise diphasic serotypes such as S. weltevreden and S. simi) Cell_free filtrate, when introduded into rabbit ligated ileal loops causes fluid exsorption, as studied 18-hr after treatment. Light microscopic histology of treated ileum shows denudation of the columnar epithelium at several places, thereby allowing the passage of the toxic principle into circulation. An important target organ, liver shows extensive centrilobular necrosis, as observed by light microscopy. Transmission electron microscopy of ileum reveals opening of membrane junctions between the adjacent cells of epithelial lining of the treated ileum at places, and focal devitalization including formation of intra-cellular membranous inclusinos. Electron microscopy of liver shows extensive damage and swelling of cytoplasmic membranes. However, the areas of darkly staining lamellae of granulated endoplasmic reticulum are also seen in stacks as will as dispersed. These studies stress that Salmonella toxic substances can cause extensive damage to intestine and liver both.

Na, K-ATPase: a molecular target for Leptospira interrogans endotoxin

On the basis of our report that a glycolipoprotein fraction (GLP) extracted from Leptospira interrogans contains a potent inhibitor of renal Na,K-ATPase, we proposed that GLP-induced inhibition of Na,K-ATPase might be the primary cellular defect in the physiopathology of leptospirosis. The present study was designed to test this hypothesis by determining whether or not 1) GLP inhibits all the isoforms of Na,K-ATPase which are expressed in the tissues affected by leptospirosis, 2) Na,K-ATPase from leptospirosis-resistant species, such as the rat, is sensitive to GLP, 3) GLP inhibits Na,K-ATPase from intact cells, and 4) GLP inhibits ouabain-sensitive H,K-ATPase. The results indicate that in the rabbit, a leptospirosis-sensitive species, GLP inhibits with similar efficiency (apparent IC5O: 120-220 mug protein GLP/ml) all isoforms of Na,K-ATPase known to be expressed in target tissues for the disease. Na,K-ATPase from rat kidney displays a sensitivity to GLP similar to that of the rabbit kidney enzyme (apparent IC50: 25-80 and 50-150 mug protein GLP/ml for rat and rabbit, respectively), indicating that resistance to the disease does not result from the resistance of Na,K-ATPase to GLP. GLP also reduces ouabain-sensitive rubidium uptake in rat thick ascending limbs (pmol mm-1 min-1 ñ SEM; control: 23.8 ñ 1.8; GLP, 88 mug protein/ml: 8.2 ñ 0.9), demonstrating that it is active in intact cells. Finally, GLP had no demonstrable effect on renal H,K-ATPase activity, even on the ouabain-sensitive form, indicating that the active principle of GLP is more specific for Na,K-ATPase than ouabain itself. Although the hypothesis remains to be demonstrated in vivo, the present findings are compatible with the putative role of GLP-induced inhibition of Na,K-ATPase as an initial mechanism in the physiopathology of leptospirosis.

Effect of enterotoxin on glutathione status in the intestinal mucosa.

The effect of luminal exposure of enterotoxins on the intestinal mucosal glutathione (GSH) was studied in rat. Cholera toxin induced fluid secretion and decreased mucosal GSH by 35% without altering oxidized glutathione (GSSG) level. Toxin induced fluid secretion was tested after mucosal GSH depletion by compounds such as diethyl maleate (DEM) and buthionine sulfoximine (BSO) and thiol supplementation with N-Acetyl cysteine (NAC). Fluid secretion was not altered by prior thiol depletion or supplementation. Exposure of intestinal lumen to bacterial endotoxin resulted in 25% decrease in mucosal GSH with two fold increase in GSSG. Luminal exposure of Shiga toxin did not alter the mucosal thiol. The level of other low molecular weight thiols, cysteine and cystine was not altered by luminal exposure of any of these toxins. These results show that although cholera toxin decreased the mucosal GSH level, prior modulation of thiol status of the mucosa may not have any effect on toxin-induced fluid secretion.

Endotoxin-induced enteric hypomotility in jaundiced loops in vitro

Biliary obstruction may be accompanied by systemic endotoxemia due to increased growth of enteric microbiota and failure of hepatic clearance mechanisms. This endotoxemia is related to increased postoperative morbidity and mortality. An increased growth of the aerobic flora has been demonstrated experimentally in the presence of biliary obstruction, and in previous studies we observed intestinal hypomotility of jaundiced loops in vitro. To determine the ileal motor response in the presence of jaundice caused by biliary obstruction and in the presence of endotoxemia, an in vitro study was carried out on ileal segments from 10 female Holtzman rats, 2-3 months old, weighing 200 to 300 g, divided into two groups (N = 5): A, washed loops of jaundiced rats, and B, washed loops of jaundiced rats to which endotoxin was added. On the seventh postoperative day, we evaluated the effect of exogenous endotoxin (E. coli 0111:B4, Sigma) on the motor response to acetylcholine of distal ileal segments isolated from both animal groups. A 4-cm ileal segment, located 10 cm from the ileal papilla, was removed and studied in an organ chamber in order to assess dose-response curves to acetylcholine. There was an increase in threshold dose in jaundiced loops with intraluminally injected endotoxin when compared with the loops without intraluminal endotoxin (291 + 188 vs 8.5 + 6.7 muM, P<0.05). The maximum contraction was reduced in jaundiced loops with intraluminal endotoxin in relation to control loops (5.3 + 1.7 vs 18.7 + 4.8 mm, P<0.05), and pD2 was also reduced in jaundiced loops with intraluminal endotoxin in relation to control loops (2.4 + 0.6 vs 3.7 + 0.5, P<0.05). There was no statistical difference between jaundiced loops with and without intraluminal endotoxin when the maximal contraction doses were compared (86 + 66 vs 48 + 22 mM, P>0.05). These results demonstrate that intraluminal endotoxin depressed enteric motility in jaundiced rats.