Targeted innovative design of Bt Cry toxin insecticidal mimics / 生物工程学报

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.

Sildenafil in endotoxin-induced pulmonary hypertension: an experimental study

Abstract Background Sepsis and septic shock still represent great challenges in critical care medicine. Sildenafil has been largely used in the treatment of pulmonary arterial hypertension, but its effects in sepsis are unknown. The aim of this study was to investigate the hypothesis that sildenafil can attenuate endotoxin-induced pulmonary hypertension in a porcine model of endotoxemia. Methods Twenty pigs were randomly assigned to Control group (n = 10), which received saline solution; or to Sildenafil group (n = 10), which received sildenafil orally (100 mg). After 30 minutes, both groups were submitted to endotoxemia with intravenous bacterial lipopolysaccharide endotoxin (LPS) infusion (4 µg.kg-1.h-1) for 180 minutes. We evaluated hemodynamic and oxygenation functions, and also lung histology and plasma cytokine (TNFα, IL-1β, IL6, and IL10) and troponin I response. Results Significant hemodynamic alterations were observed after 30 minutes of LPS continuous infusion, mainly in pulmonary arterial pressure (from Baseline 19 ± 2 mmHg to LPS30 52 ± 4 mmHg, p< 0.05). There was also a significant decrease in PaO2/FiO2 (from Baseline 411 ± 29 to LPS180 334 ± 49, p< 0.05). Pulmonary arterial pressure was significantly lower in the Sildenafil group (35 ± 4 mmHg at LPS30, p< 0.05). The Sildenafil group also presented lower values of systemic arterial pressure. Sildenafil maintained oxygenation with higher PaO2/FiO2 and lower oxygen extraction rate than Control group but had no effect on intrapulmonary shunt. All cytokines and troponin increased after LPS infusion in both groups similarly. Conclusion Sildenafil attenuated endotoxin-induced pulmonary hypertension preserving the correct heart function without improving lung lesions or inflammation.

Owlet moths (Lepidoptera: Noctuoidea) associated with Bt and non- Bt soybean in the brazilian savanna / Noctuóides (Lepidoptera: Noctuoidea) associados a soja Bt e não-Bt no Cerrado brasileiro

Abstract The use of GMO expressing Bt toxin in soybean production has increased significantly in the last years in Brazil in order to manage the damage caused by lepidopteran pests. In this study, we compared the richness and abundance of owlet moths (Noctuoidea) associated with Bt and non-Bt soybean. We determined the temporal variations as a function of phenology, and correlated the population variations of the most common species with meteorological variables. The research was conducted at the experimental area of Embrapa Cerrados. The collection method used was differentiated being suppressive and absolute. A total of 13 species were collected, of which eight occurred on Bt soybeans. The most representative taxa were Chrysodeixis includens (72.87%), Anticarsia gemmatalis (18.17%) and Spodoptera spp (5.22%). The number of larvae belonging to species targeted by the Bt technology was 10 times lower on Bt than on non-Bt soybeans. Utetheisa ornatrix and Elaphria deltoides were recorded on soybean for the first time, observing larvae of both species in non-Bt soybean and those of U. ornatrix also in Bt soybean. Only A. gemmatalis larvae correlated (p <0.05) negatively with precipitation. This study provided field information on the abundance and species richness of owlet moths on non-Bt soybeans, associated with the effects of Bt soybean. When considering the different levels of infestation between cultivars as a criterion, larvae monitoring is of substantial importance in order to develop the lost control program.

Resumo O uso de OGM que expressam toxina Bt na produção de soja tem aumentado significativamente nos últimos anos no Brasil e são utilizados para conter os danos causados ​​pelos lepidópteros pragas. Neste estudo comparamos a riqueza e a abundância de Noctuoides (Noctuoidea) associados à soja Bt e não-Bt. Determinamos as variações temporais em função da fenologia e correlacionamos às variações populacionais das espécies mais comuns com variáveis ​​meteorológicas. A pesquisa foi conduzida na área experimental da Embrapa Cerrados. O método de coleta utilizado foi diferenciado sendo supressivo e absoluto. Um total de 13 espécies foram coletadas, das quais oito ocorreram em soja Bt. Os taxa mais representativos foram Chrysodeixis includens, Anticarsia gemmatalis e Spodoptera spp. O número de larvas pertencentes às espécies alvo da tecnologia Bt foram 10 vezes menores na soja Bt do que em soja não-Bt . Utetheisa ornatrix e Elaphria deltoides foram registradas na soja pela primeira vez, observando-se larvas de ambas espécies na soja não-Bt e as de U. ornatrix também na soja Bt. Somente as larvas de A. gemmatalis se correlacionaram (p <0,05) negativamente com a precipitação. Este estudo forneceu informações em campo sobre a abundância e riqueza de espécies na soja não- Bt, associada aos efeitos da soja Bt. A importância do monitoramento das lagartas é substancial, a fim de tomar a melhor decisão de controle, considerando-se os diferentes níveis de infestação entre cultivares como critério.

Characterization of Cry toxins from autochthonous Bacillus thuringiensis isolates from Mexico / Caracterización de toxinas Cry de aislados de Bacillus thuringiensis autóctonos de México

Abstract: Background: Chemical pesticides, widely used in agriculture and vector-borne disease control, have shown toxic effects on the environment and the people in contact with them. Bacillus thuringiensis is a widely used bacterium for alternative and safer control of insect pests. Its toxins are specific for insects but innocuous for mammals and may be used as powerful adjuvants when applied with vaccines. The objective of this work was to characterize some autochthonous B. thuringiensis strains, which could be used for the control of a local pest (Diatraea considerata Heinrich) that affects sugar cane crops in Sinaloa, Mexico. Also, to evaluate these strains as a source of Cry toxins, which may be used in the future as adjuvants for some vaccines. Methods: Eight strains from field-collected dead insects were isolated. These were microbiologically identified as B. thuringiensis and confirmed by amplification and sequencing of 16S rDNA. Bioassays were performed to evaluate their pathogenicity against D. considerata, and Cry toxins were identified by proteomic analyses. Results: An increased mortality among larvae infected with strain Bt-D was observed, and its toxin was identified as Cry1Ac. Conclusions: The observed data showed that the selected strain was pathogenic to D. considerata and seemed to produce Cry1Ac protein, which has been reported as an adjuvant in different types of immunization.

Resumen: Introducción: Los pesticidas químicos, ampliamente usados en agricultura y en el control de vectores transmisores de enfermedades, han mostrado efectos tóxicos sobre el medio ambiente y las personas expuestas a ellos. Bacillus thuringiensis es una bacteria ampliamente utilizada como una alternativa segura y eficaz en el control biológico de plagas agrícolas. Sus toxinas son específicas de insectos, pero inocuas para mamíferos, e incluso poseen gran potencial para ser usadas como adyuvantes en vacunas. El objetivo de este trabajo fue caracterizar cepas autóctonas de B. thuringiensis con efectividad contra el gusano barrenador (Diatraea considerata Heinrich) de la caña de azúcar en cultivos del estado de Sinaloa, México, y como fuente de proteínas Cry, con potencial de utilizarse como adyuvantes en vacunas. Métodos: Se lograron aislar ocho cepas a partir de insectos muertos en campos agrícolas, las cuales fueron identificadas microbiológicamente como B. thuringiensis, lo que se confirmó por amplificación y secuenciación del 16S rDNA. La efectividad de los aislados para el control del gusano barrenador fue evaluada mediante bioensayos y las toxinas Cry fueron identificadas por análisis proteómico. Resultados: Se observó una mortalidad elevada en las larvas infectadas con las cepas de estudio. Particularmente, la cepa Bt-D, de la cual el análisis molecular mostró que posee una toxina tipo Cry1Ac. Conclusiones: Los resultados mostraron que la cepa Bt-D posee un elevado potencial patogénico hacia D. considerata y produce la proteína Cry1Ac, de la cual existen reportes de su aplicación como adyuvante en diferentes formas de inmunización.

Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.

Isolation of Bacillus thuringiensis strains that contain Dipteran-specific cry genes from Ilha Bela (São Paulo, Brazil) soil samples / Isolamento de linhagens de Bacillus thuringiensis contendo genes cry díptero-específicos a partir de amostras de solo oriundas de Ilha Bela-São Paulo, Brasil

The entomophatogenic bacterium Bacillus thuringiensis produces crystal proteins, named Cry proteins which are encoded by the cry genes. This bacterium is used on biological control of important economical pests, as well as in the control of disease´s vectors, such as Aedes aegypti, a mosquito that transmits the dengue viruses. Isolates of this bacterium can be characterized by the content of cry genes and this prediction helps target different insect orders. In this research, we isolated 76 colonies of B. thuringiensis from 30 soil samples that were taken from Ilha Bela (SP, Brazil), a place where simulids are already biologically controlled by B. thuringiensis, to find bacterial isolates that were capable of controlling A. aegypti. The 16S ribosomal subunit genes of the selected isolates were sequenced, and the isolates were molecularly characterized based on their Dipteran-specific cry gene contents. Eight of the 76 isolates (10.52%) contained the cry4Aa, cry4Ba or cry10Aa genes, these isolates were carried out against A. aegypti larvae on bioassay. The presence or absence of specific cry genes was associated with the observed average larval mortalities. From the 76 isolates, seven (9.2%) were potentially able to control A. aegypti larvae. Therefore these are promising isolates for the biological control of A. aegypti larvae.

Bacillus thuringiensis é entomopatogênica, por produzir proteínas cristais, denominadas proteínas Cry, as quais são codificadas pelos genes cry. Essa bactéria atua no controle biológico de insetos-praga de culturas economicamente importantes, bem como no controle de insetos vetores causadores de doenças, como o Aedes aegypti, mosquito transmissor do vírus da dengue. Os isolados dessa bactéria podem ser caracterizados pelo conteúdo de genes cry que possuem e, assim, predizer o alvo de controle dos mesmos às diferentes ordens de insetos. Com o objetivo de encontrar isolados eficientes no controle do vetor A. aegypti, o presente trabalho isolou 76 colônias de B. thuringiensis a partir de 30 amostras de solo oriundas de Ilhabela-SP, município que se caracteriza por realizar controle biológico de simulídeos com essa bactéria. Os 76 isolados foram sequenciados na região da subunidade ribossomal 16S e caracterizados molecularmente quanto ao conteúdo de genes cry díptero-específicos. No total, oito isolados (10,52% do total) apresentaram bandas para os genes cry4Aa, cry4Ba e cry10Aa, sendo os mesmos testados contra larvas de A. aegypti por meio de bioensaios. A presença e/ou ausência dos genes cry foi associada à mortalidade média de larvas. Dentre os isolados estudados, sete (9,2% do total) apresentaram elevado potencial de controle às larvas de A. aegypti, sendo assim considerados como promissores para o manejo do controle biológico de larvas de A. aegypti com a bactéria B. thuringiensis.

Treatment of an Aedes aegypti colony with the Cry11Aa toxin for 54 generations results in the development of resistance

To study the potential for the emergence of resistance in Aedes aegypti populations, a wild colony was subjected to selective pressure with Cry11Aa, one of four endotoxins that compose the Bacillus thuringiensis serovar israelensis toxin. This bacterium is the base component of the most important biopesticide used in the control of mosquitoes worldwide. After 54 generations of selection, significant resistance levels were observed. At the beginning of the selection experiment, the half lethal concentration was 26.3 ng/mL and had risen to 345.6 ng/mL by generation 54. The highest rate of resistance, 13.1, was detected in the 54th generation. Because digestive proteases play a key role in the processing and activation of B. thuringiensis toxin, we analysed the involvement of insect gut proteases in resistance to the Cry11Aa B. thuringiensis serovar israelensis toxin. The protease activity from larval gut extracts from the Cry11Aa resistant population was lower than that of the B. thuringiensisserovar israelensis susceptible colony. We suggest that differences in protoxin proteolysis could contribute to the resistance of this Ae. aegypti colony.

High mobility group box 1 as a mediator of endotoxin administration after hemorrhagic shock-primed lung injury

High mobility group box 1 (HMGB1) was discovered as a novel late-acting cytokine that contributes to acute lung injury (ALI). However, the contribution of HMGB1 to two-hit-induced ALI has not been investigated. To examine the participation of HMGB1 in the pathogenesis of ALI caused by the two-hit hypothesis, endotoxin was injected intratracheally in a hemorrhagic shock-primed ALI mouse model. Concentrations of HMGB1 in the lung of the shock group were markedly increased at 16 h (1.63 ± 0.05, compared to the control group: 1.02 ± 0.03; P < 0.05), with the highest concentration being observed at 24 h. In the sham/lipopolysaccharide group, lung HMGB1 concentrations were found to be markedly increased at 24 h (1.98 ± 0.08, compared to the control group: 1.07 ± 0.03; P < 0.05). Administration of lipopolysaccharide to the hemorrhagic shock group resulted in a notable HMGB1 increase by 4 h, with a further increase by 16 h. Intratracheal lipopolysaccharide injection after hemorrhagic shock resulted in the highest lung leak at 16 h (2.68 ± 0.08, compared to the control group: 1.05 ± 0.04; P < 0.05). Compared to the hemorrhagic shock/lipopolysaccharide mice, blockade of HMGB1 at the same time as lipopolysaccharide injection prevented significantly pulmonary tumor necrosis factor-alpha, interleukin-1beta and myeloperoxidase. Lung leak was also markedly reduced at 16 h; blockade of HMGB1 24 h after lipopolysaccharide injection failed to alter lung leak or myeloperoxidase at 48 h. Our observations suggest that HMGB1 plays a key role as a late mediator when lipopolysaccharide is injected after hemorrhagic shock-primed ALI and the kinetics of its release differs from that of one-hit ALI. The therapeutic window to suppress HMGB1 activity should not be delayed to 24 h after the disease onset.

Evaluation of Bacillus sphaericus bioinsecticide produced with white soybean meal as culture medium for the control of Culex (Culex) quinquefasciatus / Avaliação do bioinseticida de Bacillus sphaericus, produzido com meio de cultivo de farelo branco de soja no controle de Culex (Culex) quinquefasciatus

Bioinsecticides are shown to be useful in control programs to prevent several diseases, based on their specificity and efficiency against insect vectors. In the current study a bioinsecticide based on Bacillus sphaericus was produced using a white soybean culture medium and applied to larvae of Culex quinquefasciatus, the susceptible species, and Aedes aegypti, the refractory species used as the negative control. Efficacy was compared with that of the product fermented with the Luria Bertani (LB) reference medium. The experiments showed that C. quinquefasciatus was highly susceptible to the product prepared with white soybean meal, reaching 100 percent larval mortality even at 10mg/L, while A. aegypti failed to reach 70 percent mortality at a concentration of 1g/L. By comparison with the reference medium, the proposed culture medium showed high larvicidal power, reaching a LD90 of 2.26mg/L, while 4.37mg/L was needed for the LB medium to achieve the same mortality rate. Cost comparison between the formulations favored the use of the bioinsecticide produced with white soybean meal. After factoring in the LD90 value, the cost ratio favored the new raw material by nearly 1:220.

A utilização de bioinseticidas tem se mostrado útil aos programas de prevenção de diversas enfermidades devido a sua especificidade e eficiência contra insetos vetores. No presente trabalho, o bioinseticida de Bacillus sphaericus foi produzido com um meio de cultivo composto de farelo branco de soja e aplicado em larvas de Culex quinquefasciatus, espécie susce-tível, e Aedes aegypti, espécie refratária, usada como controle negativo. O desempenho foi comparado com o do produto fermentado com o meio referência Luria Bertani (LB). Os experimentos constataram que C. quinquefasciatus apresentou uma alta suscetibilidade ao produto produzido com farelo branco de soja, alcançando mortalidade de 100 por cento mesmo na diluição de 10mg/L, enquanto A. aegypti não atingiu 70 por cento na concentração de 1g/L. Quando comparado com o meio referência, a formulação proposta proporcionou um alto poder larvicida, alcançando uma DL90 de 2,26mg/L, ao passo que LB precisou de 4,37mg/L para a mesma mortalidade. A comparação do custo das formulações favoreceu o bioinseticida produzido com o meio farelo branco de soja. Considerando os valores de DL90, a relação do custo das matérias-primas ficou próxima de 1:220.

Effects of endotoxin on liver Smac apoptosis channel / 华中科技大学学报（医学）（英德文版）

To study the effect of endotoxin on liver apoptosis, L02 liver cells were cultured and passaged in vitro, and then stimulated by endotoxin at 10 mg/mL for 4, 8, 16 and 24 h respectively. Liver apoptosis was flow cytometrically and fluorescently detected. Immunohistochemistry was used to detect the delivery of smac and caspase9. The delivery of liver cell smac and the activity of caspase3 were measured by caspase3 assay kit. The hepatic failure models of rats were established by using D-galactosamine. The blood serum and liver tissues were collected for the detection of the liver function, the level of endotoxin and the activity of caspase3 by using chromogenic substrate limulus amebocyte lysate method (LAL) and caspase3 active assay kit. The expression of smac and caspase9 in liver cells was detected by Western blotting. With in vitro study, the L02 cells stimulated by LPS condensed into conglobation and formed apoptotic bodies. After those cells were stained by hoechst, the apoptotic cells displayed blue color under the fluorescent microscope. The apoptosis rate was increased over time and the apoptosis was mainly of advanced stage. Meanwhile, the rate of smac delivery and activity of caspase9 and caspase3 were increased on L02 cell membrane. In vivo, hepatic failure and obvious endotoxemia were induced by injection of more than 200 mg/kg D-GalN. Hepatic mitochondria smac was reduced with dosage of D-GalN and, on the contrary, the activity of caspase3 was increased. D-GalN at 200 mg/kg increased Caspase9 while D-GalN at 300 mg/kg decreased caspase9. Mitochondria signal channel plays an important role in the endotoxin-induced apoptosis of hepatic cells by promoting the release of smac from mitochondria to cytoplasm and activating caspase9 and caspase3 in its low-level channel.

Correlation between endotoxin tolerance in human monocyte leukemia cell line THP-1 with glucocorticoid receptor-alpha / 华中科技大学学报（医学）（英德文版）

Human monocyte leukemia cell line THP-1 was stimulated with lipopolysaccharide (LPS) to simulate the sepsis model and the expression of human glucocorticoid receptor-alpha (GR-alpha) mRNA in montocytes with endotoxin tolerance was investigated. THP-1 cells were cultured in serum-free medium, randomly divided into groups A, B, C, D and E, and stimulated with 0, 10, 10, 100, 0 ng/mL LPS for 24 h followed with 100, 100, 10, 100, 0 ng/mL LPS for another 24 h respectively. The expression of GR-alpha mRNA was detected by semi-quantitative reverse transcriptional polymerase chain reaction. Tumor necrosis factor-alpha (TNF-alpha) was determined by enzyme linked immunosorbent assay (ELISA). The results showed that the A values of GR-alpha/beta-actin in groups A, B, C, D and E was 0.607 +/- 0.006, 0.368 +/- 0.005, 0.484 +/- 0.008, 0.509 +/- 0.004 and 0.564 +/- 0.014 respectively with the difference being significant among the groups (P < 0.05). The GR-alpha mRNA expression was negatively correlated with the TNF-alpha expression (P < 0.01). It was concluded that the down-regulation of the expression of GR-alpha mRNA in endotoxin tolerance THP-1 cells might play an important role in the development of endotoxin tolerance in THP-1 cells.

Influence of endotoxin induced fever on the pharmacokinetics of intramuscularly administered cefepime in rabbits

This study examined the effect of experimentally induced fever on the pharmacokinetics of cefepime (75 mg/kg BW) administered intramuscularly to six rabbits. The study was carried out in two consecutive phases separated by a two-week washout period. An infection was induced by an intravenous inoculation of 5 x 10(8) colony-forming units of Escherichia coli 24 h before the pharmacokinetic investigation. A quantitative microbiological assay was employed to measure the plasma cefepime concentrations using an agar-gel diffusion method with Bacillus subtilis ATCC 6633 as the test organism. Twenty-four hour after the injection, the rectal temperature in the infected animals increased by 1degrees C. There was a significant reduction in the elimination halflife by 21.8% in the febrile rabbits compared to healthy animals. In addition, the infection significantly increased the peak plasma concentrations by 11.9%, the mean residence time by 19.9%, the area under the plasmaconcentration- time curve by 53.6% and the area under the moment curve by 62.3%. In conclusion, the endotoxin-induced febrile state produced significant changes in the plasma levels as well as some of the pharmacokinetic variables of cefepime in rabbits.

Effect of ascorbic acid on stimulatory status of activated mouse peritoneal phagocytes.

Mouse peritoneal macrophages (MPM) when elicited by the antioxidant ascorbic acid have been found to be significantly stimulatory, exhibiting marked alteration at the cellular and enzyme levels. Alterations recorded were as follows--cellular yield per mouse, their protein content, lysosomal acid hydrolase levels and capability to phagocyte, all were significantly enhanced. The new stimulant was observed to produce no synergistic action on MPM when thioglycollate, BCG or endotoxin along with the same stimulated the latter. Levels of antioxidants like ascorbic acid and glutathione were found to be enhanced in elicited macrophages whereas superoxide dismutase levels varied when the three above stimulators were administered. However, the ascorbic acid elicited cells showed an increase in glutathione levels and a decrease in SOD levels but no change in total intracellular ascorbic acid levels. Further, though ascorbic acid interaction enhanced the phagocytic capability of MPM as compared to resident cells, no significant boosting of phagocytic process could be observed when each of three stimulators coupled with ascorbic acid was used for macrophage elicitation.

Edwardsiella tarda endotoxin as an immunopotentiator in Singhi, Heteropneustes fossilis fingerlings.

Endotoxin of E. tarda grown in brain heart infusion broth at 30 degrees C for 18 hr was extracted by differential centrifugation. Fingerlings of H. fossilis (weighing 1-2 g) were allowed for hyperosmotic infiltration in the endotoxin at the rate of 0,2,4,8,16 and 20 mg/l. Mortality varied from 20-50% at 2 to 20 mg/ml. Toxin treated fishes were challenged 21 days post treatment with the same E. tarda strain containing 2.1 x 10(9) CFU/ml. There was 80% mortality in the control group whereas only 20% mortality in toxin treated group at 2 mg/l concentration after challenge with homologous E. tarda. Subsequently a second challenge of E. tarda was given to the survivors of fish one month after first challenge using same concentration where no mortality could be observed. It was concluded that the endotoxin could enhance percentage of survival against E. tarda infection in Singhi.

Actividad de las metaloproteinasas en el miocardio de ratas sometidas a endotoxina y su inhibición con doxiciclina / Metalloproteinase activity in myocardium of rats exposed to endotoxin: its inhibition with doxycycline

Background: The ventricular dysfunction of endotoxic shock could be secondary to the activity of myocardial metalloproteinases that degrade collagenous matrix. Metalloproteinase activity can be inhibited with doxycycline in some tissues. Aim: To study if the effect of endotoxemia on myocardial metalloproteinase activity can be inhibited with doxycycline. Material and methods: Left ventricular metalloproteinase activity was studied in four groups of rats. Group 1 received intraperitoneal dextrose in water, group 2 received 8 mg/kg intraperitoneal E coli endotoxin, group 3 received 60 mg/kg/day doxycycline for three days and group 4 received doxycycline and E coli endotoxin. Enzymatic activity was measured by Western Blot and zymography. Results: Zymography showed a higher metalloproteinase 2 (49 percent) and 9 (100 percent) activity in rats treated with endotoxin, when compared with control rats. In group 4, doxycycline reduced the activity of metalloproteinases 2 and 9 by 71 percent and 63 percent respectively, as compared with group 3. Western blot showed a 50 percent increase in the expression of metalloproteinase 1 in rats treated with endotoxin, that was reduced by 64 percent with the use of doxycycline. Conclusions: Endotoxin administration increases myocardial metalloproteinases and doxycyclin inhibits this activation. Therefore, doxycyclin could reduce the degradation of myocardial fibrillar collagen and ventricular dysfunction of endotoxic shock

Los anticuerpos monoclonales en el tratamiento de la sepsis provocada por microorganismos gramnegativos / The monoclonal antibodies in the treatment of sepsis caused by Gram-negative microorganisms

A pesar de los avances en el diagnóstico y la intervención temprana con antibióticos, es alta la morbilildad y mortalidad asociada con la sepsis por bacterias gramnegativas. Los mediadores responsables de la patogénesis de la sepsis son componentes derivados de las propias bacterias (endotoxinas) y de las células de la respuesta inmune del hospedero (factor de necrosis tumoral y algunas interleuquinas). El tratamiento que tradicionalmente se ha utilizado en la sepsis, está dirigido sobre todo contra el microorganismo, mediante el uso de antibióticos cada vez más potentes. Sin embargo, está claro que los antibióticos no constituyen una solución definitiva, ya que aun si provocan la muerte bacteriana, no tienen efectos sobre la endotoxina y pueden aumentar su liberación cuando ocurre la lisis celular. A partir de la década de los 80 se han probado nuevos y exitosos tratamientos para la sepsis, que incluyen el uso de anticuerpos policlonales y monoclonales de origen murino y humano, dirigido contra el lípido A de la endotoxina, así como anticuerpos monoclonales contra el factor de necrosis tumoral. Si bien en todos los casos no se puede hablar de una total eficacia de estas moléculas para interrumpir la cadena de acontecimientos indeseables provocados por la endotoxina, sí es válido celebrar el advenimiento de inmunoterapias como tratamiento adjunto para una condición que amenaza la vida

Endotoxina, endotoxemia y sepsis / Endotoxins, endotoxemia and sepsis

La importancia creciente atribuida a la endotoxina y al factor de necrosis tumoral (TNF) como eje en la patogenia de la sepsis nos ha inducido a realizar un estudio clínico tendiente a demostrar la presencia de endotoxina en muestras de sangre obtenidas en el pre y posoperatorio en 30 enfermos hospitalizados en el Servicio de Cirugía del Hospital Clínico Regional de Concepción. Se demostró la presencia de endotoxina circulante en 17 de los 30 pacientes estudiados en el preoperatorio y esta cifra se elevó a 24 en las determinaciones realizadas en el posoperatorio. Si se compara los 30 enfermos en base al tipo de intervención quirúrgica se demuestra que el Test del Limulus fue positivo en 23 de los 25 enfermos sometidos a laparatomía por afecciones abdominales y sólo 1 de los 5 pacientes intervenidos por afecciones extraabdominales. Creemos importante destacar que los enfermos que presentaron sintomatología propia de la respuesta inflamatoria (fiebre, leucocitosis, hipermetabolismo, alteraciones hemodinámicas) evidenciaron con mayor frecuencia Test del Limulus positivo y que los dos únicos pacientes fallecidos presentaron, además de la sintomatología ya señalada, hipotensión arterial sistémica. Estos resultados revelan la interrelación entre endotoxemia, respuesta inflamatoria e hipotensión arterial sistémica, lo que debe considerarse como de mal pronóstico en los pacientes quirúrgicos graves

Effect of tolerance to bacterial lipopolysaccharide on gastric emptying rats

This study examined whether pretreating rats with endotoxin (lipopolysaccharide, LPS) modifies the retardation of the gastric emptying of liquids induced by this some toxin. Male SPF Wistar rats (8-10 weeks old, 230-300 g) were divided into groups of eight animals each that received either the vehicle solution sterile pyrogen free saline or LPS (50 mg/kg, intravenously), 96 and 5 hours before the measurement of gastric emptying. The four groups were defined on the basis of the sequence of intravenous infections namely saline + saline, saline + LPS, LPS + saline, and LPS + LPS. All the animals were fasted for 24 hours before and after the first injection and then received food ad libitum for the next 48 hours, before being fasted again for 24 hours prior to the evaluation of gastric emptying. Throughout these periods, the rats had free access to water that was only withdrawn 1 h before the study. A saline solution containing red phenol was used as the test meal. Gastric retention (GR) was measured between 2 and 4 p.m. 10 minutes after the orogastric administration of the meal. The results showed that in the saline + LPS group the GR (mean + SEM = 61.1 + 2.6 percent) was significantly greater (P<0.05, Tukey test) than in relation to the saline + saline group (40.5 + 2.6 percent). On the other hand, there were no significant differences between the animals which received LPS + LPS (41.8 + 2.5 percent) and those receiving LPS + saline (37.5 + 1.8 percent) or between these the groups and those receiving saline on both occasions. In conclusion the pretreatment with LPS suppressed the effect of endotoxin on gastric emptying by inducing early tolerance in the motor activity of the stomach in a manner similar to that described for other systems.