Studies on IL-18 in HIV infection : effects of the cytokine on intestinal integrity, and platelets as a new source of the cytokine

L'interleukine IL-18 (IL-18), un membre de la famille de l’IL-1, est une cytokine pro-inflammatoire multifonctionnelle. Elle est produite par les monocytes, les macrophages, les cellules dendritiques, les cellules épithéliales, les kératinocytes et le cortex surrénal dans le corps humain. Cette cytokine est d'abord produite comme une protéine précurseure inactive, qui est par la suite clivée en une forme mature par la caspase-1 activée. La caspase, en elle-même, existe comme précurseur inactif dans les cellules humaines et requiert l'assemblage d'inflammasomes pour son activation. L'IL-18 pour joue un rôle clé dans la médiation des conditions inflammatoires. Notre laboratoire et d'autres ont montré que l'infection par le VIH est accompagnée d'une augmentation des taux circulants d'IL-18 avec une diminution des niveaux de son antagoniste, l'interleukine-18 binding protein (IL-18BP). Dans cette thèse, nous démontrons pour que l'IL-18 est également produite et sécrétée par les plaquettes humaines lors de leur activation. Les plaquettes contiennent des composants de l'inflammasome. Ils assemblent et activent la caspase-1, qui ensuite traite le précurseur de l'IL-18 dans sa forme mature au cours du processus d'activation des plaquettes. La cytokine est synthétisée de novo lors de l'activation des plaquettes. Contrairement à l'IL-18, les plaquettes expriment constitutivement l’IL-18BP, et la libèrent de manière constitutive, ainsi que lors de l'activation. L'IL-18 et l'IL-18BP sont colocalisés avec CD63, un marqueur pour les granules α des plaquettes. L'IL-18 libéré des plaquettes constitue la source principale de cette cytokine dans la circulation humaine chez les individus sains. Nous avons identifié des concentrations faibles de cette cytokine dans les lysats de plaquettes chez les individus infectés par le VIH par rapport à ceux en santé. D'autre part, les concentrations ont été augmentées dans le sérum et le plasma pauvre en plaquettes chez les individus infectés. Des résultats similaires ont été obtenus avec l'IL-18BP dans les lysats de plaquettes d'individus sains et infectés par le VIH. Cependant, des quantités plus faibles de cet antagoniste ont été trouvées dans le sérum et le plasma pauvre en plaquettes d'individus infectés par le VIH par rapport à ceux en santé. Nos résultats ont des implications importantes pour les maladies inflammatoires chroniques dans laquelle une activité accrue de l'IL-18 joue un rôle pathogène. Le VIH est également accompagné par une inflammation intestinale et une diminution de l'intégrité intestinale, mesurée par la réparation de la muqueuse, la régénération et la perméabilité. Cependant, on en sait peu sur la relation entre le niveau élevé de l'IL-18 associé à l'infection au VIH et la perméabilité intestinale: ceci n'a jamais été étudié. Dans cette thèse, nous démontrons le rôle du virus et sa protéine Tat à augmenter la production d'IL-18 chez deux lignées de cellules épithéliales intestinales (HT29 et Caco2) ainsi qu'une diminution de l'IL-18BP. L'IL-18 induit une hyperperméabilité de la barrière épithéliale en perturbant à la fois les jonctions serrées et adhérentes, et ce, en modulant l'expression et la distribution de l'occludine, de claudine-2 et de la bêta-caténine. Une désorganisation de l'actine F a également été observée dans les cellules lors de l'incubation avec l'IL-18. Les mêmes observations ont été faites avec la protéine Tat du VIH-1. Après une incubation prolongée, l'IL-18 a causé la mort des cellules intestinales et induit l'apoptose par l'activation de la caspase-1 et la caspase-3. Fait intéressant, les taux plasmatiques de lipopolysaccharides chez trois catégories différentes de patients au VIH (ART-naïf, ART-traitée et contrôleurs élite) sont en corrélation avec les niveaux plasmatiques de l'IL-18. Enfin, nous avons étudié la voie de signalisation à travers laquelle l'IL-18 induit une perméabilité intestinale accrue. En bref, nos études identifient les plaquettes comme une source importante d'IL-18, et leur activation lors d'une infection à VIH contribue à des concentrations accrues de cette cytokine. Le virus entraine également l'augmentation de la production de cytokines par les cellules épithéliales intestinales. L'activité biologique accrue de ces cytokines contribue à la pathogenèse du sida en augmentant la perméabilité intestinale et en causant la mort des cellules intestinales. L'IL-18 pourrait servir de cible moléculaire pour retarder la progression du sida et réduire l'inflammation chronique dans un stade précoce d'une infection à VIH.

Effects of pre-slaughter fasting on broiler welfare, meat quality, and intestinal integrity

The Brazilian Ministry of Agriculture (MAPA) regulations establish 12 hours as the maximum pre-slaughter fasting period for broilers; however, many processing plants have considered this time is not sufficient, and consequently return the birds to the farms, with consequent economic losses and welfare problems. Therefore, it is necessary to investigate the possible effects of longer pre-slaughter fasting times. The objective of the present study was to evaluate the effect of pre-slaughter fasting times longer than those established by MAPA on broiler welfare, breast meat quality, and intestinal integrity. Forty 42-d-old broilers were submitted to different pre-slaughter fasting times: group I: 6 hours, group II 9h, group III 12h, and group IV 15h. Bird welfare was assessed before slaughter. After sacrifice, intestinal samples were collected to assess their morphology and morphometrics, and the Pectoralis major muscle was analyzed for pH and color. There was no influence (p>0.05) of treatments on breast muscle pH or color.There were no significant changes in intestinal morphometrics (p<0.05). Bird behavior was affected (p<0.05), suggesting that welfare was impaired as fasting time increased, but no differences in the analyzed parameters were detected between broilers fasted for 12 or 15 hours. It was concluded that the behavioral differences between birds fasted for 12 and 15 hours are not sufficient to assert that those fasted for 15 hours were in worse welfare conditions.

Intestinal integrity and performance of broiler chickens fed a probiotic, a prebiotic, or an organic acid

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Saccharomyces Cerevisiae cell wall dietary supplementation on the performance and intestinal mucosa development and integrity of broiler chickens vaccinated against coccidiosis

This study was carried out to verify if Saccharomyces cerevisiae cell wall (SCCW) dietary supplementation (0.2%) was capable of protecting the intestinal mucosa of broiler chickens vaccinated against coccidiosis. Body weight gain, feed intake, feed conversion and intestinal mucosa morphometric parameters and epithelial loss were evaluated. In the experiment,400 day-old male chicks were distributed according to a completely randomized design in a 2x2 factorial arrangement. The following treatments were applied: T1 - no vaccination/ no SCCW supplementation; T2 - no vaccination/SCCW supplementation; T3 - vaccination/no SCCW supplementation; and T4 - vaccination/SCCW supplementation to four replicates of 25 birds each. Birds were vaccinated on the first day of age using a spray vaccine (Coccivac B®, Coopers), containing E. acervulina, E. maxima, E. mivati and E. tenella. S. cerevisiae cell wall was supplied from the first day of age. Live performance, intestinal morphometric parameters and epithelial loss were evaluated at 14, 21 and 28 days of age. Performance was affected by vaccination only at 21-days of age, when body weight gain was reduced in the vaccinated birds, but no body weight difference was observed on day 28. Vaccine also increased the crypt depth (p<0.05) in the duodenum and jejunum, suggesting a high cell activity in the crypt:villus transition area to maintain the epithelial cell turnover. Villi number/area (103,269 µm²) was not affected (p>0.05) by vaccine or cell wall supplementation, and epithelial loss was more pronounced in the duodenum and jejunum. In conclusion, the findings of this study suggest that S. cerevisiae cell wall supplementation may be an useful management tool to maintain the intestinal integrity of broilers vaccinated against coccidiosis.

Efeito do gluconato de sódio e fontes de metionina sobre o desempenho, rendimento de carcaça, morfometria intestinal e densitometria óssea de frangos de corte de 22 a 42 dias de idade

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efeito do tempo de jejum pré-abate sobre o bem estar, qualidade de carne de peito e integridade intestinal em frangos de corte

Pós-graduação em Zootecnia - FMVZ

Integridade intestinal e desempenho de frangos de corte suplementados com probióticos, prebióticos e ácidos orgânicos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of microbiological quality of drinking water in the intestinal morphology of broilers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Respostas densitométricas, morfofisiológicas e desempenho de frangos de corte tratados com água filtrada e não filtrada

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Plasma sanguíneo desidratado na alimentação da Tilápia-do- Nilo

Pós-graduação em Zootecnia - FMVZ

Emergence of enteric viruses in production chickens is a concern for avian health

Several viruses have been identified in recent years in the intestinal contents of chickens and turkeys with enteric problems, which have been observed in commercial farms worldwide, including Brazil. Molecular detection of these viruses in Brazil can transform to a big threat for poultry production due to risk for intestinal integrity. This disease is characterized by severely delayed growth, low uniformity, lethargy, watery diarrhea, delayed feed consumption, and a decreased conversion rate. Chicken astrovirus (CAstV), rotavirus, reovirus, chicken parvovirus (ChPV), fowl adenovirus of subgroup I (FAdV-1), and avian nephritis virus (ANV) were investigated using the conventional polymerase chain reaction (PCR) and the reverse transcription polymerase chain reaction (RT-PCR). In addition, the infectious bronchitis virus (IBV), which may play a role in enteric disease, was included. The viruses most frequently detected, either alone or in concomitance with other viruses, were IBV, ANV, rotavirus, and CAstV followed by parvovirus, reovirus, and adenovirus. This study demonstrates the diversity of viruses in Brazilian chicken flocks presenting enteric problems characterized by diarrhea, growth retard, loss weight, and mortality, which reflects the multicausal etiology of this disease

Agglutinating Secretory IgA Preserves Intestinal Epithelial Cell Integrity during Apical Infection by Shigella flexneri.

Shigella flexneri, by invading intestinal epithelial cells (IECs) and inducing inflammatory responses of the colonic mucosa, causes bacillary dysentery. Although M cells overlying Peyer's patches are commonly considered the primary site of entry of S. flexneri, indirect evidence suggests that bacteria can also use IECs as a portal of entry to the lamina propria. Passive delivery of secretory IgA (SIgA), the major immunoglobulin secreted at mucosal surfaces, has been shown to protect rabbits from experimental shigellosis, but no information exists as to its molecular role in maintaining luminal epithelial integrity. We have established that the interaction of virulent S. flexneri with the apical pole of a model intestinal epithelium consisting of polarized Caco-2 cell monolayers resulted in the progressive disruption of the tight junction network and actin depolymerization, eventually resulting in cell death. The lipopolysaccharide (LPS)-specific agglutinating SIgAC5 monoclonal antibody (MAb), but not monomeric IgAC5 or IgGC20 MAbs of the same specificity, achieved protective functions through combined mechanisms, including limitation of the interaction between S. flexneri and epithelial cells, maintenance of the tight junction seal, preservation of the cell morphology, reduction of NF-κB nuclear translocation, and inhibition of proinflammatory mediator secretion. Our results add to the understanding of the function of SIgA-mediated immune exclusion by identifying a mode of action whereby the formation of immune complexes translates into maintenance of the integrity of epithelial cells lining the mucosa. This novel mechanism of protection mediated by SIgA is important to extend the arsenal of effective strategies to fight against S. flexneri mucosal invasion.

Role of phospholipase A(2) and tyrosine kinase in Clostridium difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Use of scanning electron microscopy for the evaluation of intestinal epithelium integrity

Propõe-se, neste trabalho, o uso de microscopia eletrônica de varredura para avaliar perda de epitélio intestinal da mucosa intestinal de pintos machos e fêmeas submetidos a prolongado jejum pós-eclosão de água e ração. Dois segmentos do duodeno, jejuno e íleo por ave foram coletados e processados pelo método de rotina para microscopia eletrônica de varredura. Seis diferentes graus de perda de epitélio intestinal foram determinados: grau 0, vilos normais, sem extrusão; grau 1, vilos com pequenos pontos de extrusão; grau 2, vilos com perda de epitélio no ápice; grau 3, vilos com perda de epitélio na região apical; grau 4, vilos com perda de epitélio em sua metade superior; grau 5: vilos sem epitélio; grau 6: vilo quebrado. As três regiões intestinais das fêmeas apresentaram aproximadamente 90% de seus vilos normais (graus 0 e 1), enquanto nos machos ocorreu 38% de vilos normais no duodeno e jejuno e 85% no íleo. Além disso, machos apresentaram graus mais acentuados de perda de epitélio (graus 3, 4, 5 e 6) que as fêmeas (grau 3). Os dados indicaram que a mucosa intestinal de pintos machos é mais sensível a prolongado jejum pós-eclosão que a de pintos fêmeas. A microscopia eletrônica de varredura pode ser usada como um método de rotina seguro para a caracterização e quantificação de perda de epitélio intestinal.

Effects of corn replacement by sorghum in broiler diets on performance and intestinal mucosa integrity

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)