Effects of the PARP Inhibitor Olaparib on the Response of Human Peripheral Blood Leukocytes to Bacterial Challenge or Oxidative Stress.

Prior studies demonstrate the activation of poly-(ADP-ribose) polymerase 1 (PARP1) in various pathophysiological conditions, including sepsis. We have assessed the effect of olaparib, a clinically used PARP1 inhibitor, on the responses of human peripheral blood leukocytes (PBMCs) obtained from healthy volunteers in response to challenging with live bacteria, bacterial lipopolysaccharide (LPS), or oxidative stress (hydrogen peroxide, H2O2). The viability of PBMCs exposed to olaparib or to the earlier generation PARP inhibitor PJ-34 (0.1-1000 µM) was monitored using Annexin V and 7-aminoactinomycin D. To evaluate the effects of olaparib on the expression of PARP1 and its effects on protein PARylation, PBMCs were stimulated with Staphylococcus aureus with or without olaparib (1-10 µM). Changes in cellular levels of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP), as well as changes in mitochondrial membrane potential (MMP), were measured in PBMCs exposed to H2O2. Bacterial killing was evaluated in PBMCs and polymorphonuclear leukocytes (PMNs) incubated with S. aureus. Cytokine production was measured in supernatants using a cytometric bead array. Reactive oxygen species (ROS), nitric oxide (NO) production, and phagocytic activity of monocytes and neutrophils were measured in whole blood. For ROS and NO production, samples were incubated with heat-killed S. aureus; phagocytic activity was assessed using killed Escherichia coli conjugated to FITC. Olaparib (0.1-100 µM) did not adversely affect lymphocyte viability. Olaparib also did not interfere with PARP1 expression but inhibits S. aureus-induced protein PARylation. In cells challenged with H2O2, olaparib prevented NAD+ and ATP depletion and attenuated mitochondrial membrane depolarization. LPS-induced production of TNF-α, MIP-1α, and IL-10 by PBMCs was also reduced by olaparib. Monocytes and neutrophils displayed significant increases in the production of ROS and NO after stimulation with S. aureus and phagocytic (E. coli) and microbicidal activity, and these responses were not suppressed by olaparib. We conclude that, at clinically relevant concentrations, olaparib exerts cytoprotective effects and modulates inflammatory cytokine production without exerting adverse effects on the cells' ability to phagocytose or eradicate pathogens. The current data support the concept of repurposing olaparib as a potential experimental therapy for septic shock.

Gut Microbiota Imbalance Can Be Associated with Non-malabsorptive Small Bowel Shortening Regardless of Blind Loop.

INTRODUCTION: Some traditional bariatric surgery procedures may lead to functional gut shortening, which may unsettle the fine-tuned gastrointestinal physiology and affect gut microbiota balance. PURPOSE: Evaluate the gut microbiota behavior in rat models facing gut shortening due to intestinal bypass. MATERIALS AND METHODS: Wistar rats (n = 17) were randomly distributed in three groups: (1) sham group (n = 5); (2) blind loop group (n = 6); and (3) resection group (n = 6). Intestinal samples and feces were analyzed to measure bacterial concentrations (small intestinal bacterial overgrowth-SIBO) 12 weeks after the experimental procedures. Bacterial translocation (BT) was investigated in the mesenteric lymph node (MLN), liver, spleen, and lung of the animals. In addition, inflammatory aspects were investigated in their liver and small bowel through histological analysis. RESULTS: Regardless of blind loop, gut shortening groups recorded similar high level of bacterial concentrations in intestine compartments, greater than that of the sham group (p ≤ 0.05). BT was only observed in the MLN of gut shortening models, with higher percentage in the blind loop group (p ≤ 0.05). The gut and liver histopathological analysis showed similar low-grade chronic inflammation in both gut shortening groups, likely associated with SIBO/BT events. CONCLUSION: Sustained SIBO/BT was associated with proximal gut shortening in half regardless of blind loop, whereas the GI tract's ability to restore gut microbiota balance after a surgical challenge on the small bowel appears to be linked to the functional remaining gut.

Expression of aggregative adherence to hela cells by Escherichia coli strains isolated from sick horses

The virulence attributes of 56 Escherichia coli strains isolated from sick horses (secretions of uterine cervices; gastrointestinal and lung fragments of necropsy; diarrheic feces, and tracheal washings) was examined by determining their adherence pattern to HeLa cells and searching for the presence of virulence genes of the various E. coli pathotypes. Two non-adherent strains presented astA, which encodes the enteroaggregative E. coli heat-stable toxin. Twenty-seven strains (48.2 percent) adhered to HeLa cells, 21 (77.8 percent) of which presented the aggregative adherence pattern (AA) that characterize the Enteroaggregative E. coli pathotype (EAEC). Nine of the strains presenting AA were isolated from secretions of uterine cervix, including one carrying virulence genes of the EAEC pathotype (aggR,aap,irp2, and pic). This is the first description of the AA phenotype amongst E. coli strains from sick horses. Such strains should be further evaluated regarding their potential role in the pathogenesis of diverse equine diseases and as reservoirs of human infections.

Características de virulência de 56 amostras de Escherichia coli isoladas de eqüinos doentes (secreção de colo uterino, fragmentos de necrópsia do trato gastrointestinal e de pulmões, fezes diarréicas e lavado traqueal) foram examinadas para determinar o padrão de aderência em células HeLa e pesquisar a presença de genes de virulência de vários patotipos de E. coli. Duas amostras não aderentes apresentaram astA, gene que codifica a toxina termo-estável de E. coli enteroagregativa. Das vinte e sete amostras (48,2 por cento) que aderiram a células HeLa, 21 (77,8 por cento) apresentaram o padrão de aderência agregativa (AA) que caracteriza o patotipo de E. coli Enteroagregativa (EAEC). Nove destas amostras que apresentaram AA foram isoladas de secreção de colo uterino, incluindo uma que apresentava genes de virulência de patotipos de EAEC (aggR,aap,irp2 e pic). Esta é a primeira descrição do fenótipo AA em amostras de cavalos doentes. Estas amostras deverão ser melhor avaliadas em relação a sua potencial função na patogênese de diferentes doenças eqüinas, bem como à possibilidade destes animais representarem um reservatório de infecções humanas causadas por esta bactéria.