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Immunomodulation in the canine endometrium by uteropathogenic Escherichia coli.

ABSTRACT: This study was designed to evaluate the role of E. coli ?-hemolysin (HlyA) in the pathogenesis of canine pyometra, and on the immune response of canine endometrial epithelial and stromal cells. In Experiment 1, the clinical, hematological, biochemical and uterine histological characteristics of ?-hemolytic and non-hemolytic E. coli pyometra bitches were compared. More (p < 0.05) metritis cases were observed in ?-hemolytic E. coli pyometra uteri than in non-hemolytic E. coli pyometra uteri. ?-hemolytic E. coli pyometra endometria had higher gene transcription of IL-1? and IL-8 and lower gene transcription of IL-6 than non-hemolytic E. coli pyometra endometria (p < 0.01). In Experiment 2, the immune response of endometrial epithelial and stromal cells, to hemolytic (Pyo18) and non-hemolytic E. coli strains (Pyo18 with deleted hlya-Pyo18?hlyA- and Pyo14) were compared. Following 4 h of incubation, Pyo18 decreased epithelial cell numbers to 54% (p < 0.001), and induced death of all stromal cells (p < 0.0001), whereas Pyo18?hlyA and Pyo14 had no effect on cell numbers. Compared to Pyo18?hlyA and Pyo14, respectively, Pyo18 induced a lower transcription level of IL-1? (0.99 vs 152.0 vs 50.9 fold increase, p < 0.001), TNF? (3.2 vs 49.9 vs 12.9 fold increase, p < 0.05) and IL-10 (0.4 vs 3.6 vs 2.6 fold increase, p < 0.001) in stromal cells, after 1 h of incubation. This may be seen as an attempt of hemolytic E. coli to delay the activation of the immune response. In conclusion, endometrial epithelial and stromal cell damage induced by HlyA is a potential relevant step of E. coli virulence in the pathogenesis of pyometra.

SUBMITTER: Henriques S

PROVIDER: S-EPMC5103487 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Immunomodulation in the canine endometrium by uteropathogenic Escherichia coli.

Henriques Sofia S Silva Elisabete E Silva Marta F MF Carvalho Sandra S Diniz Patrícia P Lopes-da-Costa Luís L Mateus Luisa L

Veterinary research 20161109 1

This study was designed to evaluate the role of E. coli α-hemolysin (HlyA) in the pathogenesis of canine pyometra, and on the immune response of canine endometrial epithelial and stromal cells. In Experiment 1, the clinical, hematological, biochemical and uterine histological characteristics of β-hemolytic and non-hemolytic E. coli pyometra bitches were compared. More (p < 0.05) metritis cases were observed in β-hemolytic E. coli pyometra uteri than in non-hemolytic E. coli pyometra uteri. β-hem ...[more]

PMID: 27829462

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Project description:PurposeAmniotic membrane stem cells have a high capacity of proliferation, cell expansion, and plasticity, as well as immunomodulatory properties that contribute to maternal-fetal tolerance. Owing to the lack of research on human amniotic membrane at different gestational stages, the canine model is considered ideal because of its genetic and physiological similarities. We aimed to characterize the canine amniotic membrane (CAM) cell lineage in different gestational stages and evaluate the expression of immunomodulatory genes.Materials and methodsTwenty CAMs from early (20-30 days) (n=7), mid- (31-45 days) (n=7), and late gestation (46-63 days) (n=6) stages were studied. The cell features were assessed by cell viability tests, growth curve, colony-forming units, in vitro differentiation, cell labeling for different immunophenotypes, and pluripotent potential markers. The cells were subjected to RT-PCR and qPCR analysis to determine the expression of IDO, HGF, EGF, PGE2, and IL-10 genes.ResultsCAM cells exhibited a fibroblastoid morphology and adherence to plastic with an average cell viability of 78.5%. The growth curve indicated a growth peak in the second passage and we obtained an average of 138.2 colonies. Osteogenic, chondrogenic, and adipogenic lineages were confirmed by in vitro differentiation assays. Cellular immunophenotyping experiments confirmed the presence of positive mesenchymal markers (CD90 and CD105) and the low or negative expression of hematopoietic markers (CD45 and CD34). Qualitative analysis of the immunomodulatory functions indicated the expression of the IDO, HGF, EGF5, and PGE2 genes. When stimulated by interferon-gamma, CAM cells exhibited higher IDO levels throughout gestation.ConclusionThe CAMs from different gestational stages presented features consistent with mesenchymal stem cell lineage; better results were observed during the late gestation stage. Therefore, the gestational stage is a key factor that may influence the functionality of therapies when using fetal membrane tissues from different periods of pregnancy.

Project description:BackgroundAccumulation of lipid droplets (LDs) was recently observed in pyometra-affected uteri. As data about their nature and function are missing we intended to compare the localization, quality and quantity of LDs in canine healthy and pyometra-affected tissues and in an in vitro model.Methods and resultsWe characterized LDs in healthy and pyometra uterine tissue samples as well as in canine endometrial epithelial cells (CEECs) in vitro by means of histochemistry, immunohistochemistry, transmission electron microscopy, western blot, and RT-qPCR. Oil Red O (ORO) staining and quantification as well as p-phenylenediamine staining showed a higher number of LDs in epithelial cells of pyometra samples. Immunohistochemistry revealed that the amount of LDs coated by perilipin2 (PLIN2) protein was also higher in pyometra samples. Transmission electron microscopy showed an increase of LD size in surface and glandular epithelial cells of pyometra samples. In cell culture experiments with CEECs, supplementation with oleic acid alone or in combination with cholesterol lead to an increased LD accumulation. The expression of PLIN2 at protein and mRNA level was also higher upon oleic acid supplementation. Most LDs were double positive for ORO and PLIN2. However, ORO positive LDs lacking PLIN2 coating or LDs positive for PLIN2 but containing a lipid class not detectable by ORO staining were identified.ConclusionsWe found differences in the healthy and pyometra-affected endometrium with respect to LDs size. Moreover, several kinds of LDs seem to be present in the canine endometrium. In vitro studies with CEECs could show their responsiveness to external lipids. Since epithelial cells reacted only to oleic acid stimulation, we assume that the cyclic lipid accumulation in the canine endometrium is based mainly on triglycerides and might serve as energy provision for the developing early embryo. Further studies are necessary to verify the complex role of lipids in the healthy and pyometra-affected canine endometrium.

Project description:Biofilm formation enhances bacteria's ability to colonize unique niches while protecting themselves from environmental stressors. Escherichia coli that colonize the urinary tract can protect themselves from the harsh bladder environment by forming biofilms. These biofilms promote persistence that can lead to chronic and recurrent urinary tract infections (UTI). While biofilm formation is frequently studied among urinary E. coli, its association with other pathogenic mechanisms and adaptations in certain host populations remains poorly understood. Here we utilized whole genome sequencing and retrospective medical record analysis to investigate associations between the population structure, phenotypic resistance, resistome, virulome, and patient demographic and clinical findings of 104 unique urinary E. coli and their capacity to form biofilms. We show that population structure including multilocus sequence typing and Clermont phylogrouping had no association with biofilm capacity. Among clinical factors, exposure to multiple antibiotics within that past 30 days and a clinical history of recurrent UTIs were positively associated with biofilm formation. In contrast, phenotypic antimicrobial reduced susceptibility and corresponding acquired resistance genes were negatively associated with biofilm formation. While biofilm formation was associated with increased virulence genes within the cumulative virulome, individual virulence genes did not influence biofilm capacity. We identified unique virulotypes among different strata of biofilm formation and associated the presence of the tosA/R-ibeA gene combination with moderate to strong biofilm formation. Our findings suggest that E. coli causing UTI in dogs utilize a heterogenous mixture of virulence genes to reach a biofilm phenotype, some of which may promote robust biofilm capacity. Antimicrobial use may select for two populations, non-biofilm formers that maintain an arsenal of antimicrobial resistance genes to nullify treatment and a second that forms durable biofilms to avoid therapeutic insults.

Dataset Information

Immunomodulation in the canine endometrium by uteropathogenic Escherichia coli.

Publications

Immunomodulation in the canine endometrium by uteropathogenic Escherichia coli.

Similar Datasets

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