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Evaluation of Intestinal S100/calgranulin (S100A8/A9 and S100A12) Expression and Fecal Calprotectin (S100A8/A9) in Cats with Chronic EnteropathiesRiggers, Denise Sarina 05 June 2024 (has links)
No description available.
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Modeling diarrheagenic E. coli infections and co-infections: specific roles of diet and pathogenLedwaba, Solanka Ellen 03 1900 (has links)
PhD (Microbiology) / Department of Microbiology / Diarrhoea is still a major problem worldwide. Enteric pathogens such as Enteroaggregative E. coli (EAEC), Enteropathogenic E. coli (EPEC) and Enterotoxigenic E. coli (ETEC) have been reported to cause diarrhoea in children under the age of 5 years. The incidences of these pathogens are due to factors such as poor water quality, sanitation and hygiene practices. Infections with these pathogens result in diarrhoea and have been reported to result in severe disease outcomes more especially in children under 2 years of age.
EPEC infections have been well studied using in vitro analyses, with studies highlighting the adherence traits, proteins and virulence genes involved in pathogenesis and inflammatory responses. EPEC is characterized by localized adherence with microcolony formation at the site of infection. In vivo studies have reported on human EPEC infection. However, the current animal models have not been able to replicate clinical outcomes (such as diarrhoea and weigh loss) of EPEC infection similar to humans. Therefore, there is still a need for a suitable small animal model that mimic clinical outcomes of human EPEC infections in vivo.
Children living in poor environmental conditions are more susceptible to diarrhoeal pathogens. Furthermore, the incidences of children being exposed to co-infections (more than one pathogen at the same time) is relatively high. The EAEC/EPEC (A/P) and EPEC/ETEC (P/T) co-infections have been increasingly detected in children with and without diarrhoea. It has been suggested that patients infected with these co-infections might result in severe disease outcome than those infected with single pathogens. Pathogens are constantly evolving and the microbe-microbe interaction in the host can result in these pathogens competing for the same niche and thus result in increased virulence. Interaction of co-infections can lead to increased inflammatory responses thus affecting the infected host.
The first objective of this study was to develop an EPEC murine model using weaned
C57BL/6 mice that have been pretreated with antibiotic cocktail. Mice were orally infected with wild-type (WT) typical EPEC, bfp- and escN mutant strains. The WT had transient weight loss and wet stools with mucous; and the bfp- infected mice also had transient weight loss and bloody stool appearance. Increase in inflammatory biomarkers MPO, LCN-2, CRP, IL-6 and SAA were observed in the WT and bfp- infected mice. The mice infected with escN mutant did not exhibit any weight changes and the stools were similar to the uninfected mice. Furthermore, no inflammatory biomarkers were observed in mice infected with the escN mutant. Metabolic perturbations were observed in WT EPEC infected mice at day 3 post infection with the TCA cycle metabolites (reduced succinate, citrate, fumarate, cis-aconitate) being excreted at lower quantities indicating that the energy production in the infected mice was greatly affected.
The second objective of this study was to determine the interaction between the P/T coinfections using in vitro and in vivo analyses. In vitro, human colorectal tumour 8 (HCT-8) cells were infected with single strains of ETEC, EPEC and both the pathogens and incubated for 3 hours. After infection the cells were analysed for bacterial adherence using real-time PCR. The single strains adhered at the same rate similar to the P/T coinfected cells. IL-8, as a marker of inflammatory response, was measured using ELISA. The results indicated that the P/T co-infected cells had a significant increase in IL-8 response higher than the single infections. The P/T co-infections were further analysed in vivo using the EPEC murine model developed in this study. Interestingly, mice infected with P/T co-infections developed severe diarrhoea accompanied with significant increased weight loss and some mice died during the 3-day infection period. The inflammatory responses MPO, LCN-2 and SAA were higher in the co-infected mice indicating a synergistic effect. The bfp and eltA virulence genes were significantly increased in the P/T co-infections.
The third objective of this study was to determine the interaction between A/P coinfections using in vitro and in vivo analyses. HeLa cells and HCT-8 cells were infected with EAEC, EPEC and both the pathogens at the same time in order to determine adherence and inflammatory responses. EAEC adherence was higher than EPEC and A/P co-infections adherence. A/P co-infections did not have increased IL-8 response in
HCT-8 cells when compared to EAEC alone. The virulence genes involved in EPEC adherence and Type 3 Secretion System (bfp, eae, tir, ler, per, espB and espA) were significantly reduced in A/P co-infected cells. An interesting adherence trait was observed between the A/P co-infections in HeLA cells, EAEC was found to adhere around EPEC altering the localized adherence pattern. The A/P co-infections were further analysed using the EPEC murine model developed in this study. The A/P infected mice had diminished weight changes and EAEC shedding was enhanced when EPEC was present. Faecal inflammatory biomarkers MPO and LCN-2 in A/P infected mice did not have any additive effect.
The findings of this study contributed significantly to the knowledge of human EPEC infection in weaned C57BL/6 mice, highlighting clinical outcomes, inflammatory responses and metabolic perturbations. Furthermore, this study also highlighted the interaction of P/T and A/P co-infections using in vitro and in vivo analyses in order to determine the disease severity and outcomes. It was observed in this study that coinfections can result in either synergistic or antagonistic effects. Further studies are therefore, required in order to understand the underlying mechanisms that are involved during co-infections and this can further assist in the development of therapeutic interventions. / NRF
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Endothelial FasL in lymph nodes and in intestinal lymphatic tissueKokkonen, T. (Tuomo) 29 March 2016 (has links)
Abstract
The function of the transmembrane protein FasL is to complex with the Fas receptor in a target cell and induce target cell apoptosis. Fas/FasL-mediated apoptosis plays important role in immunoregulation. FasL expression is mostly seen in activated lymphocytes. We have characterized endothelial FasL expression in different functional compartments of lymph nodes and gut-associated lymphoid tissue. Furthermore, we have explored the functional role of endothelial FasL expression by analyzing correlation with apoptosis of lymphocyte subpopulations in lymph nodes and by assessing endothelial expression under different conditions by activation of immune functions in gastrointestinal mucosa.
Immunohistochemical stainings (Fas, FasL, CD3, CD20, CD19, CD23, CD56, FVIII) were performed on 20 reactive lymph node tissues (I and II), 60 pediatric endoscopy biopsy samples (III) or 60 samples from gut resections (IV). A double-staining method combining apoptosis detection with the TUNEL-method and lymphocyte classification with FasL, Fas and cell lineage markers was optimized. Patient groups included non-pathological lymph nodes, pediatric cow’s milk-sensitive enteropathy, pediatric celiac disease, appendicitis, ulcerative colitis and Crohn’s disease. Control groups included normal biopsy samples from pediatric patients and non-pathological resecate samples from the appendix, colon or ileum to correspond to patient groups. Quantitative analysis (positive vessels or cells per mm2) was performed thoroughly for each anatomical region. In a subset of patients, soluble FasL in the serum was quantified with standard enzyme-linked immunosorbent assay.
In reactive lymph nodes FasL expression was predominantly present in high endothelial venules located in the paracortical area, where apoptotic T and B lymphocytes, some expressing Fas, were subsequently found. In the gut wall vascular FasL expression was seen in high endothelial vessels near lymphoid follicles. Serum FasL was elevated in children with an abundance of mucosal lymphoid follicles. In IBD, vascular FasL was upregulated in ulcers and in the submucosa of colons affected by Crohn’s disease.
The results indicate that endothelial FasL is characteristically present in high endothelial venules of lymphoid tissues. Detection of apoptotic Fas expressing lymphocytes adjacent to such vessels supports the idea that endothelial FasL functions as a selective gatekeeper by inducing apoptosis of Fas+ lymphocytes entering from the blood stream. / Tiivistelmä
Solukalvon läpäisevän proteiinin, FasL:n, tehtävä on sitoutua kohdesolun Fas-reseptoriin ja indusoida kohdesolun apoptoosi. Fas/FasL-välitteinen apoptoosi on merkittävä tekijä immunologisessa säätelyssä. FasL ilmentyy pääsääntöisesti aktivoituneissa lymfosyyteissä. Olemme kuvanneet tutkimuksessamme FasL:n endoteelistä ilmentymistä imukudoksen eri toiminnallisissa alueissa ja suoliston lymfaattisessa kudoksessa. Lisäksi kartoitimme endoteelin FasL:n toiminnallista merkitystä analysoimalla sen yhteyttä lymfosyyttien alaryhmien apoptoosiin imusolmukkeissa ja arvioimalla FasL:n endoteelistä ilmentymistä suoliston limakalvon immunologisesti erilaisissa sairauksissa.
Teimme immunohistokemiallisia värjäyksiä (Fas, FasL, CD3, CD20, CD19, CD23, CD56 ja FVIII) 20 reaktiiviselle imusolmukkeelle (I ja II), 60 lapsen endoskooppiselle biopsianäytteelle (III) sekä 60 suoliresekaattinäytteelle (IV). Optimoimme kaksoisvärjäysmenetelmän, missä yhdistettiin apoptoosin havainnointimenetelmä TUNEL ja FasL-, Fas- tai solulinjamarkkeri. Potilasryhmiin kuului potilaita, joilla oli normaalit imusolmukkeet, sekä potilaita, jotka sairastivat lasten viivästynyttä lehmänmaitoallergiaa, lasten keliakiaa, umpilisäketulehdusta, haavaista paksusuolitulehdusta tai Crohnin tautia. Verrokkiryhmiin kuului normaaleja biopsianäytteitä lapsipotilailta sekä terveitä resekaattinäytteitä umpilisäkkeestä sekä paksu- tai sykkyräsuolesta potilasryhmien mukaisesti. Jokaiselle anatomiselle alueelle suoritimme perusteellisen määrällisen analyysin (positiivista suonta tai solua per mm2). Osalle ryhmistä suoritimme seerumin liukoisen FasL:n määrityksen entsyymivälitteisellä immunosorbenttimäärityksellä.
Reaktiivisissa imusolmukkeissa FasL:n ilmentyminen näkyi pääsääntöisesti parakortikaalialueen korkeaendoteelisissä venuleissa, missä myös apoptoottiset T- ja B-lymfosyytit (joista osa ilmensi Fasia) sittemmin näkyivät. Suoliston seinämässä havaitsimme verisuoniperäistä FasL:n ilmentymistä korkeaendoteelisissä suonissa lymfaattisten itukeskusten lähettyvillä. Niillä lapsipotilailla, joilla havaitsimme limakalvon lymfaattisten itukeskuksien lisääntymistä, oli myös seerumin FasL-pitoisuus koholla. Tulehduksellisissa suolistosairauksissa verisuoniperäinen FasL oli lisääntynyt limakalvon haavaumissa sekä Crohnin tautia sairastavien potilaiden submukoosassa.
Tulokset osoittavat verisuoniperäisen FasL:n tyypillisesti ilmentyvän imukudoksen korkeaendoteelisissa suonissa. Apoptoosin havaitseminen Fasia ilmentävissä lymfosyyteissä näiden suonien läheisyydessä tukee ajatusta siitä, kuinka verisuoniperäinen FasL toimii valikoivana portinvartijana ja aiheuttaa Fas-positiivisten lymfosyyttien apoptoosin estämällä niiden pääsyn verenkierrosta.
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