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Bacterial translocation : cause of activated intestinal macrophages in decompensated liver diseaseDu Plessis, Johannie 08 August 2012 (has links)
Background and Aim: Bacterial infections are a well described
complication of cirrhosis and occur in 37% of hospitalized patients. Culture
positive infections in addition to the presence of bacterial products and
DNA lead to loss of liver function and decompensation in cirrhosis. The
mechanisms and molecular pathways associated with Bacterial
Translocation (BT) are unknown. The aims of this study were to determine:
i. macrophage phenotype and molecular pathways associated with
bacterial translocation ii. if intestinal macrophages in liver cirrhosis are
capable of modulating intestinal permeability.iii. structural integrity of the
epithelial barrier.
Methods: Duodenal biopsies and serum samples were collected from 29
patients with decompensated cirrhosis, 15 patients with compensated and
19 controls. Duodenal macrophages were characterized by means of flow
cytometry and IHC. Gene expression analysis was performed to determine
molecular pathways involved in BT. Inflammatory cytokine determination
was done in serum and culture supernatant by means of customized
cytometric bead arrays.
Results: Patients with decompensated cirrhosis demonstrated: increased
frequency of CD33+/CD14+/TREM-1+ and iNOS+ macrophages in their
duodenum, elevated mRNA levels of nitric oxide synthase 2 (NOS2),
chemokine ligand 2 (CCL2), chemokine ligand 13 (CCL13) and interleukin
8 (IL8) and increased serum levels of interleukin 6 (IL6), IL8 and
lipopolysaccharides (LPS). Additionally, patients with decompensated
cirrhosis showed an increase in NO, IL6, IL8 and CCL2 levels in culture
supernatant after short term duodenal biopsy culture. Although the
epithelial barrier on EM seemed intact, significantly increased expression of
the “pore” forming tight junction claudin 2 was observed.
Conclusion: This study showed the presence of activated CD14+Trem-
1+iNOS+ intestinal macrophages and increased levels of NO, IL-6 and
claudin-2 levels in the duodenum of patients with decompensated liver
cirrhosis, suggesting that these factors enhance intestinal permeability to
bacterial products. / Afrikaans: Inleiding: Bakteriele infeksie is ‘n beskryfde komplikasie van lewersirrose
wat in 37% van gehospitaliseerde pasiente voorkom. Kultuur positiewe
infeksies asook die teenwoordigheid van bakteriele produkte en DNA lei tot
verlies van lewerfunksie en dekompensasie. Die molekulere meganismes
wat verband hou met bakteriele translokasie is nog onbekend. Die doel van
hierdie studie was om: i. Makrofaag fenotipe en molekulere meganismes
geassosieerd met bakteriele translokasie te beskryf, ii. te bepaal of
intestinale makrofage dermdeurlaatbaarheid beinvloed, asook iii. om die
struktruele integriteit van die dermwand te bepaal.
Methods: Serum en dunderm biopsies was verkry van 29 pasiente met
gedekompenseerde lewer sirrose, 15 pasiente met gekompenseerde
sirrose en 19 kontroles. Dunderm makrofage was gekarakteriseer met
behulp van vloeisitometrie en immunohistochemie. Molekulere meganisms
belangrik tydens bakteriele translokasie was bepaal met behulp van geneekspressie.
Serum en selkultuur supernatant sitokien bepalings was met
Bioplex assays gedoen.
Resultate: Pasiente met gedekompenseerde sirrose demonstreer: ‘n
verhoogde frekwensie van CD33+/CD14+/TREM-1+ en iNOS+ makrofage
in hul dunderm, verhoogde mRNA vlakke van NOS2, CCL2, CCL13 en IL8
asook verhoogde serum vlakke van IL6, IL8, LPS. Addisioneel het pasiente
met gedekompenseerde sirrose vehoogde supernatant vlakke van NO, IL6,
IL8 and CCL2 na kort termyn dunderm biopsie kulture. Alhoewel
elekronmikroskopie gewys het dat die dundermwand intak is, was daar
statisties-beduidend verhoogde ekspressie van die “porie” vormende vasteaansluitings-
proteien, claudin 2 sigbaar. Gevolgtrekking: Gesamentlik het die studie gewys dat geaktiveerde
CD14+/Trem-1+/iNOS+ intestinale makrofage asook verhoogde vlakke van
NO, IL-6 en claudin-2 teenwoordig is in die dunderm van pasiente met
gedekompenseerde sirrose. Dit dui daarop dat diè faktore derm
deurlaatbaarheid vir bakteriele produkte kan verhoog. / Dissertation (MSc)--University of Pretoria, 2011. / Immunology / MSc / Unrestricted
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Targeting the macrophage in equine post-operative ileusLisowski, Zofia Maria January 2018 (has links)
Post-operative ileus (POI) is the functional inhibition of propulsive intestinal motility which is a frequent occurrence following abdominal surgery in the horse and in humans. Rodent and human-derived data have shown that manipulation-induced activation of the resident muscularis externa (ME) macrophages in the intestine contributes to the pathophysiology of the disease. Most studies of the disease, specifically in the horse, have focussed on identification of risk factors, descriptive studies of the disease or the assessment of the efficacy of various therapeutic and prophylactic interventions. As a result, the proposed pathogenesis of equine POI is largely reliant on the translation of data from rodent models. The aims of this thesis were to identify macrophage populations in the normal equine gastrointestinal tract (GIT) and to study equine macrophage activation by stimulating equine bone marrow-derived macrophages (eqBMDMs) with lipopolysaccharide (LPS) as a model for intestinal macrophage activation. Firstly, the normal population of macrophages in the equine GIT was determined. Using CD163 as an immunohistochemical marker for macrophages. CD163+ve cells were present in all tissue layers of the equine intestine: mucosa, submucosa, ME and serosa. CD163+ve cells were regularly distributed within the ME, with accumulations adjacent to the myenteric plexus, and therefore to intestinal motility effector cells such as neurons and the Interstitial Cells of Cajal. The differentiation and survival of intestinal macrophages depends upon signals from the macrophage colony-stimulating factor (CSF-1) receptor. LPS translocation from the gut lumen is thought to be a key activator of ME macrophages. To provide a model for gut macrophages, a protocol was optimised to produce pure populations of equine bone marrow-derived macrophages (eqBMDMs) by cultivation of equine bone marrow in CSF-1. Macrophage functionality was assessed using microscopy, flow cytometry and phagocytosis assays. EqBMDMs responded to LPS stimulation with increases in expression of positive control genes, tumour necrosis factor alpha (TNF-α) and Indoleamine 2,3-dioxygenase (IDO1). The same mRNA was subjected to transcriptomic (RNA-Seq) analysis. Differential gene expression and network cluster analysis demonstrated an inflammatory response characterised by the production of pro-inflammatory cytokines such as interleukin 1 beta (IL-1β) and interleukin 6 (IL-6). However, in contrast to rodent macrophages, eqBMDMs failed to produce nitric oxide in response to LPS, showing species-specific variation in innate immune biology. Using these data, we compared gene expression in normal equine intestine and in intestine from horses undergoing abdominal surgery for colic (abdominal pain). Horses undergoing abdominal surgery showed evidence of increased expression of IL-1β, IL-6 and TNF-α in the mucosa and ME when compared to control tissue. Horses with post-operative reflux (POR), a clinical sign of POI, had increased gene expression of IL-1β, IL-6 and TNF-α compared to horses that did not develop POR following abdominal surgery. These preliminary data suggest that there is macrophage activation within the ME of the intestine during abdominal surgery in the horse, and that a greater activation state is present in horses that subsequently develop POR. The final part of this study was to investigate the effect of a long-acting form of CSF- 1, an Fc fusion protein (CSF1-Fc), as a potential treatment for POI using a mouse model. This work, performed in collaboration with another research group, found that mice lacking the C-C chemokine receptor type 2 (CCR2) gene, which is required for monocyte recruitment into tissues, had a longer recovery period following intestinal manipulation (IM) than wild type (WT) mice. With the administration of CSF1-Fc, infiltration of neutrophils to the ME was reduced and the number of macrophages in the ME was increased in both WT and CCR2-/- mice following IM. Administration of CSF1-Fc in CCR2-/- mice improved recovery of gastrointestinal transit three days following IM, to the same extent as WT mice. Network cluster analysis and RT-qPCR of the ME revealed clusters of genes induced and downregulated by CSF1-Fc, with increased expression of anti-inflammatory and pro-resolving genes after IM in WT and CCR2-/- mice following treatment with CSF1-Fc.
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