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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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CELLULAR AND MOLECULAR MECHANISM OF LISTERIA ADHESION PROTEIN-MEDIATED BACTERIAL CROSSING OF THE INTESTINAL BARRIERRishi Drolia (5929649) 14 January 2021 (has links)
<p>The
crossing of host barriers (intestinal, blood-brain, and placental) is a critical
step for systemic infections caused by entero-invasive pathogens. In the
intestine, the epithelial cells are the first line of defense against
enteric pathogens. <i>Listeria monocytogenes</i> is a
facultative-intracellular foodborne pathogen that first crosses the intestinal
barrier to cause a systemic infection. However, the underlying
mechanism is not well understood.</p><p><br></p>
<p>We
demonstrate that <i>Listeria</i> adhesion protein (LAP) promotes
the translocation of <i>L. monocytogenes </i>across the intestinal
barrier in mouse models (A/J and C57BL/6). Relative to the wild-type
(WT; serotype 4b) or the isogenic bacterial invasion protein
Internalin A mutant (Δ<i>inlA</i>) strain, the <i>lap<sup>─</sup></i>
strain showed significant defect in translocation across the intestinal
barrier and colonization of the mesenteric-lymph nodes, liver and
spleen in the early phase of infection (24 h and 48
h). LAP induces intestinal epithelial barrier dysfunction for
increased translocation as evidenced by increased permeability
to 4-kDa FITC-dextran (FD4), a marker of paracellular
permeability, in the serum and urine of WT and Δ<i>inlA</i>- infected
mice and across Caco-2 cell barrier, but not the <i>lap<sup>─</sup></i> mutant
strain. Microscopic examination confirmed localization of the WT
and Δ<i>inlA</i> strains in the tight junction, a crucial
barrier of intestinal paracellular permeability, in the mouse ileal tissue
but the <i>lap<sup>─</sup></i> strain remained confined in the
lumen. LAP also upregulates TNF-α and IL-6 in intestinal epithelia
of mice and in Caco-2 cells for increased permeability. </p><p><br></p>
<p>Investigation
of the underlying molecular mechanisms of LAP-mediated increase in intestinal
permeability by using <i>lap<sup>─</sup></i> mutant strain, purified
LAP and shRNA-mediated Hsp60 suppression, we demonstrate that LAP
interacts with its host receptor, Hsp60, and activates the canonical NF-κB
signaling, which in turn facilitates myosin light-chain
kinase (MLCK)-mediated opening of the epithelial barrier via the cellular
redistribution of major epithelial junctional proteins claudin-1, occludin, and
E-cadherin. Pharmacological inhibition of NF-κB or MLCK in cells or
genetic ablation of MLCK in mice (C57BL/6) prevents mislocalization of
epithelial junctional proteins, intestinal permeability and <i>L.
monocytogenes</i> translocation across the intestinal barrier.</p>
<p><br></p><p>Furthermore,
LAP also promotes <i>L. monocytogenes </i>translocation
across the intestinal barrier and systemic dissemination in a
Mongolian gerbil that are permissive to the bacterial invasion proteins;
InlA-and InlB-mediated pathways; similar to that in humans. We show
a direct LAP-dependent and InlA-independent pathway<i> </i>for <i>L.
monocytogenes</i> paracellular translocation across the intestinal
epithelial cells that do not express luminally accessible
E-cadherin. Additionally, we show a functional InlA/E-cadherin interaction
pathway that aids <i>L. monocytogenes</i> translocation by targeting
cells with luminally accessible E-cadherin such as cells at the site of
epithelial cell extrusion, epithelial folds and mucus-expelling goblet
cells. Thus, <i>L. monocytogenes</i> uses LAP to exploit
epithelial innate defense in the early phase of infection to cross the
intestinal epithelial barrier, independent of other invasion proteins.</p><p><br></p>
<p>This
work fills a critical gap in our understanding of <i>L.
monocytogenes </i>pathogenesis and sheds light to the complex interplay
between host-pathogen interactions for bacterial crossing of the crucial
intestinal barrier.</p>
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