Human peritoneal mesothelial cells respond to bacterial ligands through a specific subset of Toll-like receptors

Background. Bacterial infection remains a major cause of morbidity and mortality in peritoneal dialysis (PD) patients worldwide. Previous studies have identified a key role for mesothelial cells, lining the peritoneal cavity, in coordinating inflammation and host defense. Toll-like receptor (TLR) involvement in early activation events within the mesothelium, however, remains poorly defined. To investigate the initiation of bacterial peritonitis, we characterized TLR activation by bacterial ligands in human peritoneal mesothelial cells (HPMC)

The soluble form of Toll-like receptor 2 is elevated in serum of multiple sclerosis patients: a novel potential disease biomarker

Multiple sclerosis (MS) is an immune-mediated inflammatory demyelinating disease of the central nervous system. It was previously shown that Toll-like receptor (TLR)-2 signalling plays a key role in the murine experimental autoimmune encephalomyelitis (EAE) model of MS, and that TLR2-stimulation of regulatory T cells (Tregs) promotes their conversion to T helper 17 (Th17) cells. Here, we sought potential sources of TLR2 stimulation and evidence of TLR2 activity in MS patient clinical samples. Soluble TLR2 (sTLR2) was found to be significantly elevated in sera of MS patients (n=21), in both relapse and remission, compared to healthy controls (HC) (n=24). This was not associated with the acute phase reaction (APR) as measured by serum C-reactive protein (CRP) level, which was similarly increased in MS patients compared to controls. An independent validation cohort from a different ethnic background showed a similar upward trend in mean sTLR2 values in relapsing-remitting MS (RRMS) patients, and significant differences in sTLR2 values between patients and healthy controls were preserved when the data from the two cohorts were pooled together (n=41 RRMS and 44 HC, P=0.0006). TLR2-stimulants, measured using a human embryonic kidney (HEK-293) cell transfectant reporter assay, were significantly higher in urine of MS patients than healthy controls. A screen of several common urinary tract infections (UTI)-related organisms showed strong induction of TLR2-signalling in the same assay. Taken together, these results indicate that two different markers of TLR2-activity - urinary TLR2-stimulants, and serum sTLR2 levels - are significantly elevated in MS patients compared to healthy controls

Release of soluble vascular endothelial growth factor receptor-1 (sFlt-1) during coronary artery bypass surgery

<p>Abstract</p> <p>Background</p> <p>This study was conducted to follow plasma concentrations of sFlt-1 and sKDR, two soluble forms of the vascular endothelial growth factor (VEGF) receptor in patients undergoing coronary artery bypass graft (CABG) surgery with extracorporeal circulation (ECC).</p> <p>Methods</p> <p>Plasma samples were obtained before, during and after surgery in 15 patients scheduled to undergo CABG. Levels of sFlt-1 and KDR levels were investigated using specific ELISA.</p> <p>Results</p> <p>A 75-fold increase of sFlt-1 was found during cardiac surgery, sFlt-1 levels returning to pre-operative values at the 6^thpost-operative hour. In contrast sKDR levels did not change during surgery. The ECC-derived sFlt-1 was functional as judge by its inhibitory effect on the VEGF mitogenic response in human umbilical vein endothelial cells (HUVECs). Kinetic experiments revealed sFlt-1 release immediately after the beginning of ECC suggesting a proteolysis of its membrane form (mFlt-1) rather than an elevated transcription/translation process. Flow cytometry analysis highlighted no effect of ECC on the shedding of mFlt-1 on platelets and leukocytes suggesting vascular endothelial cell as a putative cell source for the ECC-derived sFlt-1.</p> <p>Conclusion</p> <p>sFlt-1 is released during CABG with ECC. It might be suggested that sFlt-1 production, by neutralizing VEGF and/or by inactivating membrane-bound Flt-1 and KDR receptors, might play a role in the occurrence of post-CABG complication.</p

Basic and Translational Understandings of Microbial Recognition by Toll-Like Receptors in the Intestine

Microbial recognition by multicellular organisms is initially accomplished by a group of pattern recognition receptors which are specialized to recognize microbe-associated molecular patterns (MAMPs) such as lipopolysaccharide, bacterial lipoprotein, CpG DNA motif, double strand RNA and flagellin. Toll-like receptors (TLRs) are the representative pattern recognition receptors, and microbial recognition by TLRs elicits innate and inflammatory responses. Ten TLR family members have been presently identified in human genome, and numerous studies discovered that intracellular responses from MAMPs-TLR engagements are mediated by a participation of at least 4 immediate adaptor molecules such as myeloid differentiation primary response gene-88 (MyD88), MyD88 adaptor-like (Mal) (also known as Toll/IL-1 receptor domain-containing adaptor protein [TIRAP]), Toll/IL-1 receptor domain-containing adaptor-inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) leading to activate transcription factors including nuclear factor κB, activator protein-1 and interferon-regulatory factors. Given that large amounts of commensal microbiota constantly reside in the intestinal lumen, enteric microbial recognition by TLRs at the intestinal epithelium provides a critical impact on regulating intestinal homeostasis. Indeed, aberrant TLR4 and TLR5 activations are etiologically associated with the development and progress of intestinal inflammatory diseases including inflammatory bowel disease and necrotizing enterocolitis. In this review article, we present the molecular mechanism by which TLRs elicit intracellular signal transduction, and summarize the physiological relevance of TLRs related to the gastrointestinal tract

In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

Background: Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin. Methodology/Principal Findings: We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin. Conclusions/Significance: PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico)Fundacao Butantan, BrazilFAPESP (Brazil

Toll-like receptors (TLRs) and mannan-binding lectin (MBL): On constant alert in a hostile environment

In the beginning were neither B cells nor T cells nor antibodies, but innate immune defense alone. The primary functional theme of innate immunity is the distinction between self and non-self, which is maintained by a vast number of cellular and subcellular components. In this context, the immense importance of the Toll-like receptors (TLRs) is well established. Positive (Darwinian) selection seems to be acting on the ligand-binding domains of these molecules, suggesting a selection pattern similar to that previously observed in the MHC proteins. In sharp contrast to TLRs, the biological significance of mannan-binding lectin (MBL) is controversial, and, concerning humans, it has been suggested that low concentration of MBL in serum represents a selective advantage. In this mini-review, based on a doctoral thesis, evolutionary aspects of TLRs and MBL are discussed

Plasminogen promotes influenza A virus replication through an annexin 2-dependent pathway in the absence of neuraminidase

Proteolytic cleavage of haemagglutinin (HA) is essential for the infectivity of influenza A viruses (IAVs). This is usually mediated by trypsin-like proteases present in the respiratory tract. However, the ability to use plasminogen (PLG) as an alternative protease may contribute to pathogenesis of IAV infections and virus replication outside the respiratory tract. It was demonstrated previously that neuraminidase (NA) of the IAV strain A/WSN/33 can sequester PLG, allowing this virus to replicate in a PLG-dependent fashion. However, PLG also promotes replication of other IAVs, although its mode of action is poorly understood. Here, using NA-deficient viruses, we demonstrate that NA is not required for the binding of PLG and subsequent cleavage of HA. However, we demonstrate that the cellular protein annexin 2 (A2) can bind PLG and contributes to PLG-dependent cleavage of HA and subsequent IAV replication. Collectively, these results indicate that PLG promotes IAV replication in an A2-dependent fashion in the absence of NA

Annexin II Incorporated into Influenza Virus Particles Supports Virus Replication by Converting Plasminogen into Plasmin ▿

For influenza viruses to become infectious, the proteolytic cleavage of hemagglutinin (HA) is essential. This usually is mediated by trypsin-like proteases in the respiratory tract. The binding of plasminogen to influenza virus A/WSN/33 leads to the cleavage of HA, a feature determining its pathogenicity and neurotropism in mice. Here, we demonstrate that plasminogen also promotes the replication of other influenza virus strains. The inhibition of the conversion of plasminogen into plasmin blocked influenza virus replication. Evidence is provided that the activation of plasminogen is mediated by the host cellular protein annexin II, which is incorporated into the virus particles. Indeed, the inhibition of plasminogen binding to annexin II by using a competitive inhibitor inhibits plasminogen activation into plasmin. Collectively, these results indicate that the annexin II-mediated activation of plasminogen supports the replication of influenza viruses, which may contribute to their pathogenicity