Lipopolysaccharide (LPS) the major structural component of the outer membrane of Gram-negative bacteria contributes to the cardiovascular collapse and death observed in septic patients, as well as in ...the immunocompromised host. LPS activates multiple cells to release proinflammatory cytokines, nitric oxide (NO) and other reactive molecules able to depress cardiac functions. It has been appreciated that the pattern recognition receptor, TLR4, is a signalling receptor for LPS, but its role in the embryonal cardiomyocytes is poorly understood. Here, we provide evidence for TLR4-dependent functional responses by LPS treated embryonal cardiomyocytes. It will be reported that LPS is able to induce TNF-alpha and NO release from cultured cardiomyocytes, while molecular and morphological evidence demonstrates the expression of TLR4 on surface membrane of embryonal cardiomyocytes. LPS-induced signalling was studied evaluating the expression of the extracellular signal-regulated kinase (ERK) and signal transducer and activators of transcription (STAT) protein families in response to LPS. The role of TLR4 was investigated with blocking assays using monoclonal antibody against this endotoxin receptor. Our results indicated that LPS-induced activation of signal transduction in embryonal cardiomyocytes occurs by a TLR4-dependent mechanism. In summary, chick embryonal cardiomyocytes may constitute a valid experimental model in order to study the LPS induced inflammatory responses by cardiomyocytes, useful not only to identify the signalling pathways evoked by endotoxin receptor, including TLR4, but also to suggest therapeutic targets for the control of myocardial dysfunction induced by infectious agents. In this respect, in elderly a continuous leakage of LPS from gut flora and/or external environment should be regarded as a possible cause of cardiac failure and, therefore, adequately prevented or treated.
Sjögren's syndrome (SS) is an autoimmune rheumatic disease that targets salivary and lachrymal glands, characterized by a high concentration of serum autoantibodies directed against nuclear and ...cytoplasmic antigens. It is known that autoantibodies can enter viable cells and this phenomenon has functional consequences including activation of apoptotic process. The objective of this work was to explore whether autoantibodies contained in IgG purified from Sjögren sera trigger apoptotic process in an experimental model represented by the human salivary gland cell line A-253. To define if the intrinsic or extrinsic pathways are activated, we examined which caspases are critical for inducing cell death. The results have demonstrated that morphological changes and DNA laddering, consistent with apoptotic cell death, occurred in A-253 cells treated with IgG from Sjögren sera. Sjögren IgG induced cleavage and activation of the effector caspase-3 and degradation of the caspase-3 substrate poly(ADP-ribose)polymerase. Both the intrinsic and extrinsic apoptotic pathways were activated, since both caspase-8 and caspase-9 cleavages occurred. In conclusion, autoantibodies contained in IgG purified from Sjögren sera mediate apoptosis of the A-253 cell line in a caspase-dependent manner.
The aim of this study was to evaluate cytokine expression in 22 Leishmania infantum naturally infected dogs, in order to correlate this parameter with the clinical status of infected animals. After 4 ...and 8 months from the first diagnosis of Leishmania infection, clinical and laboratory examination of dogs was performed and peripheral blood mononuclear cells (PBMC) were isolated. The cytokine profile was analysed in terms of IFN-gamma, IL-4, IL-10 and TNF-alpha mRNA expression in cultured PBMC by a semi-quantitative reverse transcriptase-PCR. Thirteen out of 22 Leishmania-infected dogs remained asymptomatic in the follow-up, while 9 showed clinical signs of leishmaniasis. IL-4, IL-10, TNF-alpha and IFN-gamma mRNA levels were not significantly different in asymptomatic compared to symptomatic animals 4 months from the diagnosis of Leishmania infection, but were significantly higher in symptomatic versus asymptomatic dogs after 8 months from diagnosis. In addition, IL-4, IL-10 and TNF-alpha mRNA levels significantly increased only in symptomatic dogs at 8 months, in comparison to the levels found at 4 months. These results show a mixed Th1 and Th2 cytokine response in Leishmania-infected dogs, with higher cytokine expression in dogs with manifest clinical disease, during the second follow-up after 8 months from the first diagnosis of infection.
Ligation of N-formyl-methionyl-leucyl-phenylalanine (fMLP) to its specific cell surface receptors triggers different cascades of biochemical events, eventually leading to cellular activation. The ...formyl peptide receptors (FPRs) are members of the seven-transmembrane, G-protein coupled receptors superfamily, expressed at high levels on polymorphonuclear and mononuclear phagocytes. The main responses elicited upon ligation of formylated peptides, referred to as cellular activation, are those of morphological polarization, locomotion, production of reactive-oxygen species and release of proteolytic enzymes. FPRs have in recent years been shown to be expressed also in several non myelocytic populations, suggesting other unidentified functions for this receptor family, independent of the inflammatory response. Finally, a number of ligands acting as exogenous or host-derived agonists for FPRs, as well as ligands acting as FPRs antagonists, have been described, indicating that these receptors may be differentially modulated by distinct molecules.
Objectives: The fibulins are a family of extracellular matrix (ECM) molecules that regulate the organ shape along with other growth factors and stromal cells and have recently been shown to be ...involved in a variety of cellular functions including proliferation, migration, differentiation, and survival. Important changes in acinar and ductal morphology and function, together with pronounced ECM remodelling, are detectable in the labial salivary glands (LSGs) of patients with Sjögren's syndrome (SS). Here we report the in vitro expression of the recently identified ECM proteins fibulin-6 and fibulin-7 by human salivary gland epithelial cells (SGECs). The ability of anti-Ro SSA autoantibodies (Abs) to modulate fibulin-6 and fibulin-7 expression was investigated.
Methods: Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR were used to analyse fibulin-6 and fibulin-7 mRNA expression. Confocal microscopy and fluorescence-activated cell sorting (FACS) were used to study expression of the proteins in primary human SGEC cultures, established from biopsies of minor LSGs, in both untreated control cells and anti-Ro SSA Abs-treated cells.
Results: The methods used show the expression of fibulin-6 and fibulin-7 in SGECs. Treatment of cells with anti-Ro SSA Abs results in a down-regulation of fibulin-6 mRNA expression whereas no significant differences were observed in fibulin-7 expression between untreated and treated cells.
Conclusion: Dysregulation of fibulin expression in SGECs by anti-Ro SSA Abs may contribute to disorganization of the ECM environment and thus cause injury to the salivary gland architecture and functionality observed in SS.
Odontoblast terminal differentiation occurs according to a tooth-specific pattern and implies both temporo-spatially regulated epigenetic signaling and the expression of specific competence. ...Differentiation of odontoblasts (withdrawal from the cell cycle, cytological polarization, and secretion of predentin/dentin) is controlled by the inner dental epithelium, and the basement membrane (BM) plays a major role both as a substrate and as a reservoir of paracrine molecules. Cytological differentiation implies changes in the organization of the cytoskeleton and is controlled by cytoskeleton-plasma membrane-extracellular matrix interactions. Fibronectin is re-distributed during odontoblast polarization and interacts with cell-surface molecules. A nonintegrin 165-kDa fibronectin-binding protein, transiently expressed by odontoblasts, is involved in microfilament reorganization. Growth factors (TGFβ1,2,3/BMP2,4, and 6), expressed in tooth germs, signal differentiation. Systemically derived molecules (IGF1) may also intervene. IGF1 stimulates cytological but not functional differentiation of odontoblasts: The two events can thus be separated. Immobilized TGFβ1 (combined with heparin) induced odontoblast differentiation. Only immobilized TGFβ1 and 3 or a combination of FGF1 and TGFβ1 stimulated the differentiation of functional odontoblasts over extended areas and allowed for maintenance of gradients of differentiation. Presentation of active molecules in vitro appeared to be of major importance; the BM should fulfill this role in vivo by immobilizing and spatially presenting TGF(3s. Attempts are being made to investigate the mechanisms which spatially control the initiation of odontoblast differentiation and those which regulate its propagation. Analysis of molar development suggested that odontoblast differentiation and crown morphogenesis are interdependent, although the possibility of co-regulation requires further investigation.
In this paper we analyze a 55-amino acid (aa) sequence which is relatively well conserved in several seven-transmembrane receptor families (from Insects to Mammals) and in some Viruses. This ...sequence, which covers the second transmembrane domain, the first extracellular loop and the third transmembrane domain, appears in its complete configuration in most of the seven-transmembrane receptor families, as well as in the protein products of some viruses. Other seven-transmembrane receptors and viruses exhibit reduced configurations of the conserved sequence, lacking either aa 31 or aa 30-31. 53-aa configurations are typically found in most chemokine receptor (CKR) subfamilies, as well as in some viral protein products. However, the CCR1, CCR3, and CCR6 subfamilies comprise a 54-aa configuration and the CKR-related protein products, ChemR23 and RDC1, include the complete 55-aa sequence. For each CKR subfamily the "modal sequence" of the conserved segment was constructed by selecting the most frequently occurring aa at each position. Then, pairwise alignments were made between: (i) the modal CKR sequences, and (ii) the sequence (53-aa) of the Yaba-like disease virus - 7L protein. From the alignments two consensus matrices were derived: (i) the consensus 1 matrix with reference to the whole conserved segment, and (ii) the consensus 2 matrix with reference to aa 22-29, which appear to be the most variable segment of the sequence. Based on the obtained consensus values and with reference to this specific conserved segment, the following conclusions are proposed: (1) ChemR23 and RDC1 are probably the more primitive CKR forms; (2) CCR1 and CCR3 may be grouped in a single cluster; (3) CCRs 2, 4, and 5 are closely related to each other and may be grouped in a cluster; CCR7 is likely to be evolutionarily related to this cluster; (4) CXCRs 2, 3, and 4 and CCX CKR appear to be evolutionarily related to each other and very likely derived from an CCR6-like gene; (5) CCR2/4/5 and CCR7 may have derived either from CCR1/3-like or CCR6-like genes; (6). The Yaba-like disease virus--7L protein most likely derived, through "molecular piracy", from a CCR8-like gene. We also discuss possible, more remote, evolutionary links between CKRs, formylpeptide receptors, and possibly the highly conserved 18S rRNA genes.
Abstract
FMLP (N-formyl-methionyl-leucyl-phenylalanine) and other N-formylpeptides are powerful "activators" of polymorphonuclear and mononuclear phagocytes, but they are also active on other cell ...types. Present knowledge about formylpeptide receptors and the relevant tools for their imaging and the study of their dynamics are briefly discussed. The main responses elicited by FMLP in granulocytes are cell polarisation, the generation of reactive oxygen species, the production of arachidonic acid metabolites, and the release of lysosomal enzymes. The transduction cascades involved and the agents able to modulate these responses are reviewed. Homologous desensitization and heterologous desensitization of the FMLP-receptor following ligation of other chemokine receptors are also outlined. Finally, the receptor expression and the pharmacological and toxic actions of FMLP upon other tissues and organs, and its actions on the developing embryo, are illustrated.
Microparticles (MPs) are small vesicles released from the membrane surface during eukaryotic cell activation or apoptosis. They originate from various cell types, displaying the typical surface cell ...proteins and cytoplasmic components of their cell origin. Their procoagulant properties are linked to phosphatidylserine exposed at their surface. Numerous reports have shown that MPs are able to mediate long-range signaling, acting on different targets from those of their own cellular origin. MPs-mediated binding to other cells occurs by integration into the membrane, by adhesion to the cell surface or by ligand-receptor interaction. Elevated levels of circulating MPs have been detected in cardiovascular and immune-mediated diseases. Despite extensive studies of the procoagulant and pro-inflammatory properties of MPs, little is known about their effect on vascular function. MPs accumulate in atherosclerotic plaques and injured vascular wall. Circulating MPs from patients with myocardial infarction induce endothelial dysfunction by impairing the endothelial nitric oxide (NO) pathway, without causing changes in endothelial NO-synthase (eNOS) expression. However, MPs from T-cells may induce endothelial dysfunction, altering gene expression of eNOS and caveolin-1. Moreover, MPs may promote the expression of pro-inflammatory proteins implicated in vascular contractility alterations. This review describes the origin of MPs and their biological role in physiological conditions and in various pathological states, with special reference to the possible linkage between their pro-inflammatory and procoagulant properties and vascular dysfunction.
RNA interference (RNAi) was used in this study for selective knockdown of TNF-alpha gene expression in anti-Ro/SSA autoantibodies (Abs)-treated human salivary gland epithelial cells. Our findings ...reveal that selective TNF-alpha gene silencing resulted in the subsequent attenuation of the pro-apoptotic effects of anti-Ro/SSA Abs; this could have therapeutic effects in autoimmune diseases.
