Streptococcus anginosus adheres to vascular endothelium basement membrane and purified extracellular matrix proteins

The mechanisms of bacterial adherence in the initial stages of native valve endocarditis are unclear, especially in patients without valve disease or the presence of a platelet-fibrin thrombus. Extracellular matrix may act as a ligand in areas of exposed basement membrane on the endothelial monolayer. In this study, adherence of 55 clinical blood and 21 oral viridans streptococcal isolates was examined using purified extracellular matrix compounds. The majority of blood and oral isolates exhibited adherence to purified laminin, fibronectin, and fibrinogen, with lesser adherence to type I and IV collagens. Adherence to laminin and fibronectin was concentration dependent, saturable, and competitively inhibited with soluble ligand. A Streptococcus anginosus isolate and other viridans strains exhibiting a strong laminin adherence phenotype bound extensively to the endothelial aspect of human and porcine valve tissue sections and were inhibited by soluble laminin and anti-laminin antibody fragments. Using a novel native porcine valve explant adherence model, we localized binding to areas of exposed basement membrane by confocal and scanning electron microscopy. These studies support the hypothesis that bacterial adherence to exposed basement membrane plays a role in the initial phase of native valve endocarditis.     

The basement membrane component of biologic scaffolds derived from extracellular matrix

The extracellular matrix (ECM) has been successfully used as a scaffold for constructive remodeling of multiple tissues in both preclinical studies and in human clinical applications. The basement membrane is a specialized form of the ECM that supports and facilitates the growth of epithelial cell populations. The morphology and the molecular composition of the ECM, including the basement membrane, vary depending upon the organ from which the ECM is harvested and the methods by which it is processed for use as a medical device. Processing steps, such as decellularization, lyophilization, disinfection, and terminal sterilization, may affect the morphology and composition of an ECM scaffold, including, but not limited to, the integrity of a basement membrane complex. The present study evaluated the presence and integrity of a basement membrane complex in processed ECM derived from three different tissues: the urinary bladder, small intestine, and liver. Immunohistochemical determination of the presence and localization of three basement membrane molecules, collagen IV, laminin, and collagen VII, was conducted for each ECM scaffold. Scanning electron microscopy (SEM) was used to further explore the surface ultrastructure of selected ECM scaffolds. The effect of a surface basement membrane presence upon the pattern of in vitro growth of two separate cell types, NIH 3T3 fibroblasts and human microvascular endothelial cells (HMEC), was also evaluated for each ECM scaffold. Results showed that the only intact basement membrane complex was found on the luminal surface of the ECM derived from the urinary bladder and that the basement membrane was an effective barrier to penetration of the scaffold by the seeded cells. We conclude that the urinary bladder ECM but not the small intestine- or liver-derived ECM contains a surface with composition and morphology consistent with that of an intact basement membrane complex, that the basement membrane complex can survive processing, and that the basement membrane structure can modulate in vitro cell growth patterns.     

The basement membrane matrix in malignancy

Cancer is the second most common cause of death among Americans, although for several age groups it ranks first. Most of these deaths are not due to the primary tumour but rather to tumour cell metastases to distant organs. There are many steps that lead to metastasis, all of which are being studied with the goal of preventing these fatalities. Normally, cells attach to the extracellular matrix to maintain tissue integrity. During cancer progression, cells become more motile and acquire invasive qualities. Tumour cells move along blood and lymph vessels or invade into them to travel to distant sites. Then, the tumour cells must attach to the vessel wall, extravasate from the vessel, invade the new tissue, proliferate, and form a secondary tumour. Angiogenesis, the formation of new blood vessels, is critical to survival of these cells at the new site and is also important for primary tumour growth and spread. Tumour cell metastasis is a complex cascade of sequential steps, each of which is not yet fully understood. Progress has been made in identifying several key activators, one of which is the extracellular matrix. A major tumour promoter is the glycoprotein laminin, which is predominantly found in the extracellular matrix produced by endothelial and epithelial cells. This review will follow the metastatic process with particular attention to the effect of laminin on tumour cells.     

Early diagnosis of SARS coronavirus infection by real time RT-PCR.

BACKGROUND: A novel coronavirus was recently identified as the aetiological agent of Severe Acute Respiratory Syndrome (SARS). Molecular assays currently available for detection of SARS-coronavirus (SARS-CoV) have low sensitivity during the early stage of the illness. OBJECTIVE: To develop and evaluate a sensitive diagnostic test for SARS by optimizing the viral RNA extraction methods and by applying real-time quantitative RT-PCR technology. STUDY DESIGN: 50 nasopharyngeal aspirate (NPA) samples collected from days 1-3 of disease onset from SARS patients in whom SARS CoV infections was subsequently serologically confirmed and 30 negative control samples were studied. Samples were tested by: (1) our first generation conventional RT-PCR assay with a routine RNA extraction method (Lancet 361 (2003) 1319), (2) our first generation conventional RT-PCR assay with a modified RNA extraction method, (3) a real-time quantitative RT-PCR assay with a modified RNA extraction method. RESULTS: Of 50 NPA specimens collected during the first 3 days of illness, 11 (22%) were positive in our first generation RT-PCR assay. With a modification in the RNA extraction protocol, 22 (44%) samples were positive in the conventional RT-PCR assay. By combining the modified RNA extraction method and real-time quantitative PCR technology, 40 (80%) of these samples were positive in the real-time RT-PCR assay. No positive signal was observed in the negative controls. CONCLUSION: By optimizing RNA extraction methods and applying quantitative real time RT-PCR technologies, the sensitivity of tests for early diagnosis of SARS can be greatly enhanced.     

Topographic characterization and protein quantification of esophageal basement membrane for scaffold design reference in tissue engineering

Basement membrane (BM) has been investigated widely and applied in the field of bio-synthesized scaffold design in tissue engineering. However, this respect of investigation has seen scarcely in the field of esophageal tissue engineering. This study reports BM's basic topographic characters and quantification of structural and major proteins involving collagen IV, laminin, entactin, proteoglycans of porcine esophagus. Several methods were adopted to strip epithelium from mucosa tissue. For example, ethylene diamine tetraacetic acid, sonication, dithiothreitol, cold sodium chloride (NaCl), mechanical force of glass coverslip, and cold trypsin and so forth. Assessed experimental results under different conditions, the optimal condition to isolate epithelium from BM was established. After the reaction of cold trypsin at 4°C for 15 h, the BM was isolated from epithelium and exposed integratedly. Hematoxylin and eosin staining and immunohistochemical staining verified the effectiveness of epithelium removal and the integrity of BM. The topographic features of the exposed BM were observed under transmission electron microscopy and scanning electron microscopy. It displayed a rugged surface and 3-D topography consisting of pores and fibers with sub-100 nm range via ImageJ software. The major proteins existing in BM were quantitatively analyzed by enzyme-linked immune sorbent assay. All these results will provide references for the design of synthesized scaffolds and protein modification in esophageal tissue-engineering research.     

Modulation of the immune response by extracellular matrix proteins

Inflammation entrains a focused and coordinated response from many different elements. Soluble factors such as chemokines and cytokines direct the recruitment, differentiation, and fate of leukocytes. Cells and pathogens are killed and consumed, yet where the response is effective, inflammation will melt away, leaving a healthy functioning tissue. All this commonly takes place in an environment known as the extracellular matrix (ECM). The ECM is not a passive partner in the process and recent work demonstrates the important role that proteins found in this environment play in connecting different parts of the immune response together. In this review we will focus on these connections and the proteins that make them. One emerging trend that we will highlight is the ability of endogenous molecules to interact with receptors that are better known as sensors of the molecular fingerprints of infection. We propose that this may be particularly relevant in the context of autoimmunity, since the provision of such signals may be crucial in breaking tolerance.     

Expression profile of extracellular matrix and its regulatory proteins during the process of interstitial fibrosis after anti-glomerular basement membrane antibody-induced glomerular sclerosis in Sprague-Dawley rats

Anti-glomerular basement membrane (GBM) nephritis in Sprague-Dawley (SD) rats was characterized by development of marked glomerular sclerosis and tubulointerstitial fibrosis. To elucidate sequential change of the glomerular sclerosis and tubulointerstitial fibrosis, accumulation and mRNA expression of extracellular matrix (ECM) components and transforming growth factor (TGF)-beta were examined in the glomerulus and cortex during the disease course by histology, immunostaining and ribonuclease protection assay. Mild proliferative and degenerative lesions appeared in the glomeruli by day 15 after anti-GBM antibody binding to GBM and progressed to glomerular sclerotic lesion thereafter. Conversely, interstitial change was first recognized by infiltration of mononuclear cells after day 20, followed by marked accumulation of ECM and tubular degeneration. The interstitial fibrosis was induced without apparent binding of anti-GBM antibody to tubular basement membrane. Accumulation of fibronectin, collagen type I and type IV was noted in the interstitium by immunofluorescence microscopy in association with enhanced expression of mRNA for these ECM components and their regulatory molecules such as matrix metalloproteinase (MMP2), tissue inhibitor of metalloproteinase (TIMP)-1 and TGF-beta1 both in glomeruli and cortex. The glomerular expression of these mRNA increased apparently by day 15 and reached a plateau or a peak at day 20. The expression of the same mRNA increased gradually from day 15 to day 29 in the cortex. These observations show that interstitial fibrosis follows glomerular sclerosis after anti-GBM antibody injection in SD rats, suggesting that at least a part of the mechanism for ECM accumulation in the glomerulus and interstitium is essentially the same in terms of composition of ECM and expression of its regulatory molecules.     

Dexamethasone attenuates oxidation of extracellular matrix proteins by human monocytes

In response to infection or in immune complex-mediated diseases, inflammatory cells may oxidatively damage extracellular matrix (ECM) proteins. In this study we evaluated whether human monocytes could oxidize ECM and whether this could be modulated by exposure to LPS, IgG complexes, and dexamethasone (DEX). Wells in tissue culture plates were coated with the ECM preparation Matrigel. Porous inserts with or without the human monocyte cell line THP-1 were placed into ECM-containing wells and cells were exposed to control conditions or to LPS (10 ng/ml), IgG complexes (200 and 500 microg/ml), or DEX (10(-7) and 10(-6) M). ECM was then subjected to Western blot analysis using an antibody to oxidized protein. In addition, Western blot analysis was carried out on DEX-treated cells to evaluate expression of the NADPH oxidase components p67-phox and gp91-phox. THP-1 cells enhanced ECM oxidation and this effect was augmented by LPS and by IgG aggregates. Preincubation of cells with DEX attenuated ECM oxidation and was also associated with decreased expression of p67-phox and gp91-phox. These findings suggest that human monocytes can oxidize ECM proteins and that this may be modulated by IgG complexes and LPS. Dexamethasone appears to attenuate ECM oxidation and a better understanding of this mechanism might allow for interventions to minimize oxidative damage to ECM proteins by monocytes in infectious and inflammatory states.     

Ultrastructural characterization of an artificial basement membrane produced by cultured keratinocytes

A recent study in our laboratories on the growth of keratinocytes at the culture medium/air interface has led to the identification of a novel thin sheet-like matrix that supports adherent cells. This novel matrix consists of components secreted by keratinocytes, including type IV collagen, and laminins 1 and 5, that self-assembled to a membrane structure. In the present study, a detailed ultrastructural characterization of this membrane was done with high-resolution electron microscopy after negative staining. The basic organization of the membrane was found to be a dense network of 8- to 10-nm-wide irregular rod-like elements. High-resolution examination and immunolabeling showed that type IV collagen filaments form the core of these elements, and other components including heparan sulfate proteoglycan in the form of 4.5- to 5-nm-wide ribbon-like "double tracks" are aggregated around it. These detailed features of the membrane strikingly resembled those of the basement membrane in vivo. These ultrastructural similarities indicate that the membrane may also have basement membrane-like functional properties, and suggest that it should be considered for testing in future medical applications.     

Modulation of mesenchymal stem cell genotype and phenotype by extracellular matrix proteins

ABSTRACT

Aim: To investigate the effect of extracellular matrix (ECM) proteins on characteristics of mesenchymal stem cells (MSCs) and tendon-derived cells (TDCs). Materials and Methods: MSCs and TDCs, cultured in a monolayer (2D) or hydrogels (3D), with or without ECM protein supplementation, and on a non-viable native tendon (NNT) matrix were assayed for adhesion, proliferation, gene expression, and integrin expression. Results: MSCs exhibited a fibroblastic, spindle-shaped morphology on 2D matrices except in the presence of fibronectin. In 3D matrices, MSCs displayed a rounded phenotype except when cultured on NNTs where cells aligned along the collagen fibrils but, unlike TDCs, did not form inter-cellular cytoplasmic processes. MSC proliferation was significantly (p < 0.01) increased by collagen type I in 2D culture and fibronectin in 3D culture. TDC proliferation was unaffected by substrata. MSCs and TDCs differentially expressed α2 integrin. Adhesion to substrata was reduced by RGD-blocking peptide and β1 integrin antibody. The presence of collagen I or fibronectin upregulated MSC expression of collagen type I and collagen type III, COMP, decorin, osteopontin, and fibronectin. Conclusions: The morphology, gene expression, and adhesion of both MSCs and TDCs are sensitive to the presence of specific ECM components. Interaction with the ECM is, therefore, likely to affect the mechanism of action of MSCs in vitro and may contribute to phenotypic modulation in vivo.     

Recognition of extracellular matrix proteins by Paracoccidioides brasiliensis yeast cells.

The adhesion of microorganism to host cells or extracellular matrix (ECM) proteins is the first step in the establishment of an infectious process. Interaction between Paracoccidioides brasiliensis yeast cells and ECM proteins has been previously noted. In vivo, in the chronic phase of experimental paracoccidioidomycosis (PCM), laminin and fibronectin have been detected on the surface of yeast cells located inside granulomatous lesions. The aim of the present study was to examine the ability of P. brasiliensis yeast cells to interact with extracellular matrix proteins (laminin, fibrinogen and fibronectin) and to establish which molecules were involved in this interaction. Immunofluorescence microscopy and flow cytometry demonstrated that all three ECM proteins tested were able to bind to the surface of P. brasiliensis yeast cells. Treatment with trypsin, chymotrypsin, chitinase, proteinase K or different sugars resulted in no change in laminin binding. In addition, ligand affinity assays were performed using different yeast extracts (total homogenates, beta-mercaptoethanol, SDS extracts). These assays demonstrated the presence of 19 and 32-kDa proteins in the cell wall with the ability to bind to laminin, fibrinogen and fibronectin. This interaction could be important in mediating attachment of the fungus to host tissues and may consequently play a role in the pathogenesis of PCM.     

Quantitation of defective and ecotropic viruses during LP-BM5 infection by real time PCR and RT-PCR

Murine AIDS (MAIDS) is a pathology induced by the LP-BM5 murine leukaemia virus mixture in susceptible strains of mice such as C57BL/6J resulting in lymphoproliferation and progressive immunodeficiency. The etiologic agent of this pathology is BM5d, a replication defective virus. BM5e is a replication competent virus in the viral mixture that functions as a helper virus. This paper describes real time PCR and RT-PCR assays for quantitation of the proviral DNA and viral RNA of BM5d and BM5e. Data is presented describing the change in BM5d and BM5e proviral DNA levels and viral RNA levels in both blood and spleen in the first 8 weeks of infection. Infected mice have increasing levels of BM5d and BM5e viral DNA and RNA detectable from as early as 2 weeks post infection. Similar levels of proviral DNA was found for BM5d and BM5e in PBMC and spleen, however higher levels of BM5e viral RNA were observed in both tissues throughout infection. The assays described can be used as both a diagnostic tool and to investigate the direct effect of treatments on the BM5d and BM5e viruses and MAIDS development.     

Development of a real time RT-PCR to detect and type ovine pestiviruses

A real time one-step RT-PCR was designed to detect and type border disease virus (BDV), bovine viral diarrhea virus (BVDV) type 1 and BVDV type 2 in ovine samples. The real time RT-PCR was shown to behave in a linear manner and had limits of detection of 100-1000 copies of viral RNA as judged by in vitro transcribed RNA. The real time RT-PCR was validated on 50 clinical samples from UK flocks and was more sensitive than a virus isolation and a classical nested RT-PCR (nRT-PCR). The results of real time RT-PCR virus typing agreed completely with sequencing. The majority of ovine isolates were BDV; a small proportion were BVDV type 1. BVDV type 2 was not detected in any sample. This test appears reliable and can be used for the typing of ovine pestiviruses in the UK.     

Collagenous and basement membrane proteins of chordoma: immunohistochemical analysis

Tissue localization of collagenous and basement membrane proteins in the extracellular matrix of five sacro-coccygeal chordomas and human fetal notochords was examined immunohistochemically to assess the implications for the histogenesis and histological diagnosis of chordoma. Human fetal notochords and conventional chordomas both exhibited basement membrane proteins (such as type IV collagen and laminin) and type VI collagen on the surfaces of cellular cords. Type II collagen, a main structural protein of cartilage, was also present in both tissues. In the chordomas, however, type II collagen was not so widespread as it was in the notochords, and the predominant collagenous protein was type I. In contrast, an altered deposition of these proteins was noticed in a recurrent tumour which, histologically, showed considerable atypia and eventually metastasized to the liver. The characteristic cartilage-type and basement membrane proteins disappeared and unusual collagen types, such as types III and V, appeared in the stroma. The results further support the notochordal origin of chordoma and suggest that the immunohistochemistry of collagenous and basement membrane proteins may be a helpful criterion for the histological diagnosis and prediction of the biological aggressiveness of chordomas.     

Partial characterization of collagenous and noncollagenous basement membrane proteins synthesized by the 14.5-day rat embryo parietal yolk sac in vitro

Parietal yolk sacs isolated from 14.5-day rat embryos and incubated in vitro with either [14C]proline, [3H]mannose or 3H-labeled amino acid mixture synthesized and secreted basement membrane collagenous and noncollagenous glycoprotein components with relative molecular weights of 350,000 (350K), 220,000 (220K), 185,000 (185K), 175,000 (175K) and 150,000 (150K). The 185K and 175K components appeared to be similar to the pro-alpha 1 (IV) and pro-alpha 2(IV) chains, respectively, which have been isolated from other sources. These components were completely susceptible to bacterial collagenase, but were only partially susceptible to alpha-chymotrypsin digestion. The 350K and 220K components appeared to be similar to subunits of laminin (or PYS A and PYS B, respectively) which have been characterized by others, while the 150K component may be similar to entactin (or PYS C). These components were completely resistant to bacterial collagenase and completely susceptible to alpha-chymotrypsin digestion. In addition, the basement membrane of the parietal yolk sac (Reichert's membrane) stained intensely with antibodies directed against either rat laminin or mouse basement membrane procollagen. The results of these experiments suggest that the 14.5-day rat embryo parietal yolk sac is a useful system for studying the structure, biosynthesis and deposition of basement membrane components.     

Binding of outer membrane preparations of Campylobacter jejuni to INT 457 cell membranes and extracellular matrix proteins

Binding of outer membrane (OM) preparations of the thermophilic Campylobacter species C. jejuni to epithelial cell membranes and extracellular matrix proteins were studied in an in vitro model system using enzyme-linked immunosorbent assay. The OM preparations exhibited significant binding to INT 407 intestinal cell membranes. The process of adhesion was modulated by enzymatic, chemical or immunological pretreatment of the bacteria. Following oxidation of the lipopolysaccharide (LPS) with sodium meta-periodate, the OM preparations essentially retained their binding properties. After pretreatment with proteinase K, the OM preparations lost their binding capacity and the apparent molecular mass of the major OM protein shifted from 42 to 24 kDa. Preincubation of C. jejuni bacteria with C. jejuni-specific antiserum reduced adhesion significantly; preincubation with LPS-specific monoclonal antibodies only to a minimal extent. The OM preparations also bound significantly to the extracellular matrix proteins collagen and fibronectin; however, they bound virtually no bovine serum albumin or horse serum.     

Interactions between HIV-infected monocytes and the extracellular matrix: HIV-infected monocytes secrete neutral metalloproteases that degrade basement membrane protein matrices.

The frequency of human immunodeficiency virus (HIV)-infected monocytes that spread on a model basement membrane was about twofold greater than that of an equal number of uninfected control cells through the initial 12 to 18 h of culture. By 24 h, virtually all HIV-infected and uninfected control cells spread on the basement membrane gel. The frequency of spread cells in the uninfected control population was less than 10% of total cells by 12 days. In contrast, 30 to 40% of HIV-infected monocytes remained spread through this time interval and formed a dense interdigitated network of cell processes on and into the gel matrix. Invasion of the basement membrane matrix by HIV-infected monocytes suggested increased secretion of proteases able to digest the gel. Indeed, levels of neutral protease activity in culture fluids from HIV-infected monocytes were significantly higher than those from equal numbers of uninfected control cells. High levels of protease activity in culture fluids of HIV-infected monocytes required productive virus infection and were not observed with cells exposed to T cell-tropic HIV isolates. The predominant protease activity in these cultures was a 92-kd neutral metallogelatinase. HIV-induced changes in monocyte metalloprotease activity may be important for extravasation of infected cells to tissue or for the development of AIDS-associated neuropathology, carcinogenesis, and opportunistic infection.     

Binding of Haemophilus ducreyi to extracellular matrix proteins

We developed an enzyme-linked immunosorbent assay-based assay to assess Haemophilus ducreyi binding to extracellular matrix (ECM) proteins. H. ducreyi 35000HP bound to fibronectin, laminin, and type I and III collagen but not to type IV, V, or VI collagen or elastin. Isogenic strains with mutations in ftpA or losB bound as well as the parent, suggesting that neither pili nor full-length lipooligosaccharide is required for H. ducreyi to bind to ECM proteins.     

Herpes gestationis--bullae from hormones that cause increased extracellular matrix (autoantibodies to epidermal basement membrane as a secondary phenomenon)

Herpes gestationis is a bullous skin disease clearly secondary to the hormones of pregnancy and other hormonal influences. It is the result multiple hormones. No one hormone is specific. The hormones increase ground substance viscosity in the skin and this induces edema and bullae formation. Only 10 to 20% of the patients demonstrate IgG formation by indirect immunofluorescence. By direct immunofluorescent studies 30 to 40% of the patients demonstrate deposition of IgG in the basement membrane zone. Other variations of immune factors are present in most cases but they are weak by titers usually present in most so called autoimmune disorders. Many bullous diseases of the skin, including herpes gestationis, are considered autoimmune. It is known that trauma to cells and tissues can induce autoantibodies to form. It is proposed that autoantibodies can form as a secondary phenomenon in bullous skin disorders. The authors who have proposed an autoimmune etiology of herpes gestationis do not attempt to explain the clear association of the disease with the hormones of pregnancy, other female hormones, chorio-carcinoma, or hydatidiform mole.