CD44 plays a role in adhesive interactions between glioma cells and extracellular matrix components

To evaluate how individuals infected with the human immunodeficiency virus (HIV) became aware of their infection, when they first suspected they were infected with HIV and factors associated with suspecting HIV infection, we surveyed 227 patients at an urban outpatient HIV clinic. Though nearly all patients acknowledged risk factors for HIV, 60% reported that they did not suspect that they were infected until they received a positive HIV antibody test result. Non-white patients were less likely to suspect HIV infection prior to testing than white subjects (p < 0.03). Subjects not suspecting infection more often received HIV testing through a screening program or during a medical encounter (p = 0.02) and were less likely to be told by others that they might be infected (p = 0.001) than patients suspecting infection prior to testing. Forty-eight percent of subjects who suspected HIV infection prior to testing waited one year or more before obtaining their HIV antibody test. Interventions to reduce faulty personal HIV risk perception are needed to promote earlier HIV diagnosis.     

Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate

STUDY DESIGN: Cells from normal rabbit nucleus pulposus (NP) and anulus fibrosus (AF) were cultured in alginate beads for as long as 14 days to allow them to reform a matrix made up of two compartments: the cell-associated matrix (CM) and further removed matrix (FRM). At different time points, the CM and FRM made by each cell population were analyzed using histologic, biochemical, and immunologic assays. OBJECTIVES: To study the metabolism of normal rabbit NP and AF cells in alginate by characterizing the CM and FRM formed by each cell population, and to identify metabolic properties that may shed light on mechanisms at play in disc degeneration. SUMMARY OF BACKGROUND DATA: Little is known about the metabolism of intervertebral disc cells, in part because of the lack of microculture systems appropriate for the study of these cells in vitro. In recent studies from our laboratories, it was suggested that articular chondrocytes cultured in alginate beads remain phenotypically stable and reform a matrix similar to the one they populate in vivo. This culture system appears ideally suited for the study of intervertebral cells available only in limited numbers. METHODS: Rabbit NP and AF cells released from the matrix by sequential enzyme digestion were encapsulated in alginate beads (20,000 cells/bead) and cultured for as long as 14 days. At selected time points, beads were solubilized with calcium chelating agents, and the CM and FRM were isolated. The rate of 35S-sulfate incorporation into proteoglycans, and the contents of various extracellular matrix molecules (total sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, collagen, and pyridinium crosslinks) were measured. RESULTS: Both NP and AF cells remained phenotypically stable in the alginate gel throughout the culture period and reestablished a matrix composed of CM and FRM compartments. The two cell populations exhibited numerous differences in their metabolic activities in vitro. Nucleus pulposus cells synthesized fewer proteoglycan and collagen molecules and were less effective in incorporating these into the CM than AF cells. CONCLUSIONS: Intervertebral disc cells, especially NP cells, are extremely sluggish in reforming a CM, a protective shell rich in proteoglycans and collagen molecules. This may help explain why damage to the NP often is accompanied by progressive degeneration of the disc in vivo.     

HIV-I induced destruction of neocortical extracellular matrix components in AIDS victims

Neurological dysfunction is not uncommon in patients suffering from acquired immunodeficiency syndrome (AIDS) and, when manifested, intimates involvement of the central nervous system. Here, the human immunodeficiency virus (HIV) infects preferentially microglial cells, which thereby release substances known to interfere with neuronal function. One class of agents set free in this manner are proteases; these degrade certain components within, and thereby undermine the integrity of, the extracellular matrix (ECM) compartment, which plays a vital role in cell-to-cell communication. We wished to ascertain whether the ECM compartment is indeed disrupted in the brains of AIDS victims. We examined the neocortical areas of 27 AIDS autopsy cases, including 9 with diagnosed HIV-encephalopathy (HIVE); 8 HIV-seronegative cases with various types of brain lesion, including viral infections, were also included in this study. HIV-antigens and DNA were identified by use of immunohistochemistry and in situ hybridization, and ECM components by lectin staining and immunohistochemistry. Of the 27 AIDS cases examined, each of the 9 with HIVE was completely devoid of labeled ECM components; 8 of the 18 without HIVE had incurred substantial losses, and only 2 manifested a normal complement of constituents within this compartment. With respect to stratal and topographic variations, layers II and III were less affected than layers V to VII, as was the frontal cortex relative to other areas. These findings confirmed our expectations of the brain's ECM undergoing degradation following HIV infection, and these changes may well underlie the neurological disturbances manifested in AIDS patients.     

Vitamin D3 and ceramide reduce the invasion of tumor cells through extracellular matrix components by elevating protein phosphatase-2A

Increasing phosphorylation reactions by protein kinase A (PKA) or reducing dephosphorylation reactions of protein phosphatase-2A (PP-2A) increases the invasiveness of Lewis lung carcinoma (LLC) cells, as measured by their capacity to traverse extracellular matrix (ECM)-coated filters. Metastatic LLC-LN7 variants have reduced PP-2A activity when compared to nonmetastatic LLC-C8 variants. Immunoblotting showed that this reduced level of PP-2A activity was not due to reduced levels of the PP-2A catalytic (C) subunit. The cellular PP-2A activity could be stimulated by addition of C2-ceramide to LLC-LN7 lysates, or by incubating cells with either C2-ceramide or with a noncalcemic analog of vitamin D3, which has previously been shown to stimulate the release of ceramide. These treatments to elevate PP-2A activity in metastatic LLC-LN7 cells resulted in a decline in their capacity to invade through select (ECM) components, particularly through vitronectin and laminin. Underscoring the importance of PP-2A in limiting the invasiveness of tumor cells was the demonstration that LLC-LN7 cell transfectants overexpressing the PP-2A C alpha subunit were less invasive through ECM components than the wild-type cells. Invasion by these cells was further reduced by additionally increasing PP-2A activity by incubation with C2-ceramide or the vitamin D3 analog. These results suggest a role of a vitamin D3/ceramide/PP-2A pathway in limiting the invasiveness of tumor cells through select ECM components.     

Regulation of survival and death of mesangial cells by extracellular matrix

BACKGROUND: Cell-matrix interactions exert major effects on such phenotypic features as cell growth and differentiation. Apoptosis is an active form of cell death that is crucial for maintaining the appropriate number of cells as well as the organization of tissue. Recently, it has been suggested that apoptosis of the mesangial cells (MC) is important in glomerular remodeling after injury. The MC are surrounded by an extracellular matrix (ECM) in vivo. Since in disease conditions the mesangial matrix is altered quantitatively and qualitatively, it is of interest to determine whether cell-matrix interactions may influence apoptosis of the MC. METHODS: We first investigated the differences in the susceptibility to apoptotic stimuli of the MC cultured on various ECM components (type I collagen, fibronectin, basement membrane matrix). We then determined whether the inhibition of MC-matrix interactions would affect apoptosis. Finally, interactions between MC and matrix were disrupted by the inhibition of beta1-integrin expression with antisense oligonucleotides (ODN). RESULTS: When MC were cultured on type I collagen or fibronectin and deprived of serum for eight hours, the extracted DNA from the MC demonstrated an internucleosomal ladder pattern on gel electrophoresis that constituted the biochemical characteristic of apoptosis. However, no ladder pattern was apparent when MC were cultured on basement membrane matrix. The attachment of cells was completely inhibited when the MC were cultured on agarose-coated dishes for 24 hours. Gel electrophoresis of DNA extracted from these cells showed a ladder pattern. However, the MC attached to the substratum did not show any apoptosis. MC showed an increase in apoptotic cell death after treatment with antisense ODN against beta1-integrin molecule. CONCLUSIONS: These results indicate that normal ECM may prevent the MC from undergoing apoptosis and serve as a survival factor for MC. Signals from ECM that prevent apoptosis may be mediated by beta1-integrin molecules.     

Uteroglobin (UG) suppresses extracellular matrix invasion by normal and cancer cells that express the high affinity UG-binding proteins

Uteroglobin (UG) is a steroid-inducible, multifunctional, secreted protein with antiinflammatory and antichemotactic properties. Recently, we have reported a high affinity UG-binding protein (putative receptor), on several cell types, with an apparent molecular mass of 190 kDa (Kundu, G. C., Mantile, G., Miele, L., Cordella-Miele, E., and Mukherjee, A. B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2915-2919). Since UG is a homodimer in which the 70 amino acid subunits are connected by two disulfide bonds, we sought to determine whether UG monomers also interact with the 190-kDa UG-binding protein and if so, whether it has the same biological activity as the dimer. Surprisingly, we discovered that in addition to the 190-kDa species, another protein, with an apparent molecular mass of 49 kDa, binds reduced UG with high affinity and specificity. Both 49- and 190-kDa proteins are readily detectable on nontransformed NIH 3T3 and some murine cancer cells (e. g. mastocytoma, sarcoma, and lymphoma), while lacking on others (e.g. fibrosarcoma). Most interestingly, pretreatment of the cells, which express the binding proteins, with reduced UG dramatically suppresses extracellular matrix (ECM) invasion, when such treatment had no effect on fibrosarcoma cells that lack the UG-binding proteins. Tissue-specific expression studies confirmed that while both 190- and 49-kDa UG-binding proteins are present in bovine heart, spleen, and the liver, only the 190-kDa protein is detectable in the trachea and in the lung. Neither the 190-kDa nor the 49-kDa protein was detectable in the aorta. Purification of these binding proteins from bovine spleen by UG-affinity chromatography and analysis by SDS-polyacrylamide gel electrophoresis followed by silver staining identified two protein bands with apparent molecular masses of 40 and 180 kDa, respectively. Treatment of the NIH 3T3 cells with specific cytokines (i.e. interleukin-6) and other agonists (i.e. lipopolysaccharide) caused a substantially increased level of 125I-UG binding but the same cells, when treated with platelet-derived growth factor, tumor necrosis factor-alpha, interferon-gamma, and phorbol 12-myristate 13-acetate, did not alter the UG binding. Taken together, these findings raise the possibility that UG, through its binding proteins, plays critical roles in the regulation of cellular motility and ECM invasion.     

Migratory pattern of fetal rat brain cells and human glioma cells in the adult rat brain

The migratory behavior of two human glioma cell lines (D-54MG and GaMG) and fetal rat brain cells grafted into the adult rat brain was studied. To trace the implanted cells, they were stained with the carbocyanine vital dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate before injecting them into the white matter above the corpus callosum. The animals were sacrificed 2 h and 7 and 21 days after injection, and the brains were removed and cryosectioned. Fluorescence microscopy showed that both the 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-stained fetal and tumor cells had the same migratory pattern. Implanted cells were found along myelinated fibers in the corpus callosum and in the perivascular space. After immunostaining for several extracellular matrix (ECM) components (laminin, fibronectin, collagen type IV, and chondroitin sulfate), laminin deposits were observed in the border zone between the host tissue and implanted tumor cells as well as fetal cells. By using two different types of antibodies against fibronectin, it is shown that the fibronectin expression observed in the tumor matrix may be host derived. This was further supported by the fact that tumor spheroids obtained from the two glioma cell lines were negative when immunostained for these ECM components. Several of the ECM components may be host derived. This can be caused by neovascularization and repair synthesis or by a local production of guiding substrates which are important for tumor cell locomotion. The present data suggest that the migratory patterns of fetal and glioma cells are indistinguishable when transplanted into the adult rat brain. Thus, glioma cells may be routed by the same ECM components that play a major role during brain development.     

Exogenous nitric oxide inhibits mesangial cell adhesion to extracellular matrix components

Interactions of mesangial cells (MCs) with components of the extracellular matrix (ECM) profoundly influence the MC phenotype, such as attachment, contraction, migration, survival and proliferation. Here, we investigated the effects of exogenous nitric oxide (NO) on the process of MC adhesion to ECM molecules. Incubation of rat MCs with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) dose- and time-dependently inhibited MC adhesion and spreading on various ECM substrata, being more pronounced on collagen type I than on collagen type IV, laminin or fibronectin. In contrast, SNAP did not inhibit MC adhesion to L-polylysine-coated plates. The inhibitory effects of SNAP were reduced by hemoglobin and enhanced by superoxide dismutase. The anti-adhesive action of SNAP was mimicked not only by other NO donors but also by 8-bromo-cGMP, and significantly reversed by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ). Moreover, SNAP and 8-bromo-cGMP decreased the adhesion-induced phosphorylation of focal adhesion kinase (pp125FAK). In the presence of SNAP or 8-bromo-cGMP, adherent MCs exhibited disturbed organization of alpha-actin filaments and reduced numbers of focal adhesions, as shown by immunocytochemistry. In additional experiments with adherent MCs, it was found that exposure to SNAP or 8-bromo-cGMP for 12 and 24 hours induced detachment of MCs. The results indicate that exogenous NO interferes with the establishment and maintenance of MC adhesion to ECM components. This inhibitory NO effect is mediated predominantly by cGMP-signaling. Disturbance of MC attachment to ECM molecules could represent an important mechanism by which NO affects MC behavior in vitro and in vivo.     

Stimulation of HCO3- secretion across cystic fibrosis pancreatic duct cells by extracellular ATP

Colonic endocrine cells from prediabetic and diabetic non-obese diabetic mice as well as of the sister strain, BALB/cJ, were investigated by immunocytochemistry and computer image analysis. In prediabetic mice, enteroglucagon-and serotonin-immunoreactive cells were significantly increased in number, whereas the cell secretory index of these two cell types was significantly reduced. No significant differences were found in numbers or cell secretory index of peptide YY (PYY)-immunoreactive cells. In diabetic mice, PYY-immunoreactive cells were significantly fewer, but there were no significant differences in the numbers of enteroglucagon-and serotonin-immunoreactive cells. Whereas the cell secretory index was reduced in serotonin-producing cells, no significant differences were found between diabetic and control mice regarding the cell secretory index of PYY- and enteroglucagon-immunoreactive cells. Nor was any statistically significant difference found between controls, prediabetic and diabetic non-obese diabetic mice, regarding the thickness of submucosa, of circular and longitudinal-muscle layers, or of the mucosal area/microm baseline. The present study showed that abnormalities in colonic endocrine cells do occur, in both prediabetic and diabetic mice, but they are different in nature and can be divided into primary and secondary to the diabetes onset. The present findings of abnormal colonic endocrine cells in non-obese diabetic mice, an animal model for human insulin-dependent diabetes mellitus, might help explain the gastrointestinal disorders observed in patients with diabetes. The study also showed that the change in the colonic endocrine cells is dynamic and started before the onset of the diabetic condition.     

Stimulation of G-CSF gene expression in the macrophage cell line by contact with extracellular matrix proteins and a pre-B leukaemia cell line

Freshly prepared dentine specimens of human teeth were perfused with either horse serum or physiologic saline. After application of AllBond2, ART Bond, Syntac or an experimental dentine bonding agent called P-Bond, a composite cylinder was added and cured at the same time. After 1500 thermal cycles with constant imitation of intrapulpal pressure, the shear bond strengths were measured. Resulting shear bond strength values were analysed with Students t-test or Mann-Whitney Test. The values for AllBond2 were not significantly different. The values for ART Bond (P < 0.05) and for Syntac (P < 0.05) were significantly higher if the dentine was perfused with horse serum. For P-Bond (P < 0.001) the values were significantly higher if the dentine was perfused with physiologic saline. According to these results it does not seem to be appropriate to take a clear decision as to which of the two perfusing media investigated might be more suitable to imitate in vivo conditions.     

Altered deposition of extracellular matrix components in the skeletal muscle and lymph node of the MDX dystrophic mouse

1. MDX mice derived from a colony of C57BL/10ScSn mice develop an X-linked recessive muscular dystrophy, thus providing an adequate model to study the pathogenesis of muscular dystrophy. 2. Skeletal myofibers of MDX mutant mice were heterogeneous, with disorganization of myofilaments and the absence of immunolabelling for dystrophin with monoclonal antibody DY4/6D3. 3. Marked deposition of reticulin, collagenic fiber (types, I, IV) and laminin (LN) were consistently present mostly around lesioned and necrotic myofibers associated with an intense inflammatory reaction, whereas strong immunolabelling for TIII-C, TIV-C and FN was often associated with regenerated fibers. 4. During the onset (3 weeks of postnatal life) of disease and height of myonecrosis (5-6 weeks of postnatal life), popliteal lymph nodes showed dense argyrophilic meshwork, intense immunolabelling for collagens types I and IV, FN, LN and enlargement of the hili which were packed with mononuclear cells. Such alterations, albeit less intense, were still observed in MDX mice with 20 weeks of postnatal life. 5. The results support the view that ECM components might be influencing the migration of inflammatory cells and the process of myonecrosis in the skeletal muscle of MDX dystrophic mice.     

Function of proteoglycans in the extracellular matrix

Estimating phylogenies from DNA sequence data has become the major methodology of molecular phylogenetics. To date, molecular phylogenetics of the vertebrates has been very dependent on mtDNA, but studies involving mtDNA are limited because the several genes comprising the mt-genome are inherited as a single linkage group. The only apparent solution to this problem is to sequence additional genes, each representing a distinct linkage group, so that the resultant gene trees provide independent estimates of the species tree. There exists the need to find novel gene sequences which contain enough phylogenetic information to resolve relationships between closely related species. A possible source is the nuclear-encoded introns, because they evolve more rapidly than exons. We designed primers to amplify and sequence the 7 intron from the beta-fibrinogen gene for a recently evolved group, the woodpeckers. We sequenced the entire intron for 10 specimens representing five species. Nucleotide substitutions are randomly distributed along the length of the intron, suggesting selective neutrality. A preliminary analysis indicates that the phylogenetic signal in the intron is as strong as that in the mitochondrial encoded cytochrome b (cyt b) gene. The topology of the beta-fibrinogen tree is identical to that of the cyt b tree. This analysis demonstrates the ability of the 7 intron of beta-fibrinogen to provide well resolved, independent gene trees for recently evolved groups and establishes it as a source of sequences to be used in other phylogenetic studies.     

Contraction of extracellular matrix by transdifferentiated retinal pigment epithelial cells, inducers and inhibitors

Adhesion molecules relate to cell invasion of autoimmune thyroid disease. We studied plasma soluble P-Selectin (platelet activation-dependent granule-external membrane protein), E-Selectin (endothelial leukocyte adhesion molecule) and L-Selectin (leukocyte endothelial cell adhesion molecule-1) levels in patients with Graves' disease before and during methimazole treatment. Plasma P-, E- and L-Selectin levels in patients with untreated Graves' disease were significantly higher than those in normal subjects. Plasma P-Selectin levels decreased when their thyroid functions were normal for more than 6 months after the start of methimazole treatment. No significant change in plasma E- and L-Selectin levels in patients with Graves' disease was found between hyperthyroid state and euthyroid state after the start of methimazole treatment, but plasma L-Selectin levels in patients with untreated Graves' disease were significantly lower than those in the patients in the first euthyroid state. There was no significant correlation between plasma P-Selectin levels and serum FT4 levels, nor between plasma P-Selectin levels and serum FT3 levels. These results suggested that thyroid hormones might reflect expression of P-, L- and E-Selectin from endothelial cells, or lymphocytes, or platelets in patients with Graves' disease.     

Versican V2 is a major extracellular matrix component of the mature bovine brain

We have isolated and characterized the proteoglycan isoforms of versican from bovine brain extracts. Our approach included (i) cDNA cloning and sequencing of the entire open reading frame encoding the bovine versican splice variants; (ii) preparation of antibodies against bovine versican using recombinant core protein fragments and synthetic peptides; (iii) isolation of versican isoforms by ammonium sulfate precipitation followed by anion exchange and hyaluronan affinity chromatography; and (iv) characterization by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining or immunoblotting. Our results demonstrate that versican V2 is, together with brevican, a major component of the mature brain extracellular matrix. Versicans V0 and V1 are only present in relatively small amounts. Versican V2 migrates after chondroitinase ABC digestion with an apparent molecular mass of about 400 kDa, whereas it barely enters a 4-15% polyacrylamide gel without the enzyme treatment. The 400-kDa product is recognized by antibodies against the glycosaminoglycan-alpha domain and against synthetic NH2- and COOH-terminal peptides. Our preparations contain no major proteolytic products of versican, e.g. hyaluronectin or glial hyaluronate-binding protein. Having biochemical quantities of versican V2 available will allow us to test its putative modulatory role in neuronal cell adhesion and axonal growth.     

Assessment of the fibrogenetic activity in chronic pancreatitis. The role of circulating levels of extracellular matrix components

Determination of circulating levels of extracellular matrix components has been proposed as a reliable method to assess the activity of fibrogenetic processes. Therefore, we aimed to analyze circulating levels of laminin, fibronectin, and procollagen III peptide (PIIIP) in patients with chronic pancreatitis (CP) and to correlate them with the morphological and functional stage, and duration of the disease. Thirty patients with CP and 18 healthy controls were studied. Serum PIIIP concentrations (RIA), but not fibronectin (RID) and laminin (RIA), were abnormally high in 8 patients with CP. No correlation was found between circulating levels of extracellular matrix components and both functional and morphological stage and duration of CP. Nevertheless, patients with elevated serum PIIIP levels tend to have a more advanced CP (morphological and functional changes) than those with normal levels after a similar duration of the disease. We hypothesize that whereas functional and morphological findings reflect the cumulative effect of chronic inflammation on the pancreas, serum PIIIP concentrations would reflect the activity of the fibrogenetic process within the gland at the time of sampling. The results shown in the present study may be considered a starting point for longitudinal studies that examine the relationship between serum PIIIP or other markers for fibrogenetic activity and evolution of CP.     

Abnormal adhesion of T cells to extracellular matrix proteins in hemodialysis patients

MOTIVATION: The BioCatalog is a database of information on software of interest in molecular biology and genetics. The programs are grouped by domain of interest. AVAILABILITY: The BioCatalog is freely distributed as ASCII files. It can be searched through its Web interface at http://www.ebi.ac.uk/biocat. CONTACT: biocat@ebi. ac.uk