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PubMed1993

Fibronectin and proteolytic fragments of fibronectin interfere with the adhesion of Staphylococcus epidermidis to plastic.

The adhesion of five strains of slime-positive Staphylococcus epidermidis to plastic microwells was significantly diminished (P < 0.005) in a concentration-dependent fashion when wells were previously coated with increasing concentrations (1.6-13.1 micrograms cm-2) of human fibronectin (FN). The adhesion of four of five strains was significantly reduced when wells were coated with 3.2 micrograms cm-2 of FN and at concentrations > or = 6.5 micrograms cm-2 the adhesion of all slime-positive strains was significantly reduced. The coating of microwells with chymotryptic fragments of FN containing the heparin-binding, gelatin-binding, or cell-binding domains also reduced bacterial adhesion but none of the fragments exceeded the anti-adhesive activity of intact FN. A comparison of FN-coated or albumin-coated microwells showed that both proteins caused a significant reduction in the adhesion of test strains to plastic but that the anti-adhesive activity of FN was greater than albumin at all concentrations tested. The adhesion of the slime-negative phase variant of one of the test strains to plastic was neither enhanced nor reduced by FN coating indicating that the production of an exopolysaccharide by Staph. epidermidis influences interactions with protein-coated surfaces. These results support the contention that FN does not mediate the adhesion of all strains of Staph. epidermidis to plastic surfaces.

Bacterial Adhesion↗
PubMed1994

Clustering of fibronectin adhesins toward Treponema denticola tips upon contact with immobilized fibronectin.

Treponema denticola has been shown to bind to immobilized fibronectin (Fn) by its tips. Yet labeling of cells in suspension with an Fn-gold conjugate to localize the Fn adhesins shows that they are distributed in patches along the entire cell length. Subsequent experiments have shown that the number and proportion of tip-oriented cells increase with time, suggesting that Fn contact stimulates T. denticola to rearrange adhesins toward its tips. To test this hypothesis, T. denticola cells were allowed to migrate in a 2% methylcellulose column toward nitrocellulose filters coated with Fn, laminin, bovine serum albumin, or phosphate-buffered saline. Cells close to and distant from the filters were collected, labeled with Fn-gold probes, and examined by transmission electron microscopy. The number of gold particles on each of 20 cells was counted, dividing each cell into thirds along its length: the end third with the most label (end 1), the middle third, and the end with the least label (end 2). The mean number (+/- standard deviation) of gold probes per third was calculated. Fn-gold probes clustered toward one end of T. denticola cells when in contact with Fn-coated nitrocellulose, with > 55% of probes in end 1. In contrast, no clustering toward T. denticola ends occurred with laminin-, bovine serum albumin, or phosphate-buffered saline-coated filters or in the absence of a filter. Blocking access of the T. denticola cells to the Fn-coated nitrocellulose filter by placing an uncoated filter between them prevented clustering of Fn-gold. Removal of T. denticola cells from direct contact with the Fn-coated filter did not promote redistribution of clustered probes. These data suggest that T. denticola is stimulated to cluster Fn adhesins irreversibly toward its tips when it migrates into contact with immobilized Fn. This might be significant for establishing multiple adhesive interactions with host cells and ligands.

Bacterial Adhesion↗
PubMed1999

SFS, a novel fibronectin-binding protein from Streptococcus equi, inhibits the binding between fibronectin and collagen.

The obligate parasitic bacterium Streptococcus equi subsp. equi is the causative agent of strangles, a serious disease of the upper respiratory tract in horses. In this study we have, using shotgun phage display, cloned from S. equi subsp. equi and characterized a gene, called sfs, encoding a protein termed SFS, representing a new type of fibronectin (Fn)-binding protein. The sfs gene was found to be present in all 50 isolates of S. equi subsp. equi tested and in 41 of 48 S. equi subsp. zooepidemicus isolates tested. The sfs gene is down-regulated during growth in vitro compared to fnz, a previously characterized gene encoding an Fn-binding protein from S. equi subsp. zooepidemicus. Sequence comparisons revealed no similarities to previously characterized Fn-binding proteins, but high scores were obtained against collagen. Besides similarity due to the high content of glycine, serine, and proline residues present in both proteins, there was a nine-residue motif present both in collagen and in the Fn-binding domain of SFS. By searching the Oklahoma S. pyogenes database, we found that this motif is also present in a potential cell surface protein from S. pyogenes. Protein SFS was found to inhibit the binding between Fn and collagen in a concentration-dependent way.

Adhesins, Bacterial↗
PubMed2000

Synthetic peptide immunogens elicit polyclonal and monoclonal antibodies specific for linear epitopes in the D motifs of Staphylococcus aureus fibronectin-binding protein, which are composed of amino acids that are essential for fibronectin binding.

A fibronectin (Fn)-binding adhesin of Staphylococcus aureus contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn. Plasma from patients with S. aureus infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F. Casolini, L. Visai, D. Joh, P. G. Conaldi, A. Toniolo, M. Höök, and P. Speziale, Infect. Immun. 66:5433-5442, 1998). To eliminate the influence of Fn binding on antibody development, we used synthetic peptide immunogens D1(21-34) and D3(20-33), which each contain a conserved pattern of amino acids that is essential for Fn binding but which cannot bind Fn without N- or C-terminal extensions. The D3(20-33) immunogen promoted the production of polyclonal antibodies that were 10-fold more effective as inhibitors of Fn-binding to the D3 motif than antibodies obtained by immunizing with an extended peptide D3(16-36), which exhibits functional Fn binding. The D3(20-33) immunogen also facilitated the production of a monoclonal antibody, 9C3, which was highly specific for the epitope SVDFEED, and abolished Fn binding by the D3 motif. When mixed with polyclonal anti-D1(21-34) immunoglobulin G, 70% inhibition of Fn binding to the three tandem D motifs was achieved compared to no more than 30% inhibition with either antibody preparation alone. Therefore, by immunizing with short synthetic peptides that are unable to bind Fn, we have effectively stimulated the production of antibodies specific for epitopes comprised of amino acids that are essential for Fn binding. Although these epitopes occur within a conserved pattern of amino acids that is required for Fn binding, the antibodies recognized specific linear epitope sequences and not a conserved structure common to all repeated motifs.

Adhesins, Bacterial↗
PubMed2001

Fibronectin attachment protein homologue mediates fibronectin binding by Mycobacterium avium subsp. paratuberculosis.

Attachment of Mycobacterium avium subsp. paratuberculosis to host tissue and penetration of mucosal surfaces are pivotal events in the pathogenesis of Johne's disease. Fibronectin (FN) binding is required for attachment and internalization of several mycobacteria by epithelial cells in vitro. The objective of this study was to further characterize the FN binding activity of M. avium subsp. paratuberculosis. Although the bacteria bound FN poorly at pH above 7, brief acid pretreatment greatly enhanced FN binding within the pH range (3 to 10) studied. A 4.6-kbp fragment from an M. avium subsp. paratuberculosis genomic library was found to contain a 1,107-bp open reading frame that shows very high nucleotide sequence identity with that of the FN attachment protein (FAP) gene of M. avium subsp. avium. Pretreatment of FN with an FN-binding peptide from M. avium subsp. avium FAP abolished FN binding, indicating that M. avium subsp. paratuberculosis binds FN in a FAP-dependent manner. Pretreatment of M. avium subsp. paratuberculosis with anti-FAP immunoglobulin G did not abrogate FN binding; blocking occurred only when anti-FAP was added together with FN. FAP was detected by immunofluorescence only in lipid-extracted M. avium subsp. paratuberculosis. Western blotting and immunoelectron microscopy revealed that FAP is located near the interior of the cell envelope of M. avium subsp. paratuberculosis. The results indicate that a FAP homologue mediates the attachment of FN to M. avium subsp. paratuberculosis. Further, given the subcellular location of FAP, it is considered that this protein operates at the terminus of a coordinated FN binding system in the cell envelope of M. avium subsp. paratuberculosis.

Adhesins, Bacterial↗
PubMed2001

Variance in fibronectin binding and fnb locus polymorphisms in Staphylococcus aureus: identification of antigenic variation in a fibronectin binding protein adhesin of the epidemic CMRSA-1 strain of methicillin-resistant S. aureus.

The fnbA and fnbB genes of Staphylococcus aureus 8325-4 encode fibronectin (Fn) binding proteins FnBPA and FnBPB, which promote adherence to host tissues. Each adhesin contains three copies of a repeated D motif that binds Fn and is a target for vaccine development. In this study, we assess variability within the Fn-binding domain of the FnBP adhesins and evaluate factors that promote variance in Fn binding among clinical isolates. Based on variation in the number of fnb genes or the number of D motifs, we identified five polymorphism groups. S. aureus 8325-4 and 91% of methicillin-resistant S. aureus (MRSA) isolates belong to polymorphism group I, with two fnb genes and three copies of the D motif. Polymorphism group II contained one fnb gene with only two D motifs and was associated with the epidemic CMRSA-4 strain, which exhibited high protease activity and low Fn binding. Polymorphism group III was unique to the epidemic CMRSA-1 strain, defined by the presence of a fourth D motif that exhibited antigenic variation within a conserved sequence that is essential for Fn binding. However, the sequence of the D motifs was otherwise highly conserved among the other polymorphism groups. Variation in Fn binding among MRSA isolates was inversely related to protease activity but not to the number of fnb genes or the number of D motifs. Therefore, the fnb locus is polymorphic in a small number of strains, but this does not contribute to variation in Fn binding. The antigenic variation that was observed only in the epidemic CMRSA-1 strain may have evolved in response to a host immune response encountered during successive cycles of colonization, transmission, and infection in the nosocomial environment.

Adhesins, Bacterial↗
PubMed2004

Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein facilitates M-cell targeting and invasion through a fibronectin bridge with host integrins.

Efficient attachment and ingestion of Mycobacterium avium subsp. paratuberculosis by cultured epithelial cells requires the expression of a fibronectin (FN) attachment protein homologue (FAP-P) which mediates FN binding by M. avium subsp. paratuberculosis. Invasion of Peyer's patches by M. avium subsp. paratuberculosis occurs through M cells, which, unlike other intestinal epithelial cells, express integrins on their luminal faces. We sought to determine if the interaction between FAP-P of M. avium subsp. paratuberculosis and soluble FN enabled targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo via these surface integrins. Wild-type and antisense FAP-P mutant M. avium subsp. paratuberculosis strains were injected alone or coinjected with blocking peptides or antibodies into murine gut loops, and immunofluorescence microscopy was performed to assess targeting and invasion of M cells by M. avium subsp. paratuberculosis. Nonopsonized M. avium subsp. paratuberculosis preferentially invaded M cells in murine gut loops. M-cell invasion was enhanced 2.6-fold when M. avium subsp. paratuberculosis was pretreated with FN. Invasion of M cells by the antisense FAP-P mutant of M. avium subsp. paratuberculosis was reduced by 77 to 90% relative to that observed for the control strains. Peptides corresponding to the RGD and synergy site integrin recognition regions of FN blocked M. avium subsp. paratuberculosis invasion of M cells by 75 and 45%, respectively, whereas the connecting segment 1 peptide was noninhibitory. Antibodies against the alpha5, alphaV, beta1, and beta3 integrin subunits inhibited M-cell invasion by 52 to 73%. The results indicate that targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo is mediated primarily by the formation of an FN bridge formed between FAP-P of M. avium subsp. paratuberculosis and integrins on M cells.

Adhesins, Bacterial↗
PubMed1992

Rebound elevation of fibronectin after tissue injury and ischemia: role of fibronectin synthesis.

Plasma fibronectin (pFn) stimulates macrophage phagocytosis of tissue debris; pFn deposition in tissues may influence vascular integrity. Although the acute depletion of pFn after surgery and/or injury has been described, less attention has been given to the rebound hyperfibronectinemia presumably "triggered" by the early pFn depletion. Using a model that compartmentalized the site of tissue injury and thus attenuated the initial pFn depletion, we studied this rebound elevation of pFn in anesthetized rats (250-350 g) after the surgical trauma of groin dissection alone (sham group) or surgery coupled with 4 h of hindlimb ischemia (experimental group). Nonoperated control rats were also anesthetized. Shams had baseline (preoperative) 6-, 8-, and 22-h postoperative pFn levels of 573 +/- 61, 598 +/- 62, 695 +/- 57, and 929 +/- 87 micrograms/ml, respectively. In the surgery-ischemia group, pFn also elevated to 1,117 +/- 40 micrograms/ml at 22 h postsurgery. Nonoperated control rats (only anesthetized) had no elevation of pFn. Intravenous infusion of gelatin-coated lipid particles (50 mg/100 g) depleted pFn by 89.3% but was unable to prevent the rebound elevation of pFn. The blood clearance of 125I-labeled pFn was very similar in control, sham, and experimental rats. In contrast, pFn synthesis over the 22-h period was dramatically altered and equal to 2.12 +/- 0.16, 3.40 +/- 0.56, and 4.49 +/- 0.17 mg pFn synthesized/100 g body wt, in control, sham, and experimental rats respectively. Thus a rapid increase in pFn synthesis contributes to the rebound hyperfibronectinemia after sublethal surgical injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Induction of fibronectin mRNA by urokinase- and tissue-type plasminogen activator in human skin fibroblasts: differential role of u-PA and t-PA at the fibronectin protein level.

Plasminogen activators of the urokinase- and tissue-type and fetal calf serum (u-PA, t-PA, FCS) exert their mitogenic effect on quiescent human dermal fibroblasts and modulate the mRNA expression of cell-cycle related genes. The present study deals with the effects of PAs on the expression of fibronectin (FN), a heterodimeric extracellular matrix (ECM) protein that can be modulated in different ways by various mitogens. The kinetics of FN gene response was examined in quiescent fibroblasts upon PA stimulation (30 min -24 h). The results obtained evidenced that: (i) all mitogens tested (u-PA, t-PA and FCS) led to an increase of FN mRNA expression in early G1, as shown by the analysis of two sequences, III-9, common to all FN mRNAs, and EDA+, present only in the EDA+FN isoform; (ii) the kinetic profiles of FN mRNA stimulation were comparable for the three mitogens, although the effects on the FN-ECM assembly were distinct; (iii) t-PA and FCS led to FN assembly in the ECM, which was absent or decreased in u-PA-treated cultures. Immunobiochemical analysis of total FN and EDA+ FN showed that FN induced by t-PA was mainly dimeric (450-500 kDa), whereas FN induced by u-PA was mainly monomeric (230-250 kDa). These differences are probably due to the differential enzymatic action of t-PA and u-PA on FN, which might be related to a differential role of the two PAs in several physiopathological conditions.

Extracellular Matrix↗
PubMed1996

Fibronectin fragments cause an underestimation of plasma fibronectin levels in severe pre-eclampsia.

Elevated fibronectin (Fn) concentrations in plasma from pregnant women have been suggested to be predictive for the development of pre-eclampsia. However, reported correlations between Fn concentration with gestational age or disease severity appear to be confounded by several variables. Our finding of degradation products of Fn in plasma from patients with severe pre-eclampsia led to a study of whether their presence could influence immunological and functional measurements of intact plasma Fn. Plasma samples (10 control pregnant and 10 severe pre-eclampsia patients) were studied for the presence of intact Fn and fragments by SDS-PAGE and Western blotting. Percentages of immunoreactive Fn fragmentation in control plasma (9.37 +/- 6.7%) and severe pre-eclampsia plasma (62.57 +/- 7.0%) were determined by densitometric scanning. Immunoassays by ELISA were performed on normal plasma samples and on purified Fn in the absence or presence of plasma digests of pure Fn. Artefactual underestimations of Fn concentration occurred in the presence of Fn fragments. The percentage underestimation increased with increasing digestion times (1 h, 46.52 +/- 3.69%; 3 h, 77.5 +/- 7.18%; 4 h, 100%) and with increasing concentrations of Fn digest products (1h) added to normal plasma samples (n = 10) (40 micrograms, 23 +/- 8.1%; 125 micrograms, 36.33 +/- 5.1%; 250 micrograms, 43.66 +/- 6.5%). The underestimation of Fn concentration in the presence of Fn fragments suggests that ELISAs for Fn may be unreliable and thus lose their predictive value in these patients. Opsonic activities of control pregnant and severe pre-eclamptic plasma were determined by gelatin-latex agglutination assay. The ratios of opsonic activity to Fn concentration in plasma from patients with pre-eclampsia were significantly lower than those of control pregnant plasma samples (p < 0.008). The impaired gelatin-latex agglutination of Fn in pre-eclamptic plasma suggests that Fn fragmentation could have biological sequelae in vivo.

Blotting, Western↗
PubMed2002

Mice lacking the EDB segment of fibronectin develop normally but exhibit reduced cell growth and fibronectin matrix assembly in vitro.

Fibronectins (FNs) are major cell-adhesive proteins in the extracellular matrix and are essential for embryonic development. FNs are encoded by a single gene, but heterogeneity is introduced by alternative pre-mRNA splicing. One of the alternatively spliced segments, extra domain B (EDB), is prominently expressed during embryonic development and in tumor tissues, although it is mostly eliminated from FN in normal adult tissues. To examine the function of the EDB segment in vivo, we generated mice lacking the EDB exon using the Cre-loxP system. Although EDB-containing FNs are highly expressed throughout early embryogenesis, EDB-deficient mice developed normally and were fertile. Despite the absence of any significant phenotypes observed in vivo, however, fibroblasts obtained from EDB-deficient mice grew slowly in vitro and deposited less FN in the pericellular matrix than fibroblasts from wild-type mice. These results indicate that expression of EDB-containing isoforms is dispensable during embryonic development, yet may play a modulating role in the growth of connective tissue cells via the FN matrix.

Amino Acid Sequence↗
PubMed2005

Fibronectin preadsorbed on hydroxyapatite together with rough surface structure increases osteoblasts' adhesion "in vitro": the theoretical usefulness of fibronectin preadsorption on hydroxyapatite to increase permanent stability and longevity in spine implants.

OBJECTIVE: The aim of this study was to investigate the contribution of fibronectin (FN) preadsorption to enhance osteoblast adhesion and strength on hydroxyapatite (HA) used either as osteoconductive bone substitute or precoating of pedicle screws and cages in spine surgery. METHODS: HA substrata with two different surface roughness values (rough HA180 and smooth HA1200) were produced, and human osteoblasts were seeded after culturing on them. Prior to osteoblast seeding, the HA substrata were immersed in FN solution. Osteoblast attachment on each of the two HA substrata was evaluated by recording the number of cells, whereas osteoblast adhesion strength was determined by measuring the shear stress required to detach the cells from the HA substrates. RESULTS: FN preadsorption increased the number of attached osteoblasts on smooth and rough HA substratum at 40% and 62%, respectively, whereas it increased osteoblast attachment strength on the smooth and rough substratum at 165% and 73%, respectively. CONCLUSIONS: This study showed that FN preadsorption and rough HA surface texture synergistically increased "in vitro" both the number and the adhesion strength of human osteoblasts. Further studies in primates and humans should be carried out to disclose the clinical relevance (increase implant's stability and longevity) of the above-mentioned observations.

Adsorption↗
PubMed1986

Interaction of culture-derived macrophages with the fibroblast-binding domain of fibronectin is a necessary but inefficient signal for fibronectin enhancement of CR1-mediated phagocytosis.

Human plasma fibronectin (Fn) enhances ingestion of opsonized particles through its interaction with phagocytic cells. To better characterize the site or sites on Fn responsible for this effect, we subjected Fn to limited proteolytic cleavage by chymotrypsin and used affinity and gel filtration chromatography to isolate a 110,000 dalton cell-binding fragment, a 60,000 dalton fragment that bound both heparin and gelatin, and 50,000 and 45,000 dalton fragments that bound to gelatin but not heparin. The cell-binding fragment mediated adhesion and spreading of fibroblasts on glass slides, whereas the heparin-gelatin and gelatin-binding fragments failed to cause fibroblast spreading. At high concentrations, the cell-binding fragment doubled phagocytosis of C4b-coated sheep erythrocytes by human monocyte-derived macrophages, whereas equal concentrations of the other fragments had minimal enhancing effect on phagocytosis. Interestingly, the effect of the cell-binding fragment on CR1-mediated phagocytosis was always less than the effect of intact Fn, even when a 40-fold higher molar concentration of the cell-binding fragment was used. Fab of a monoclonal anti-Fn, HFn 7.1, which recognizes the 110,000 dalton cell-binding fragment of Fn and inhibits fibroblast binding, blocked enhancement of CR1-mediated phagocytosis by intact Fn. Fab of Fn 8, a monoclonal anti-Fn that binds the heparin-gelatin-binding fragment, failed to inhibit the Fn effect. These data suggest that interaction of the macrophage with the cell-binding domain of Fn is critical for the Fn effect on CR1-mediated phagocytosis. In addition, there may be other domains of the Fn molecule that have a role in augmenting the Fn-phagocyte interaction.

Antibodies, Monoclonal↗
PubMed1987

Modulation of polyclonal activation by plasma fibronectin and fibronectin fragments.

Modulation of activation of polyclonal IgM, IgG and IgM anti-DNA antibodies by plasma fibronectin (Fn) was studied because in some autoimmune diseases there appears to be a correlation between the increased level of Fn in the affected tissues and increased polyclonal B-cell activation. Fn caused a dose-dependent polyclonal activation of IgM, IgG and IgM anti-DNA antibody-secreting cells in cultures of mouse splenocytes. Fn significantly inhibited the generation of polyclonal antibodies by Fn-binding stimulants and did not significantly change the generation of polyclonal antibodies by the stimulants that do not bind Fn. Plasmin or trypsin digestion of Fn abolished both the polyclonal activating properties of Fn and the inhibitory effects of Fn that were selective for the Fn-binding polyclonal activators. Digestion of Fn with trypsin also generated immunosuppressive Fn fragments that inhibited polyclonal activation by both Fn-binding and non-binding bacteria. Under our culture conditions Fn or Fn digests were not mitogenic and had no effect on the mitogenicity of Fn-binding and non-binding stimulants. These results indicate that Fn can act as a polyclonal activator and that it can also modulate lymphocyte activation induced by other activators.

Animals↗
PubMed1987

Fibronectin and cellular cytotoxicity: evidence against a role for fibronectin in natural killer activity.

We investigated a possible role for fibronectin (Fn) in natural killer (NK) cell activity against K562 tumor cells. Plastic plates that had been incubated with Fn at 60 micrograms/ml bound 49.6% +/- 12.8% (mean +/- SEM) of K562 cells; plates coated with three other proteins bound essentially no tumor cells (P less than 0.01). In similar experiments, 12.6% +/- 4.7% of nonadherent lymphocytes bound to plates that had been coated with 60 micrograms/ml Fn; plates coated with other proteins bound less than or equal to 10% of these cells. Lymphocytes that bound to Fn-coated plates appeared to be slightly enriched for NK cells as assessed by morphology and by cytotoxic activity. Despite these findings, neither NK cytotoxic activity nor binding to K562 targets was affected by the addition of any of three anti-Fn antibody preparations, nor by the addition of exogenous Fn at concentrations found in plasma (300 micrograms/ml). Although Fn appears to bind both to K562 targets and to lymphocyte preparations that contain NK cells, it does not appear to have a functional role in mediating NK cell activity against K562 tumor cells.

Cell Adhesion↗
PubMed1984

[Changes in fibronectin concentration during therapeutic plasmapheresis. Therapeutic effectiveness of selective removal of fibronectin in immune complex pathology].

Variation in the concentration of plasma fibronectin (FN) seen during 65 sessions of therapeutic plasmapheresis was studied in 20 inpatients with the following diagnoses: acute leukemias (4), paraproteinemic hemoblastoses (5), Sjögren's syndrome (2), protracted septic endocarditis (1), systemic lupus erythematosus (17), acute polyradiculoneuritis (1), multiple sclerosis (3). The patients' age ranged within 19 to 64 years. There were 10 men and 10 women. The concentration of FN in 218 plasma samples was measured by the ELISA. It was discovered that plasma of healthy donors contained 200 to 400 micrograms/ml FN (M +/- 1.5 sigma). Prior to plasmapheresis the glycoprotein content in all the patients was on the average within normal (300 micrograms/ml). After the session the concentration of FN decreased almost two-fold (130 micrograms/ml); after 6 hours it was 175 micrograms/ml on the average, whereas after 24-48 hours it reached the initial level. Repeated plasmapheresis sessions carried out once every 3-4 days did not lead to the depletion of the FN pool, since its level reached the initial one over the first 24-48 hours. Such a mechanism was unchanged regardless of the initial level of FN recorded in the patients blood (high, normal or low). No relationship was found between FN deficiency which developed after plasmapheresis and infectious complications. It is assumed that FN deficiency per se cannot be responsible for the patients' decreased resistance to infection. Three patients received a series of selective plasmapheresis. Sjögren's syndrome and immune complex vasculitis was diagnosed in one female patient, hemorrhagic vasculitis due to chronic hepatitis in another female patient, the third female patient manifested gammapathy with cryoglobulinemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Platelet interactions with fibronectin: divalent cation-independent platelet adhesion to the gelatin-binding domain of fibronectin.

Divalent cation-dependent platelet adhesion to fibronectin (FN) is mediated by the integrin receptors alpha 5 beta 1 (GP Ic-IIa) and alpha IIb beta 3 (GP IIb-IIIa), which recognize the RGD (Arg-Gly-Asp) sequence in the cell-binding domain. However, FN can also support divalent cation-independent platelet adhesion. To determine which domain of FN mediates divalent cation-independent adhesion, proteolysis with thermolysin and affinity chromatography were used to isolate the cell-binding, gelatin-binding, and heparin-binding domains of FN. Unactivated and thrombin-activated platelets adhered to intact FN and the 45-Kd gelatin-binding domain in the presence of either Ca2+ or EDTA. Platelet spreading was mediated only by the 105-Kd cell-binding domain and required divalent cations. The heparin-binding domains did not support platelet adhesion. Reduction of intrachain disulfide bonds or removal of carbohydrate side chains on the gelatin-binding domain did not alter the ability to support platelet adhesion. Divalent cation-independent adhesion to the 45-Kd gelatin-binding domain was not inhibited by RGDS (Arg-Gly-Asp-Ser) synthetic peptides or monoclonal antibodies (MoAbs) directed against known platelet receptors. We conclude that platelets can adhere but not spread on the gelatin-binding domain of FN by a novel divalent cation-independent mechanism.

Amino Acid Sequence↗
PubMed1993

Increase production of fibronectin by glomerular cultures from rats with nephrotoxic nephritis. Macrophages induce fibronectin production in cultured mesangial cells.

BACKGROUND: The participation of monocytes-macrophages and their products in the pathogenesis of several types of glomerulonephritis has become increasingly evident. One of the most important aspects is the potential stimulation of monocyte-macrophages of the extracellular matrix. Therefore, production of fibronectin (FN) by glomeruli of rats with nephrotoxic nephritis was studied. In addition, the effect of macrophage-conditioned medium and interleukin-1 on FN production by cultured mesangial cell (MC) was tested. EXPERIMENTAL DESIGN: Nephrotoxic nephritis was induced in rats by injection of nephrotoxic serum and glomeruli obtained at different periods of time from nephritic and normal animals were cultured. FN in glomerular supernatants was determined by enzyme-linked immunosorbent assay and newly synthesized FN by incorporation of [35S]-methionine. Macrophage-conditioned medium was obtained from cultures of peritoneal resident macrophages and elicited macrophages by different stimuli. MC were cultured with or without macrophage supernatant or interleukin-1 and FN content from MC supernatants was determined by enzyme-linked immunosorbent assay. RESULTS: These data show increase amounts of FN in nephritic cultures when compared with saline controls (time of nephritis; day 4: 3.9-, day 8: 4.35-, and day 18: 2.68-fold increase in FN) and experiments of newly synthesized FN by incorporation of [35S]-methionine had similar results. Macrophage-conditioned medium had FN stimulatory effect on cultured MC but interleukin-1 did not. CONCLUSIONS: These data suggest: (a) that there is an increased newly synthesized FN production in glomeruli from rats with nephrotoxic nephritis, (b) that macrophage produce a FN-stimulatory factor(s) for MC, and (c) that this stimulatory factor probably is not interleukin-1.

Animals↗