Suero autólogo en el tratamiento del síndrome de ojo seco. Aspectos tecnológicos / Autologous serum in the treatment of dry eye sindrome. Technological aspects

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Foot-and-mouth disease virus is a ligand for the high-affinity binding conformation of integrin alpha5beta1: influence of the leucine residue within the RGDL motif on selectivity of integrin binding.

Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors for internalization, whereas strains that are adapted for growth in cultured cell lines appear to be able to use alternative receptors like heparan sulphate proteoglycans (HSPG). The ligand-binding potential of integrins is regulated by changes in the conformation of their ectodomains and the ligand-binding state would be expected to be an important determinant of tropism for viruses that use integrins as cellular receptors. Currently, alphavbeta3 is the only integrin that has been shown to act as a receptor for FMDV. In this study, a solid-phase receptor-binding assay has been used to characterize the binding of FMDV to purified preparations of the human integrin alpha5beta1, in the absence of HSPG and other RGD-binding integrins. In this assay, binding of FMDV resembled authentic ligand binding to alpha5beta1 in its dependence on divalent cations and specific inhibition by RGD peptides. Most importantly, binding was found to be critically dependent on the conformation of the integrin, as virus bound only after induction of the high-affinity ligand-binding state. In addition, the identity of the amino acid residue immediately following the RGD motif is shown to influence differentially the ability of FMDV to bind integrins alpha5beta1 and alphavbeta3 and evidence is provided that alpha5beta1 might be an important FMDV receptor in vivo.

Entamoeba histolytica contains a beta 1 integrin-like molecule similar to fibronectin receptors from eukaryotic cells.

Entamoeba histolytica trophozoites do interact with extracellular matrix components in order to invade and finally destroy tissue. An important step in this interaction involves the binding of a 140-kDa membrane protein that binds to fibronectin. The similarity of this amoebic receptor to fibronectin receptors from higher eukaryotic cells was defined by indirect immunofluorescence, western blot and immunohistochemistry, using polyclonal monospecific antibodies raised against the amoebic protein. These results suggest that lower eukaryotic cells have and use a beta 1 integrin-like molecule as well as mechanisms similar to those present in higher eukaryotic cells during interaction with extracellular matrix components.

Evidence for a beta 1 integrin fibronectin receptor in Candida tropicalis.

The binding of Candida tropicalis to fibronectin (FN) was studied in order to characterize the FN receptor in this species. FN binding was saturable at a concentration of 1.8 x 10(-9) M and exhibited a Kd of 2.3 x 10(-9) M and a receptor density of 854 receptors per cell. Extracts of C. tropicalis cell membrane at dilutions of 1:100-1:1000 significantly inhibited the binding of 3H-labeled FN to C. tropicalis cells (P < .03). Purified FN, antibodies to the integrin alpha 5 beta 1 (FN receptor on human placenta), and antibodies specific for the integrin beta 1 subunit recognized a C. tropicalis membrane protein of 125 +/- 25 kDa on immunoblots. Immunoprecipitation of radiolabeled proteins from C. tropicalis with purified human FN yielded a protein of 105 +/- 15 kDa. Thus, C. tropicalis expresses a protein with antigenic and functional similarity to the vertebrate beta 1 integrin FN receptor.

A 31-kDa recombinant fibronectin cell-binding domain fragment: its binding to receptor, cell adhesive activity, and fusion proteins.

The binding of fibronectin to fibronectin receptor was studied using a recombinant 31-kDa cell-binding domain fragment of fibronectin (C279), which consisted of three type III repeats (III8-III9-III10). Fibronectin receptor in several cell lysates was bound to a column of C279-immobilized Sepharose HP and obtained in a highly purified form by elution with a synthetic peptide, GRGDSP. alpha 5 beta 1-Integrin was detected in the GRGDSP-eluted fraction by immunoblotting. The cell-adhesive activity of C279 was inhibited by GRGDSP peptide, an anti-integrin a5 subunit antibody, and an anti-integrin beta 1 subunit antibody. The cell adhesion of fusion proteins of the 31-kDa fragment with biologically interesting polypeptides (heparin-binding domain of fibronectin, and basic fibroblast growth factor) was also studied. In the presence of an anti-integrin a5 subunit antibody, human fibrosarcoma HT-1080 cells attached to the fusion protein containing fibroblast growth factor, giving rise to changes the morphology of the attached cells. The cell adhesion of C279 was inhibited by GRGDSP peptide but that of the fusion protein with the heparin-binding domain of fibronectin was not completely inhibited by the peptide. These results suggest that these biologically interesting polypeptides contribute to the cell adhesion of the fusion proteins.

Presence of multiple laminin- and fibronectin-binding proteins in cell wall extract of Candida albicans: influence of dialysis.

Candida albicans has been reported to express only one to three proteins that bind extracellular matrix proteins, such as laminin and fibrinogen. In those reports, cell wall extracts were subjected to various processing steps, such as dialysis and lyophilization, prior to Western blot analysis. Here, we demonstrate that dialysis for only 2 h of cell wall protein extracts results in a substantial loss (40-60%) of protein. With overnight dialysis, the loss was increased further. After 2 h of dialysis, wall extracts contained fewer laminin- and fibronectin-reactive proteins. In addition, the number of wall proteins in the extracts detected by a polyclonal anti-human fibronectin receptor antiserum decreased after dialysis. These results demonstrate that the C. albicans yeast cell wall contains multiple proteins capable of binding laminin and fibronectin and many of these proteins are not functionally detectable following dialysis.

Regulation of integrin alpha 5 beta 1-fibronectin interactions by divalent cations. Evidence for distinct classes of binding sites for Mn2+, Mg2+, and Ca2+.

Integrin-ligand interactions are known to be dependent on divalent cations, although the precise role of cations in ligand binding is still unclear. Using the interaction between alpha 5 beta 1 and fibronectin as a model system, we have performed a comprehensive analysis of the effects of Mn2+, Mg2+, and Ca2+ on ligand binding. Each cation had distinct effects on the ligand-binding capacity of alpha 5 beta 1:Mn2+ promoted high levels of ligand binding, Mg2+ promoted low levels of binding, and Ca2+ failed to support binding. Studies of the effects of different combinations of cations on ligand binding indicated that the cation-binding sites within alpha 5 beta 1 are not all identical, or of broad specificity, but instead each site shows a distinct preference for one or more cations. Ca2+ strongly inhibited Mn(2+)-supported ligand binding, but this inhibition was noncompetitive, suggesting that Ca2+ recognizes different cation-binding sites to Mn2+. In contrast, Ca2+ acted as a direct competitive inhibitor of Mg(2+)-supported ligand binding, implying that Ca2+ can displace Mg2+ from the integrin. However, low concentrations of Ca2+ greatly increased the apparent affinity of Mg2+ for its binding site, suggesting the existence of a distinct high affinity Ca(2+)-binding site. Taken together, our results imply that the ligand-binding capacity of alpha 5 beta 1 can be regulated in a complex manner through separate classes of binding sites for Mn2+, Mg2+, and Ca2+.

Analysis of fibronectin and vitronectin receptors on human fetal skeletal muscle cells upon differentiation.

The role of fibronectin (FN) and vitronectin (VN) receptors for cell adhesion and matrix assembly was analyzed during human fetal myogenesis in vivo and in vitro. In human fetal muscle at 10 weeks gestational age FN and laminin are present in the extracellular matrix. Analysis of the integrin repertoire at this developmental stage reveals that the differentiated muscle cells in vivo express alpha 5 and alpha 6 integrins, but not alpha v, alpha 1, and alpha 3 integrins. However, in vitro cultured myoblasts (G6) isolated from the same gestational age express alpha v, alpha 1, and alpha 3 integrins in addition to alpha 5 and alpha 6 integrins. A more detailed analysis of FN and VN receptors in vitro shows that the localization of different alpha v heterodimers into focal contacts is differently regulated. Alpha v beta 1, and alpha v beta 3, are present at focal contacts throughout in vitro myogenesis whereas alpha v beta 5 appears to depend on an endogenously produced factor to localize to focal contacts. The alpha v beta 1, alpha v beta 5, and alpha 3 beta 1 heterodimers, often reported not to focalize, did form focal contacts in G6 cells, indicating that these myoblasts possess components facilitating the formation of cytoskeletal linkages containing these integrins. Alpha 5 beta 1 colocalized with FN in myoblast cultures, whereas myotubes lacked both FN and alpha 5 beta 1 on the cell surface. In summary, we show that concomitant with in vitro differentiation of G6 cells, FN matrix contacts are abolished, but vitronectin receptors continue to fulfill an anchoring function during the differentiation process in vitro. Further studies are needed to assess the relative importance of the FN and VN binding integrins for the differentiation process in comparison with the laminin-binding integrins alpha 6 and alpha 7, also present on these cells.

Fibronectin binding by Streptococcus milleri group strains and partial characterisation of the fibronectin receptor of Streptococcus anginosus F4.

The Streptococcus milleri group were shown to bind fibronectin (Fn) to their cell-surface and this binding increased the adhesion of cells to hydroxyapatite. The binding of Fn to Streptococcus anginosus F4 was studied in more detail. Fn binding to bacterial cells increased the association of the bacteria with the polymorphonuclear leukocytes obtained from the peritoneal cavity of rats but did not increase killing of the bacteria. The cell-surface receptor was a protein of M(r) 14,000 which was released from cells after mutanolysin digestion. The binding was specific, with cells having a maximum number of binding sites per cell of 770. Electron microscopy, using gold-labelled Fn, localised the receptor to areas between daughter cells.

Candida albicans expresses a fibronectin receptor antigenically related to alpha 5 beta 1 integrin.

Cell adhesion molecules, by regulating host-micro-organism interaction, play a major role in the pathogenesis of infectious diseases. The present study was undertaken to investigate the expression of the fibronectin (FN) receptor prototype, alpha 5 beta 1 integrin, on Candida albicans and its involvement in the adhesion to FN. By immunofluorescence and fluorescence activated cell sorter (FACS) analysis, several monoclonal antibodies (mAbs) directed against human alpha 5 or beta 1 integrin subunits, or two different antisera to FN receptor positively stained C. albicans yeast and germ tube phases, this immunoreactivity increasing upon germ tube transition. Twenty-five to thirty per cent of [3H]glucose-labelled Candida yeasts specifically adhered to FN and this adhesion was increased upon germ tube transition. C. albicans yeast and germ tube forms bound to an RGD-containing 120 kDa tryptic fragment of FN and adhesion to FN was markedly inhibited by GRGDSP, but not GRGESP peptides. Moreover, binding of both C. albicans phases to FN was strongly inhibited by anti-alpha 5 SAM-1 mAb, or both anti-fibronectin receptor (FNr) antisera. Overall these results indicate that C. albicans yeast and germ tube phases express a receptor antigenically related to alpha 5 beta 1 integrin which mediates their adhesion to FN. The alpha 5 beta 1 integrin-like receptor expression on C. albicans could be relevant for fungus-host interaction and in the dissemination process of Candida infection.

Isolation of Pneumocystis carinii gp120 by fibronectin affinity: evidence for manganese dependence.

Pneumocystis carinii is a major opportunistic lung pathogen and a leading cause of death among patients with the human immunodeficiency virus. Adherence of P. carinii to type I alveolar epithelial cells is essential for growth and replication and has been shown to be mediated in part by fibronectin (Fn). To better understand the mechanisms underlying this attachment, P. carinii-Fn interaction was characterized with respect to divalent and monovalent ion concentration and pH using an 125I-Fn binding assay to P. carinii. The results suggest that P. carinii has a receptor for Fn that was partially dependent on Ca2+, enhanced by Mn2+, and diminished somewhat by Mg2+. Additional data demonstrated that P. carinii-Fn interaction was sensitive to ionic strength. The pH profile revealed that P. carinii-Fn interaction increased with decreasing pH. The results from the binding assay provided the rationale for a simple isolation of the Fn receptor from P. carinii using a Fn-affinity column involving nondenaturing conditions. The isolated receptor appeared highly purified by SDS-PAGE analysis, with apparent molecular weights of 114 to 118 kD and 66 kD. Western blot analysis indicated that this receptor was gp120, a major surface glycoprotein of P. carinii. Furthermore, the isolated receptor inhibited Fn binding to P. carinii. Finally, a monoclonal antibody raised against the affinity-purified gp120 blocked Fn binding to P. carinii.

Functional role of N-glycosylation in alpha 5 beta 1 integrin receptor. De-N-glycosylation induces dissociation or altered association of alpha 5 and beta 1 subunits and concomitant loss of fibronectin binding activity.

Fibronectin (FN)-mediated cell adhesion is controlled mainly by alpha 5 beta 1 (recognizing the RGD sequence) and alpha 4 beta 1 (recognizing the CS-1 peptide sequence of FN) integrin receptors. Integrin-dependent cell adhesion to FN is greatly promoted by optimal GM3 concentration at the surface membrane (Zheng, M., Fang, H., Tsuruoka, T., Tsuji, T., Sasaki, T., and Hakomori, S. (1993) J. Biol. Chem. 268, 2217-2222), and cell adhesion mediated by alpha 4 beta 1 (to FN) or alpha 6 beta 1 (to laminin) is inhibited by modifying N-glycosylation processing of the integrin receptor (e.g. Akiyama, S. K., Yamada, S. S., and Yamada, K. M. (1989) J. Biol. Chem. 264, 18011-18018). We therefore studied the specific role of N-glycosylation in alpha 5 beta 1 function. Key findings of the present study were as follows. (i) Adhesion of K562 cells to FN-coated plates, which is mediated solely by alpha 5 beta 1, was inhibited when cells were treated with a mixture of endo-N-acetylglucosaminidase F and peptide -N4-(N-acetylglucosaminyl)asparagine amidase F (endo-F/PNGase-F). (ii) The alpha 5 beta 1 receptor at the K562 cell surface tended to dissociate into alpha 5 and beta 1 subunits when an extract of cells treated with endo-F/PNGase-F was precipitated by integrin subunit-specific antibodies, i.e. the alpha 5 subunit was preferentially precipitated by anti-alpha 5 monoclonal antibody ZH5, and the beta 1 subunit was preferentially precipitated by anti-beta 1 monoclonal antibody ZH1. When intact cells were extracted and treated with either ZH5 or ZH1, both alpha 5 and beta 1 were coprecipitated, indicating that the two subunits are normally tightly associated with each other. (iii) Adhesion of alpha 5 beta 1-containing liposomes (phosphatidylcholine:cholesterol liposomes incorporating purified alpha 5 beta 1) to FN-coated plates was abolished by treatment of liposomes with endo-F/PNGase-F. Liposomes incorporating alpha 5 beta 1 pretreated with endo-F/PNGase-F also did not bind to FN. When purified alpha 5 beta 1 receptor was treated with endo-F/PNGase-F followed by ZH5 or ZH1, the alpha 5 or beta 1 subunit was precipitated separately, respectively. In contrast, both subunits were always coprecipitated when intact purified alpha 5 beta 1 receptor was directly treated with ZH5 or ZH1. These findings indicate that N-glycosylation of both the alpha and beta subunits of the alpha 5 beta 1 integrin receptor is essential for association of these subunits and for optimal binding to FN.

Rous sarcoma virus-transformed cells develop peculiar adhesive structures along the cell periphery.

Alteration of the cell/substratum adhesive structures of rat fibroblasts (3Y1 cells) upon transformation by Rous sarcoma virus (RSV) was investigated by immunofluorescence microscopy. In serum-containing culture medium, 3Y1 cells developed focal adhesions as their main adhesive structures, while BY1 cells expressed peculiar close contacts along the cell periphery with the vitronectin receptor integrin, in addition to podosomes. These peripheral close contacts are referred to as the peripheral adhesions. The peripheral adhesions were observed as a darker region than podosomes by interference reflection microscopy. They were more easily destroyed by incubating the cells with RGD-containing peptide than were the focal adhesions. In contrast to focal adhesions and podosomes, actin bundles were not detected within the peripheral adhesions, where pp60v-src and tyrosine-phosphorylated proteins accumulated. Expression of the integrin was determined by the substratum composition when BY1 cells were cultured in serum-free culture medium. Under such conditions, BY1 cells expressed the peripheral adhesions within 3 hours on adhesion molecule-coated glass. On the other hand, in serum-containing medium, they first developed focal adhesions transiently at their early stage of adhesion, and then the peripheral adhesions were predominantly expressed within 12 hours. Podosomes were formed in a time course similar to that of the peripheral adhesions. These findings suggest that the peripheral adhesion is a class of stable adhesive structure distinct from the focal adhesion or podosome of BY1 cells. Similar close contact-type peripheral adhesions with the integrin were also observed in a variety of cultured cells such as normal fibroblasts at their logarithmic growth phase, phorbol ester-treated fibroblasts, and several malignant tumor cells, with poorly organized focal adhesions and stress fibers. These findings further suggest that the peripheral adhesions may be widely involved in the adhesion of cells that inadequately develop stress fibers and focal adhesions.

Purification of a mycobacterial adhesin for fibronectin.

Previous studies have demonstrated that mycobacteria attach to fibronectin (FN). The attachment of mycobacteria to FN is considered to be biologically important in Mycobacterium bovis BCG therapy for superficial bladder cancer, initiation of delayed hypersensitivity to mycobacterial antigens, and the phagocytosis of mycobacteria by epithelial cells. Therefore, we purified the mycobacterial receptor for FN. Culture supernatants from 3-week cultures of Mycobacterium vaccae, which contained proteins that bound FN and inhibited the attachment of both M. vaccae and BCG to FN, were used as a source of receptor. Lyophilized M. vaccae supernatants were reconstituted in 0.02 M bis-Tris (pH 6.0) and applied sequentially to an ACA 54 gel filtration column and a DEAE-Sephacel anion-exchange column. A purified inhibitory protein of 55 kDa (p55) was obtained. The purified p55 protein was observed to bind to FN and to inhibit 125I-FN binding to viable BCG in a dose-dependent manner. Polyclonal and monoclonal antibodies to the protein were generated. The resulting polyclonal antiserum blotted a single protein band at 55 kDa in crude M. vaccae supernatants, cross-reacted with a 55-kDa BCG protein by Western blot (immunoblot), and recognized a 55-kDa band that was associated with the BCG cell wall, which is consistent with its function as a FN receptor. A monoclonal immunoglobulin M(lambda) was isolated from mice immunized with purified M. vaccae p55 protein that was not functional in Western blots but inhibited the attachment of viable BCG to FN. These studies demonstrate that a protein or antigenically related proteins with M(r)s of 55,000 function as FN receptors for at least two distinct mycobacteria.

Identification of integrin cell-substratum adhesion receptors on cultured rat bone cells.

The interactions of bone cells with their extracellular matrix is of major importance in bone development, repair, and disease. We examined the ability of rat calvarial bone cells to adhere to various matrix proteins and to define the role of integrin cell-substrate adhesion receptors in these interactions. Isolated newborn rat calvarial bone cells prelabeled with 3H-thymidine and plated on plastic wells that had been precoated with serial dilutions of various substrates showed typical dose-response adherence curves to fibronectin, fibrinogen, laminin, vitronectin, and collagen I and IV. Cell adherence to poly-D-lysine, a nonspecific cell adherent, was high at all substrate concentrations > 0.0001 micrograms/ml. A polyclonal anti-rat integrin antibody blocked cell adhesion to all substrates tested except poly-D-lysine. Isolated rat calvarial bone cells were surface labeled with 125I, extracted, and immunoprecipitated with polyclonal antibodies made against the rat integrin complex and peptides derived from the cytoplasmic domains of the alpha 2, alpha 3, and alpha 5 subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (nonreduced) identified four bands representing a mixture of integrins including the alpha 1 beta 1 laminin/collagen receptor, the alpha 5 beta 1 fibronectin receptor, and the alpha V beta 3 (or possibly alpha V beta 5) vitronectin receptor. These experiments show that bone cells adhere to a wide variety of extracellular matrix proteins via specific integrins. Increased knowledge about the regulation of these receptors and the mechanisms by which they transmit information to the cell will be important for a more complete understanding of bone physiology and pathophysiology.

Changes in cell attachment by RSV transformation in rat liver epithelial cells.

BRL, a non-malignant rat liver epithelial-like cell line, possessed the ability to adhere through fibronectin to a solid substrate. Oncogenical transformation of these BRL cells with RSV induced a significant decrease in the fibronectin molecules in the extracellular matrix and reduction in its ability to adhere to fibronectin. The alpha 5 and beta 1 subunits of integrin (fibronectin receptor) were quantitatively diminished during RSV transformation in BRL cells. These results suggest that adhesive reduction of BRL cells to a substrate by RSV transformation may be caused by a decrease in cell surface fibronectin and fibronectin receptor molecules.

The attachment of nematocytes from the primitive invertebrate Hydra to fibronectin is specific and RGD-dependent.

The transient attachment of cells to components of the extracellular matrix is an important step in the complex molecular mechanisms involved in amoeboid cell locomotion. We have analyzed the attachment of nematocytes from the freshwater cnidarian Hydra to fibronectin which is a constituent of the mesoglea, the extracellular matrix, of the polyps. The percentage of attaching cells increased gradually in a concentration-dependent manner and reached a plateau value at a fibronectin concentration of 50 micrograms/ml. Attachment was inhibited by exposure of the fibronectin-coated surfaces to antibodies against the cell binding domain of fibronectin or by incubating the cells with peptides containing the recognition sequence Arg-Gly-Asp (RGD) known from vertebrate cells. This, together with data obtained by affinity chromatography, indicates that RGD-dependent binding to fibronectin, mediated by a receptor which possibly belongs to the integrin family, already occurs in Hydra, a member of an evolutionary low invertebrate phylum.