Antimicrobial activity of octenidine against multidrug-resistant Gram-negative pathogens.

Multidrug-resistant (MR) Gram-negative (GN) pathogens pose a major and growing threat for healthcare systems, as therapy of infections is often limited due to the lack of available systemic antibiotics. Well-tolerated antiseptics, such as octenidine dihydrochloride (OCT), may be a very useful tool in infection control to reduce the dissemination of MRGN. This study aimed to investigate the bactericidal activity of OCT against international epidemic clones of MRGN. A set of five different species (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, and Pseudomonas aeruginosa) was studied to prove OCT efficacy without organic load, under "clean conditions" (0.3 g/L albumin) and under "dirty conditions" (3 g/L albumin + 3 mL/L defibrinated sheep blood), according to an official test norm (EN13727). We used five clonally unrelated isolates per species, including a susceptible wild-type strain, and four MRGN isolates, corresponding to either the 3MRGN or 4MRGN definition of multidrug resistance. A contact time of 1 min was fully effective for all isolates by using different OCT concentrations (0.01% and 0.05%), with a bacterial reduction factor of >5 log10 systematically observed. Growth kinetics were determined with two different wild-type strains (A. baumannii and K. pneumoniae), proving a time-dependent efficacy of OCT. These results highlight that OCT may be extremely useful to eradicate emerging highly resistant Gram-negative pathogens associated with nosocomial infections.

Adhesive properties of the beta 3 integrins: comparison of GP IIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells.

Glycoprotein IIb-IIIa (alpha IIb beta 3) and the vitronectin receptor (alpha v beta 3), two integrins that share the common beta 3 subunit, have been reported to function as promiscuous receptors for the RGD-containing adhesive proteins fibrinogen, vitronectin, fibronectin, von Willebrand factor, and thrombospondin. The present study was designed to establish a cell system for the expression of either GP IIb-IIIa or the vitronectin receptor in an otherwise identical cellular environment and to compare the adhesive properties of these two integrins with those of native GP IIb-IIIa and the vitronectin receptor constitutively expressed in HEL cells or platelets. M21 human melanoma cells lack GP IIb-IIIa and use the vitronectin receptor to attach to vitronectin, fibrinogen, fibronectin, and von Willebrand factor. To study the functional properties of GP IIb-IIIa in these cells, we transfected GP IIb into M21-L cells, a variant of M21 cells (Cheresh, D.A., and R.C. Spiro. 1987. J. Biol. Chem. 262:17703-17711), which lack the expression of functional alpha v and are therefore unable to attach to vitronectin, fibrinogen, and von Willebrand factor. Transfectants expressing GP IIb were isolated by immunomagnetic beads and surface expression of the GP IIb-IIIa complex was documented by FACS analysis and immunoprecipitation experiments performed with 125I-labeled M21-L/GP IIb cells. Comparative functional studies demonstrated that GP IIb-IIIa expressed in M21-L/GPIIb cells as well as native GP IIb-IIIa constitutively expressed in HEL-5J20 cells (an HEL variant lacking alpha v beta 3) mediated cell attachment to immobilized fibrinogen, but not to vitronectin or von Willebrand factor, whereas the vitronectin receptor expressed in M21 cells and HEL-AD1 cells (an HEL variant expressing alpha v beta 3) mediated cell attachment to fibrinogen, vitronectin, and von Willebrand factor. Similarly, PGl2-treated resting platelets attached to immobilized fibrinogen but not to vitronectin or von Willebrand factor, and this attachment could be inhibited by mAb A2A9 (directed against a functional site on the GP IIb-IIIa complex). However, in contrast to platelets, which adhered to vitronectin and von Willebrand factor after stimulation by thrombin or PMA, activation of the protein kinase C pathway in M21-L/GP IIb or HEL cells did not induce cell adhesion to vitronectin or von Willebrand factor.(ABSTRACT TRUNCATED AT 400 WORDS)

The inoculum effect of Escherichia coli expressing mcr-1 or not on colistin activity in a murine model of peritonitis.

OBJECTIVES: Colistin often remains the last resort antibiotic active against carbapenemase-producing Enterobacteriaceae. However, while in vitro inoculum effect has been reported, therapeutic relevance of this phenomenon remains questioned. METHODS: Ten E. coli strains were used that included the wild-type CFT073 and its transconjugant CFT073-MCR-1 and eight susceptible clinical isolates. Mice with peritonitis were treated for 24 h with colistin sulfate. Bacterial loads were determined in peritoneal fluid (PF) and spleen and colistin-resistant mutants were detected. RESULTS: MICs of colistin against the eight susceptible clinical strains and CFT073 ranged from 0.125 to 0.5 mg/L with an inoculum of 105 CFU/mL and from 2 to 4 mg/L with a 107 CFU/mL inoculum; 5/9 strains with an MIC of 4 mg/L were considered resistant according to EUCAST breakpoint (resistance, > 2 mg/L). When the bacterial load of wild-type CFT073 inoculated in mice increased from 107 to 108 CFU: i) mean log10 CFU reduction generated by colistin in PF and spleen decreased from 5.8/mL and 3.1/g, respectively, (p < 0.01) to 0.9/mL and 0.8/g, respectively (NS); ii) mice survival rate decreased from 15/15 (100%) to 6/15 (40%) (p = 0.017); and iii) proportion of mice with selection of colistin-resistant mutants increased from 4/15 to 15/15 (p < 0.01). These results were comparable to those obtained when peritonitis was produced with a 107 CFU bacterial load of E. coli CFT073 expressing mcr-1, for which the mean log10 CFU reductions were 3.5/mL and 0.6/g in PF and spleen, respectively (NS), and survival rate was 8/15 (53%) (p < 0.01 versus survival of mice infected with wild-type CFT073). CONCLUSIONS: Phenotypic colistin resistance in wild-type E. coli due to an increase in inoculum size had a therapeutic impact in mice with peritonitis that was comparable to that observed when the mcr-1 gene was expressed.

Membrane-bound hemagglutinin mediates antibody and complement-dependent lysis of influenza virus-treated human platelets in autologous serum.

Influenza A virus-treated human platelets were lyzed in autologous serum. Lysis required the presence of antibody and occurred predominantly through activation of the classical complement pathway. Binding of the virus followed by its elution at 37 degrees C resulted in a dose-dependent desialation of the cells with a maximal release of 45% of total platelet sialic acid. In contrast, platelets that had been treated with Vibrio cholerae neuraminidase and from which 55% of total sialic acid had been removed were not lyzed in autologous serum and did not bind C3 as shown in binding assays using radiolabeled monoclonal anti-C3 antibody. Thus, the immune-mediated lysis of virus-treated platelets in autologous serum did not involve neoantigens expressed by desialated cells. To assess the effect of viruses on the platelet surface, treated platelets were incubated with galactose oxidase and sodium [3H]borohydride prior to separation and analysis of the labeled glycoproteins by SDS-PAGE. Viral treatment resulted in a desialation of each of the surface glycoproteins. At the same time, a labeled component of Mr 72,000 (nonreduced) and Mr 55,000 (reduced) was observed that was not present when V. cholerae-desialated platelets were examined in the same way. Immunoblotting experiments performed using antiwhole virus and anti-hemagglutinin antibodies demonstrated this component to be viral hemagglutinin. Involvement of membrane-bound hemagglutinin in antibody and in complement-mediated lysis of virus-treated platelets in autologous serum was supported by the increased lytic activity of a postvaccinal serum containing an elevated titer of complement fixing anti-hemagglutinin antibodies. Binding of a viral protein to the platelet surface provides a model for immune thrombocytopenias occurring during acute viral infections at the time of the specific immune response.

Platelet surface glycoproteins. Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery.

The accurate definition of surface glycoprotein abnormalities in circulating platelets may provide better understanding of bleeding and thrombotic disorders. Platelet surface glycoproteins were measured on intact platelets in whole blood and platelet membrane microparticles were assayed in cell-free plasma using 125I-monoclonal antibodies. The glycoproteins (GP) studied were: GP Ib and GP IIb-IIIa, two of the major intrinsic plasma membrane glycoproteins; GMP-140, an alpha-granule membrane glycoprotein that becomes exposed on the platelet surface following secretion; and thrombospondin (TSP), an alpha-granule secreted glycoprotein that rebinds to the platelet surface. Thrombin-induced secretion in normal platelets caused the appearance of GMP-140 and TSP on the platelet surface, increased exposure of GP IIb-IIIa, and decreased antibody binding to GP Ib. Patients with adult respiratory distress syndrome had an increased concentration of GMP-140 and TSP on the surface of their platelets, demonstrating in vivo platelet secretion, but had no increase of platelet microparticles in their plasma. In contrast, patients after cardiac surgery with cardiopulmonary bypass demonstrated changes consistent with membrane fragmentation without secretion: a decreased platelet surface concentration of GP Ib and GP IIb with no increase of GMP-140 and TSP, and an increased plasma concentration of platelet membrane microparticles. These methods will help to define acquired abnormalities of platelet surface glycoproteins.

Surface modifications in the platelets of a patient with alpha-N-acetyl-D-galactosamine residues, the Tn-syndrome.

The Tn-syndrome is an acquired disorder characterized by the polyagglutination of blood cells and the pathological exposure of alpha-N-acetyl-D-galactosamine residues (Tn-antigen) at the cell surface. We now report studies on the platelet of a patient (Ba.) of which 81% reacted positively with a fluorescein conjugate of Helix pomatia agglutinin (HPA). The surface proteins of Ba. platelets were labeled with 125I by the lactoperoxidase-catalyzed procedure; single and two-dimensional electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels was followed by autoradiography that revealed normal 125I-labeling of the major membrane glycoproteins (GP) but that GP Ib had a faster than normal migration. the abnormal GP Ib of Ba. platelets was strongly labeled when platelet suspensions were treated sequentially with neuraminidase, galactose oxidase, and sodium [3H]borohydride. Unlike the GP Ib of normal human platelets, it was also strongly labeled when Ba. platelets were treated with galactose oxidase and sodium [3H]borohydride alone. Both the alloantigen, PlA1, and quinidine-dependent antibody receptor activity were normally expressed by Ba. platelets, which also bound a monoclonal antibody (AN51) to GP Ib. Analysis of Ba. platelets by crossed immunoelectrophoresis using a rabbit anti-human platelet antibody preparation revealed the presence of an immunoprecipitate in the GP Ib position that had an abnormal appearance and migration in the second dimension. An altered position of the precipitate given by Factor VIIIR:Ag was also noted. Incorporation of HPA into the agarose gel during the first dimension electrophoresis resulted in the specific precipitation of the abnormal GP Ib of Ba. platelets. Our studies show that circulating Tn-platelets contain GP Ib with a modified oligosaccharide chain structure responsible for the platelet expression of Tn-antigen activity.

CHO cells expressing the high affinity alpha(IIb)beta3 T562N integrin demonstrate enhanced adhesion under shear.

BACKGROUND: Alpha(IIb)beta3-mediated platelet adhesive interactions in the vasculature, which are dependent on the functional state of this receptor, may be sensitive to shear forces. OBJECTIVES: To evaluate the influence of the alpha(IIb)beta3 affinity state on cell attachment under flow, we compared Chinese hamster ovary cells expressing the low affinity alpha(IIb)beta3 wild-type (wt) receptor to those expressing the high affinity alpha(IIb)beta3 T562N receptor. MATERIALS AND METHODS: We designed a real-time videomicroscopy adhesion assay for von Willebrand factor (VWF) or fibrinogen under flow conditions. RESULTS: At 50 s(-1), alpha(IIb)beta3 T562N supported higher cell adhesion to fibrinogen (63.3 +/- 2.9 cells/field) than alpha(IIb)beta3 wt (38.7 +/- 2.4 cells/field, P < 0.0001). At 100 s(-1), alpha(IIb)beta3 T562N mediated cell adhesion (40.5 +/- 3.8 cells/field), while alpha(IIb)beta3 wt did not (5.3 +/- 1.4 cells/field, P < 0.001), allowing to discriminate the efficiency of each receptor. Similar findings were observed for adhesion to VWF. Complete inhibition of cell adhesion to fibrinogen was achieved with 800 microM fibrinogen gamma-chain dodecapeptide [HHLGGAKQAGDV (H12)], while Arg-Gly-Asp-Ser (RGDS) peptide (10-1000 microM) induced a dose-dependent cell detachment. These results suggest that the H12 motif allows initial attachment, in contrast to the RGDS site, which strengthens the stability of adhesion. Interestingly, compared with wt, a 10-fold lower concentration of RGDS was required to reach a similar reduction of cell adhesion mediated by alpha(IIb)beta3 T562N. CONCLUSIONS: Our data show that alpha(IIb)beta3 activation is associated with a stabilization of integrin binding to fibrinogen or VWF under shear.

New hematopoietic differentiation antigens detected by anti-K562 monoclonal antibodies.

Following immunizations of BALB/c mice with K562 cells, we have obtained seven original monoclonal antibodies (MoAbs): (a) One MoAb, GA3, defines an antigen essentially restricted to the red cell series. This antigen is expressed on immature erythroblasts but is not detectable on the surface of early and late erythroid progenitors. GA3 MoAb immunoprecipitates a Mr 105,000 glycoprotein on K562 cells. (b) Two MoAbs, 14B6 and 12B1, react with cells of the monocytic series. MoAb 14B6, which also faintly stains platelets, is reactive with immature myeloid cells and the majority of hematopoietic progenitors. The 14B6 antigen has been immunoprecipitated from 12-O-tetradecanoylphorbol-13-acetate treated K562 cells as a Mr 130,000-100,000 protein. Antigen 12B1 is expressed only on cultured monocyte/macrophages and is restricted to a subpopulation of monocytes and to follicular dendritic cells. It is not detected on hematopoietic progenitors. Immunoprecipitation experiments performed on 12-O-tetradecanoylphorbol-13-acetate treated K562 cells revealed a glycoprotein with a molecular weight of 93,000-86,000. (c) Two anti-K562 MoAbs, CF4 and HE10, recognize a myeloid differentiation antigen expressed from the granulomonocytic colony forming unit stage to polymorphonuclear neutrophils. These MoAbs detect an apparently original glycolipid moiety distinct from LeX. (d) Two MoAbs recognize antigens expressed on the granulomonocytic series. 2E1 recognizes the monocyte low affinity Fc receptor (Mr 40,000) and defines a new cluster of myeloid differentiation (CDw32). The antigen is expressed on a small portion of immature hematopoietic progenitors. 8F5 identifies a Mr 95,000 protein which is also present on plasma cells. In some experiments, it is detected on erythroid colony forming unit analysis. Immunizations with K562 cells thus resulted in the production of antibodies recognizing antigens of the monocytic, granulocytic, as well as erythroid series. However, three of them are also detected on hematopoietic progenitors.

Cloning of an isoform of integrin-linked kinase (ILK) that is upregulated in HT-144 melanoma cells following TGF-beta1 stimulation.

We have shown previously that integrin-linked kinase (ILK) is upregulated in human HT-144 melanoma cells following TGF-beta1 stimulation. Using mRNA from TGF-beta1 stimulated HT-144 cells and reverse transcriptase polymerase chain reaction, we have isolated a cDNA encoding a protein highly homologous to ILK. Sequencing of the full-length 1359 base pair cDNA and polypeptide translation revealed that this protein, designated ILK-2, differs from the known ILK (hereafter called ILK-1) by only four amino acids, while the cDNA sequence diverges by 102 nucleotides, thus excluding that ILK-2 is an allelic variant of ILK-1. Expression of ILK-2 mRNA was observed in metastatic human HT-144 melanoma and HT-1080 fibrosarcoma cell lines, but not in normal human tissues. Moreover, stimulation of HT-144 cells with TGF-beta1, but not with EGF, PDGF-AB or insulin, induced a selective overexpression of ILK-2 mRNA as compared to ILK-1 mRNA. Bacterially-expressed GST/ILK-2 autophosphorylated and labeled myelin basic protein as well as a recombinant GST/beta3 integrin cytoplasmic tail peptide. Transfection of either ILK-2 or ILK-1 cDNA into the non-metastatic melanoma cell line SK-Mel-2, expressing exclusively ILK-1, induced anchorage independent cell growth and cell proliferation, as demonstrated by growth in soft agar. Our data provide evidence that ILK-2 is a new isoform of ILK-1 that is expressed in some highly invasive tumor cell lines but not in normal adult human tissues and whose expression is regulated by TGF-beta1.

Identification of platelet membrane thrombospondin binding molecules using an anti-thrombospondin antibody.

A rat monoclonal IgG2a antibody, 5G11, was raised against native human platelet thrombospondin (TSP). Western blot analysis revealed that 5G11 bound (i) to TSP before and after disulfide reduction, and (ii) to a 15-kDa fragment released after prolonged trypsin digestion. Crossed immunoelectrophoresis confirmed that the binding epitope was expressed in the presence of Ca2+ and after treatment of TSP with EDTA. Since 5G11 had no effect on platelet aggregation, the antibody was used to immunoprecipitate Ca2+-dependent and Ca2+-independent TSP-binding molecules on the surface of thrombin-activated surface-labeled 125I-platelets. The experimental basis was that ligand-receptor interactions are of high affinity and that anti-ligand antibodies should precipitate the ligand-receptor complex. With platelets activated in the presence of EDTA, 5G11 predominantly precipitated a 125I-labeled band of Mr 88,000, identified as glycoprotein (GP) IV. In contrast, in the presence of 2 mM Ca2+ and 1 mM Mg2+, 5G11 precipitated a complex of five radiolabeled proteins, among which GPIIb, GPIIIa and GPIV were the most prominent.

Monoclonal antibodies specific for human platelet membrane glycoproteins bind to monocytes by focal absorption of platelet membrane fragments: an ultrastructural immunogold study.

The membrane labeling of monocytes by monoclonal antibodies directed against platelet glycoproteins Ib (AN51), IIb (Tab), IIIa (C17), IIb-IIIa complex (J15) and to antigens common to platelets and monocytes (anti-monocyte platelet antigen and FA6 152) has been investigated by an ultrastructural immunogold method. Only with FA6 152, which identifies a structure shared by erythroblasts, platelets, and monocytes, was labeling obtained on membranes of both platelets and monocytes from normal blood. With all the other monoclonal antibodies, platelets were highly labeled but monocytes lacked quantitatively significant label; however, focal microparticles which exhibited gold particles were adherent to the membrane of monocytes. This localized labeling, interpreted as resulting from the fragmentation of platelet membranes during monocyte isolation with adhesion of the fragments to monocyte surfaces, was verified by two approaches. First, double staining with C17 visualized by an anti-IgG coupled to 40 nm gold particles and MO2 recognizing exclusively a surface monocyte antigen, as visualized by an anti-IgG coupled to 15 nm gold particles, was performed. The absence of colocalization of large and small gold particles either on monocytes or on microparticles confirmed the exclusive cell origin. Second, when analyzed by quantitative x-ray analysis, monocyte associated gold following C17 treatment was restricted to platelet pseudopods and fragments on whole mount spread cells. Finally, when monocytes were spread immediately after blood collection in the absence of sedimentation and centrifugation to prevent platelet activation, platelet rosetting was avoided and the number of microparticles markedly decreased. Thus, the attachment to monocyte membranes of microparticles originating from platelets may be confused with true labeling of monocytes by antibodies to platelet glycoproteins if analysis is limited to immunofluorescence.

Expression of platelet glycoproteins by erythroid blasts in four cases of trisomy 21.

In four patients with trisomy 21 (three constitutional, one acquired) with a morphological undifferentiated leukemia, diagnosis of erythroid leukemia was established by both immunophenotyping and ultrastructural studies. Indeed, a majority of blasts from three patients expressed several erythroid markers such as carbonic anhydrase 1, spectrin beta chain, and glycophorin A. In addition, band 3 and hemoglobin were immunologically detected in a fraction of the blast cells from two cases. At ultrastructural level, a majority or all blast cells exhibited erythroid differentiation features such as theta granules and ferritin molecules. However, platelet glycoproteins GP Ib, GP IIb, and GP IIIa were also immunologically detected in a fraction (from 14-82%) of the blasts. Since the ultrastructural study indicated that some promegakaryoblasts were also present in three patients, double labeling between erythroid markers (glycophorin A or carbonic anhydrase I) and platelet glycoprotein (Ib or IIIa) was performed and showed a clear overlap between the two kinds of markers. A similar approach was performed at ultrastructural level and indicated that blast cells with ultrastructural erythroid features of differentiation may have three distinct phenotypes, i.e., presence of glycophorin A without platelet glycoproteins or, conversely, the presence of platelet glycoproteins without glycophorin A and coexpression of glycophorin A and platelet glycoproteins. Expression of glycophorin A correlated directly with the differentiation level of the erythroid blasts, whereas platelet glycoproteins were essentially expressed in the more primitive leukemic erythroid cells. The GP Ib synthesized by these blasts was subsequently studied. The GP Ib alpha mRNA analyzed by Northern blot from these erythroid cells was identical in size with that from megakaryocytic cells as was the molecular weight of the GP Ib molecule from both after immunoprecipitation by a monoclonal antibody. Therefore, "in vivo" erythroid leukemic cells may express the main platelet glycoproteins including GP Ib.

Human megakaryocytopoiesis: in vitro regulation and characterization of megakaryocytic precursor cells by differentiation markers.

Understanding of human megakaryopoiesis has been improved by in vitro culture techniques, characterization of platelet proteins as differentiation markers and megakaryocyte purification. A continuum of megakaryocytic cells ranging from the CFU-MK (cell capable of proliferation) to the platelet has been demonstrated. CFU-MK is a heterogeneous population of cells which do not express platelet proteins other than platelet GP IIb and IIIa which may be present in low concentration. The main platelet proteins are synthesized later during differentiation of a 2N cell, just before the polyploidization process. Several homogeneous growth factors GM-CSF, interleukin 3 and erythropoietin are able to sustain human megakaryocyte colony formation. However, no specific MK-CSF has yet been purified. Purification of factors active on late stages of differentiation has proved difficult when using in vivo techniques to test their activity. However, recently such a factor has been purified to homogeneity by testing its action on the biosynthesis of PF4 by megakaryocytes. A negative regulation of megakaryopoiesis has been suggested by the inhibition of MK-colony formation by platelet products especially TGF-beta. A better understanding of human megakaryopoiesis may be of importance as it may allow modifications of platelet production in a clinical setting.

The 5' flanking region and chromosomal localization of the gene encoding human platelet membrane glycoprotein Ib alpha.

The human blood platelet membrane glycoprotein Ib (GPIb) functions as a receptor for von Willebrand factor and thrombin. The gene (gpIb alpha) encoding the GPIb alpha-chain was cloned from a genomic cosmid library. The promoter region of this gene was characterized by sequencing two BamHI fragments including 2.8 kb of the 5' flanking region where several Alu repeated elements and purine-rich sequences were found. Possible cis-regulatory elements were identified by comparing the gpIb alpha gene with established consensus sequences known to function as binding sites for transcription factors. To obtain further information on possible megakaryocyte-specific promoter or enhancer sequences, the gpIb alpha promoter region was compared with other genes expressed in platelets that are known so far. The gpIb alpha gene was found to be located on chromosome 17 in region 17p12-ter, by in situ hybridization.

alphaIIb beta 3-integrin mediated adhesion of human platelets to a fibrinogen matrix triggers phospholipase C activation and phosphatidylinositol 3',4'-biphosphate accumulation.

This study focused on the variations in phosphoinositide metabolism depending upon alphaIIbbeta3-integrin/fibrinogen interaction without previous activation of platelet agonist receptors. We found that adhesion of resting human platelets to immobilized fibrinogen stimulates phosphatidic acid production and a concomitant decrease in phosphatidylinositol 4',5'-bisphosphate. These results, and the absence of a transphosphatidylation reaction, argue in favor of the activation of a phospholipase C. Moreover, we observed the accumulation of phosphatidylinositol 3',4'-bisphosphate in adherent platelets as a consequence of the activation of a phosphatidylinositol 3-kinase. This effect was inhibited by ADP scavengers. Our results demonstrate that in adherent platelets, whereas phosphatidylinositol 3-kinase activation is controlled by both alphaIIbbeta-integrin engagement and released ADP, phospholipase C stimulation is triggered only by alphaIIbbeta-integrin/fibrinogen interaction.

Evidence from site-directed mutagenesis that the cytoplasmic domain of the beta3 subunit influences the conformational state of the alphaVbeta3 integrin ectodomain.

In order to explore the mechanisms leading to conformational changes of the vitronectin receptor alphavbeta3 following ligand or divalent cation binding, we have investigated the expression of epitopes known as ligand-induced binding sites (LIBS) on beta3 cytoplasmic tail mutants expressed in CHO cells. Truncation of the entire beta3 cytoplasmic domain induced constitutive LIBS exposure on alphavbeta3 and alphaIIbeta3. Deletion of the C-terminal NITY759 sequence or disruption of the NPLY747 motif by a Y747A substitution impaired extracellular conformational changes on alphavbeta3 following RGDS, echistatin or Mn2+ binding, whereas the substitutions Y747F, Y759A or Y759F allowed normal LIBS exposure. Furthermore, metabolic energy depletion totally prevented Mn2+-dependent LIBS exposure, but had only a minor effect on RGDS-induced conformational changes. Our results demonstrate that the structural integrity of the NPLY747 motif in the beta3 cytoplasmic domain, rather than potential phosphorylation of Tyr747 or Tyr759, is a prerequisite for conformational changes within the alphavbeta3 ectodomain, and suggest that two different mechanisms are responsible for RGDS- and Mn2+-dependent conformational changes.

A naturally occurring point mutation in the beta3 integrin MIDAS-like domain affects differently alphavbeta3 and alphaIIIbbeta3 receptor function.

We have investigated the effect of a new Leu196Pro mutation, identified in the MIDAS-like domain of the beta3 integrin subunit in a patient with type II Glanzmann thrombasthenia, on beta3 integrin receptor function. Expression of the mutant beta3Pro196 subunit in CHO cells, either associated with recombinant human alphaIIb or alphav, resulted in normal biosynthesis of beta3 and heterodimerization with alphav or alphaIIb, but selectively interfered with alphaIIbbeta3 maturation and transport to the cell surface. Functional analysis of the beta3 mutant receptors revealed strong inhibition of alphavbeta3-mediated cell spreading on immobilized fibrinogen, focal contact formation, p125FAK phosphorylation and fibrin clot retraction, as opposed to normal alphaIIbbeta3-mediated cell interaction with immobilized fibrinogen, focal contact translocation and signaling. In contrast, antibody- or DTT-activated mutant aIIbbeta3 was unable to bind soluble fibrinogen or the ligand mimetic PAC-1 monoclonal antibody, but underwent a conformational change following RGD peptide binding as demonstrated by AP5-LIBS epitope expression. These results suggest that (1) the highly conserved TL196T motif in the beta3 integrin subunit is located in a domain structurally important for the exposure of a functional binding site for soluble fibrinogen; and (2) that the MIDAS-like contact site in beta3 is not involved in alphaIIbbeta3-mediated cell adhesion to immobilized fibrinogen, while it is essential for alphavbeta3-mediated interaction with this ligand.

Male pseudohermaphroditism of the testicular feminizing type in a horse.

Features characteristic of the hereditary syndrome of testicular feminization (tfm) were observed in a 7-year-old Quarter Horse. The horse had female body habitus and male psychosexual behaviour. Gonads located in the abdomen were testes and the uterus and cervix were absent. The vagina was normal in depth but ended as a blind pocket. The sex chromosome composition of testicular fibroblast and leucocyte cultures was XY. Construction of a family pedigree revealed a pattern of hereditary transmission similar to that reported for tfm in other mammalian species.

[Schizophrenia: association of the paranoid form and antigens HLA-A9 and B5]. / Schizophrénie: association de la forme paranoïde aux antigènes HLA-A9 et B5.

30 unrelated Schizophrenic patients (15 Hebephrenic and 15 Paranoïd forms) were Typed for 14 A and 16 B Specificities by microlymphocytotoxicity. We note: 10 (5H + 5P) HLA--A1 (33.3 % / controls 23.4 %) 5 (2H + 3P) HLA--A3 (16.6 % / controls 26.2 %) 6 (5H + 1P) HLA--B8, 0 HLA--B27, 0 HLA BW16 and more specially: 10 (3H + 7P) HLA--A9 (23 + 24) 8 (2H + 6P) HLA--B5 These two last antigens are more often found in the Paranoïd patients of whom 46.6% are A9 Tcontrols: 24 %) and 40 % are B5 (controls 13.2 %. The Pc values are not significant in these too short seria but the strongly increased frequencies of A9 and B5 in the Paranoïd group seem to open a new biological aspect in the nosographic distinction of the Schizophrenia and so confirm previous results of C. L. CAZZULLO et al.