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F H Bach

Publications and source records attributed to F H Bach.

At least 55 records · Page 3Linked to original sources
PubMed1998

Alpha-galactosyl epitope-mediated activation of porcine aortic endothelial cells: type II activation.

BACKGROUND: Xenoreactive natural antibodies (XNAs) and complement mediate hyperacute rejection of discordant xenografts. Inhibition of complement alone results in some prolongation of graft survival, but delayed xenograft rejection still precludes long-term graft survival. In vitro data provide evidence for the direct proinflammatory activation of endothelial cells (ECs) by XNAs. These antibodies are primarily directed against galactose alpha(1-3)-galactose (alpha-gal), the major xenoantigen in the pig to primate xenotransplant model. Previous studies have shown EC activation by XNAs but failed to address the question of whether alpha-gal-specific ligands can induce EC activation. The aim of this study was to investigate whether agonist binding to the alpha-gal epitope by alpha-gal-specific lectins as compared with XNAs or elicited xenoreactive antibodies can directly elicit type II porcine aortic EC (PAEC) activation (i.e., activation that requires protein synthesis). METHODS AND RESULTS: The tetravalent, alpha-gal-binding Bandeiraea simplicifolia lectin I (BS-I), the wholly alpha-gal-specific BS-I isolectin B4, and elicited primate anti-pig xenoreactive antibodies (decomplemented cynomolgus monkey anti-porcine serum) induced E-selectin protein expression in PAECs. This induction was alpha-gal-specific, as preincubation with synthetic alpha-gal carbohydrate or adsorption of lectin or serum to rabbit, but not human, red blood cells removed the activating component. E-selectin expression, induced by BS-I, was inhibited in the presence of genistein, a tyrosine kinase inhibitor, and by mepacrine, an inhibitor of phospholipase A2. Human and primate XNAs lacked this activity when tested at relevant concentrations; however, stimulation of PAECs with affinity-purified human XNA (IgM and IgG) resulted in slightly increased interleukin-8 and P-selectin mRNA levels but had no apparent effects on E-selectin transcription. BS-I strongly induced E-selectin, P-selectin, intercellular adhesion molecule-1, and interleukin-8 mRNA in an NF-kappaB-dependent manner. CONCLUSIONS: Several agonists that specifically bind to alpha-gal can evoke type II EC activation. Hence, anti-Gal antibodies may contribute directly to xenograft rejection in the absence of complement activation.

Animals↗
PubMed1998

Complement-fixing elicited antibodies are a major component in the pathogenesis of xenograft rejection.

Hamster to rat cardiac xenografts undergo delayed rejection as compared with the hyperacute rejection of discordant xenografts. Elicited xenoreactive Abs (EXA) are thought to initiate hamster to rat cardiac xenograft rejection. In this study, we demonstrate that following transplantation of a hamster heart, rats generated high levels of EXA. Adoptive transfer into naive recipients of purified IgM, IgG2b, or IgG2c, but not IgG1 or IgG2a EXA, induced xenograft rejection in a complement-dependent manner. Ability of EXA to cause rejection correlated with complement activation, platelet aggregation, and P-selectin expression in the xenograft endothelium. Cyclosporin A (CyA) administration, after transplantation, totally suppressed IgG1, IgG2a, IgG2b, and IgG2c EXA, and inhibited IgM EXA production, but failed to overcome rejection. Administration of cobra venom factor (CVF), 1 day before and at the time of transplantation, resulted in complement inhibition during 3 days after transplantation, which failed to overcome rejection. Combination of CyA and CVF, which we have previously shown to overcome rejection, resulted in suppression of IgG EXA production and in the return of IgM XNA to preimmunization serum levels, 3 to 7 days after xenotransplantation, while complement remained inhibited. Thus, under CyA/CVF treatment, complement activation by hamster cells was suppressed following xenotransplantation, and presumably for this reason xenograft rejection did not occur. In conclusion, our data demonstrate that EXA play a pivotal role in the pathogenesis of xenograft rejection and that CyA and CVF suppress xenograft rejection by preventing exposure of xenograft endothelial cells to complement activation by EXA.

Adoptive Transfer↗
PubMed1998

A20 inhibits NF-kappaB activation in endothelial cells without sensitizing to tumor necrosis factor-mediated apoptosis.

Expression of the NF-kappaB-dependent gene A20 in endothelial cells (EC) inhibits tumor necrosis factor (TNF)-mediated apoptosis in the presence of cycloheximide and acts upstream of IkappaBalpha degradation to block activation of NF-kappaB. Although inhibition of NF-kappaB by IkappaBalpha renders cells susceptible to TNF-induced apoptosis, we show that when A20 and IkappaBalpha are coexpressed, the effect of A20 predominates in that EC are rescued from TNF-mediated apoptosis. These findings place A20 in the category of "protective" genes that are induced in response to inflammatory stimuli to protect EC from unfettered activation and from undergoing apoptosis even when NF-kappaB is blocked. From a therapeutic perspective, genetic engineering of EC to express an NF-kappaB inhibitor such as A20 offers the mean of achieving an anti-inflammatory effect without sensitizing the cells to TNF-mediated apoptosis.

Adenoviridae↗
PubMed1998

Alpha-galactosyl epitope-mediated activation of porcine aortic endothelial cells: type I activation.

BACKGROUND: The galactose alpha(1-3)galactose (alpha-gal) epitope associated with membrane glycoproteins and glycolipids represents a major determinant recognized on porcine cells by human xenoreactive natural antibodies (XNA). Together, bound XNA and complement rapidly induce porcine aortic endothelial cell (PAEC) activation; this process is associated with cellular shape changes, transient development of intercellular gaps, and loss of ATDPase and thrombomodulin, with release of heparan sulfate. The aim of this study was to evaluate patterns of type I endothelial cell activation (i.e., activation that does not require protein synthesis) following ligation of alpha-gal epitopes with anti-Gal antibodies and alpha-gal-specific lectins. METHODS AND RESULTS: PAEC incubated in the presence of the alpha-gal binding, Bandeiraea simplicifolia lectin (BS-I) underwent cellular shape changes associated with the formation of intercellular gaps. PAEC exposure to BS-I was also associated with the tyrosine phosphorylation of a protein (apparent molecular mass of approximately 130 kDa), not observed following lipopolysaccharide, tumor necrosis factor, or XNA stimulation. This lectin-induced tyrosine phosphorylation was not affected by cytochalasin D (inhibitor of actin filament polymerization), by genistein (inhibitor of tyrosine kinases), or by staurosporine (inhibitor of tyrosine phosphorylation and protein kinase C). In addition, incubation of PAEC with BS-I and monoclonal anti-Gal IgM induced p42/44 map kinase and activated the transcription factor NF-kappaB. CONCLUSIONS: Agonist binding of alpha-gal can evoke endothelial cell activation independently of complement activation. These observations have implications for the survival of xenografts.

Actins↗
PubMed1998

Expression of heme oxygenase-1 can determine cardiac xenograft survival.

The rejection of concordant xenografts, such as mouse-to-rat cardiac xenografts, is very similar to the delayed rejection of porcine-to-primate discordant xenografts. In concordant models, this type of rejection is prevented by brief complement inhibition by cobra venom factor (CVF) and sustained T-cell immunosuppression by cyclosporin A (CyA). Mouse hearts that survive indefinitely in rats treated with CVF plus CyA express the anti-inflammatory gene heme oxygenase-1 (HO-1) in their endothelial cells and smooth muscle cells. The anti-inflammatory properties of HO-1 are thought to rely on the ability of this enzyme to degrade heme and generate bilirubin, free iron and carbon monoxide. Bilirubin is a potent anti-oxidant, free iron upregulates the transcription of the cytoprotective gene, ferritin, and carbon monoxide is thought to be essential in regulating vascular relaxation in a manner similar to nitric oxide. We show here that the expression of the HO-1 gene is functionally associated with xenograft survival, and that rapid expression of HO-1 in cardiac xenografts can be essential to ensure long-term xenograft survival.

Animals↗
PubMed1998

Tyrosine phosphorylation following lectin mediated endothelial cell stimulation.

Terminal alpha (1,3) galactosyl galactoside epitopes (alpha-gal) on membrane glycoproteins expressed by vascular endothelial cells represent the major xenoreactive antigens in pig to primate xenotransplantation. In other discordant xenotransplantation combinations, such as from guinea pig to rat, carbohydrate epitopes other than alpha-gal may be targeted by xenoreactive antibodies (XNA). We have shown that agonist binding to alpha-gal epitopes induces proinflammatory activation of porcine aortic endothelial cells (PAEC). Binding of alpha-gal epitopes by Bandeiraea simplicifolia isolectin B4 results in both type I and type II PAEC activation. This includes the phosphorylation of tyrosine residue(s) of a protein with an apparent molecular weight of 130 kDa (p130). In order to investigate whether binding of other carbohydrate epitopes could induce a similar phosphorylation event, several lectins with different carbohydrate specificities were used to stimulate PAEC and human umbilical endothelial cells (HUVEC). In addition to BS-IB4 binding to alpha-gal, lectins binding to sialic acid isolated from Sambucus nigra (SNA), Maackia amurensis (MAA), Wheat germ agglutinin (WGA), and lectin from jack bean (Concanavalin A, ConA), that binds to mannose residues within the core structure of N-glycosylated proteins all induced the phosphorylation of the p130 protein(s). Lectins with affinity to alpha bound N-acetylgalactosamine, Dolichos biflorus (DOB), and Sophora japonoca (SOJ) did not induce this phosphorylation event. A similar negative result was obtained with Ulex europaeus lectin I, which binds to fucose residues. Conclusively, endothelial cell activation can be observed upon binding of various lectins to the glycosylated moiety of surface glycoproteins. These carbohydrate epitopes against which XNA may exist in certain models might represent minor xenoantigens from porcine to primates or may comprise the major xenoepitopes in other discordant xenograft models. Binding of XNA and subsequently the elicited xenoreactive antibodies to carbohydrate epitopes may therefore contribute to xenograft rejection even in the absence of complement inactivation.

Animals↗
PubMed1998

Xenotransplantation: problems and prospects.

Research in xenotransplantation has increased enormously in the last eight years. As the shortage of allogeneic organs has intensified, the possibility of using organs from pigs has become more attractive. Current data suggest that hyperacute rejection can be overcome in a clinically acceptable manner. However, additional likely rejection factors, probably related to endothelial cell activation, are being identified that likely lead to delayed xenograft rejection, a phenomenon that can occur in the absence of T lymphocytes. Reviewed here are various genetic engineering approaches that might help overcome these rejection factors, resulting eventually in a multi-transgene donor pig. Other concerns and current controversies in the field are also discussed.

Acute Disease↗
PubMed1998

Regulation of monocyte tissue factor activity by allogeneic and xenogeneic endothelial cells.

The regulation of tissue factor (TF) activity by the cell associated tissue factor pathway inhibitor (TFPI) during monocyte (Mo) and endothelial cell (EC) interactions is not fully understood. This report describes co-ordinate induction of TF antigen (TF-Ag) and membrane-associated TFPI-Ag on human Mo following coculture with human aortic (HAEC) or porcine aortic EC (PAEC) or after stimulation with TNFalpha. We show that both allo- and xenogeneic EC induce human Mo-TF antigen in short-term culture. However, the TF activity of TNFalpha-primed Mo is suppressed when these cells are cocultured with HAEC [by 40.3 +/- 6.3% (p<0.02)] or PAEC [by 50.5 +/- 10.6% (p<0.001)]. Antibody (Ab) blocking studies confirm that TFPI is the principal anticoagulant associated with this suppression of TF-activity. Our data indicate that anti-TF activity originates, at least in part, from the activated human Mo in the coculture; additionally, specific generation of TFPI by Mo is observed under the xenogeneic culture conditions. As Mo associated TF, induced by allo- or xenogeneic EC interactions, is regulated by cell-associated TFPI, we propose that infiltrating Mo may modulate the thrombotic process at sites of vascular injury in association with both allo- and xenograft rejection.

Animals↗
PubMed1997

Thrombin activates nuclear factor-kappaB and potentiates endothelial cell activation by TNF.

Thrombin is the central bioregulatory enzyme in hemostasis and is generated in vascular beds in which inflammatory responses are ongoing. In this study, we examined the effect of thrombin, both alone and in combination with TNF, on gene expression in porcine aortic endothelial cells (EC). Thrombin (1-10 U/ml) induced increased mRNA levels of E-selectin, monocyte chemoattractant protein-1, IL-8, plasminogen activator inhibitor-1, and IkappaB-alpha. These effects were mimicked by a thrombin receptor-activating peptide; preincubation of thrombin with hirudin blocked the induction of mRNA, suggesting that the increased gene expression was due to thrombin-specific activity. Because these genes are known to contain nuclear-factor-kappaB (NF-kappaB)-binding elements in their promoter region, we next examined the ability of thrombin to activate this transcription factor. As detected by electrophoretic mobility shift assay, thrombin (10 U/ml) or thrombin receptor-activating peptide (100 microM) stimulated increased NF-kappaB-binding activity. Supershift analysis revealed that these complexes were comprised principally of the RelA (p65) and NF-kappaB1 (p50) Rel family members. Thrombin alone did not substantively increase protein levels of E-selectin despite the increase in E-selectin mRNA levels. However, thrombin (3-10 U/ml) stimulated a 10-fold enhancement in the ability of TNF (0.3-1.0 ng/ml) to induce E-selectin surface expression. Similar potentiation of TNF-induced NF-kappaB activity and E-selectin transcription by thrombin was observed in experiments utilizing luciferase reporter constructs expressed in bovine aortic EC. The ability of thrombin to potentiate TNF-induced EC activation thus provides an important mechanism by which products of the coagulation cascade may enhance cytokine-mediated inflammatory responses.

Animals↗
PubMed1997

Xenogeneic endothelial cells activate human prothrombin.

BACKGROUND: Delayed xenograft rejection is characterized by platelet activation and fibrin deposition and is thought to occur independently of complement activation. We have therefore investigated the potential for xenogeneic endothelial cells (EC) to regulate the conversion of prothrombin to thrombin, a central component of the final common pathway of coagulation and an important platelet agonist. METHODS AND RESULTS: Quiescent porcine aortic EC (PAEC) were found to convert high levels of human prothrombin to thrombin (0.234+/-0.019 IU/ml) when compared with human aortic EC (0.017+/-0 IU/ml, 30-min time point, chromogenic assay; P<0.001). PAEC activation by human complement resulted in comparable levels of thrombin generation. Prothrombin conversion by PAEC as determined by generation of F1+2 (1.909+/-0.119 nmol/L) and formation of thrombin-antithrombin III complexes (125.611+/-6.373 microg/L) was significantly greater than the matched human aortic EC values (F1+2: 1.539+/-0.03 nmol/L, P<0.001; thrombin-antithrombin III: 1.833+/-0.104 microg/L, P<0.001). Sequential analysis of prothrombin activation by PAEC indicated generation of the intermediate meizothrombin followed by autolytically accelerated thrombin formation. Subsequent experiments established important cross-species' incompatibilities with respect to porcine thrombomodulin interaction with human thrombin and protein C in that PAEC had a reduced capacity to generate activated human protein C in vitro. CONCLUSION: These observations indicate a potentially important molecular barrier involving blood coagulation that may impact on the planned clinical application of porcine transgenic organs.

Animals↗
PubMed1997

Arachidonic acid influences proinflammatory gene induction by stabilizing the inhibitor-kappaBalpha/nuclear factor-kappaB (NF-kappaB) complex, thus suppressing the nuclear translocation of NF-kappaB.

Arachidonic acid (AA), through its myriad metabolites, is involved in inflammation in a number of ways. AA is produced and released by several cell types, including endothelial cells (EC), and acts on a variety of cells. EC activation plays a key role in inflammation presumably by modulating the immune response through up- or down-regulation of several genes. We have previously shown that AA and its nonmetabolizable analogue, 5,8,11,14-eicosatetraynoic acid (ETYA), inhibit up-regulation of proinflammatory genes in EC. In the present study we identify a mechanism to explain the inhibitory effects: AA and ETYA both inhibit the translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus by blocking the degradation of the inhibitor of NF-kappaB (IkappaB) and thus stabilizing the IkappaB/NF-kappaB complex. To investigate the mechanism whereby AA inhibits up-regulation of genes encoding proinflammatory mediators, we examined the ability of ETYA to inhibit tumor necrosis factor-alpha (TNF-alpha) mediated phosphorylation and degradation of IkappaBalpha. Western blot analysis revealed that preincubation of EC with ETYA for 40 min prior to stimulation with TNF-alpha inhibits the phosphorylation and degradation of IkappaBalpha. These findings establish a mechanism by which AA inhibits nuclear translocation of NF-kappaB and thereby explaining its modulatory role in the induction of proinflammatory genes.

Animals↗
PubMed1997

Effects of leflunomide and deoxyspergualin in the guinea pig-->rat cardiac model of delayed xenograft rejection: suppression of B cell and C-C chemokine responses but not induction of macrophage lectin.

BACKGROUND: If complement (C) activation is prevented or the host is C depleted, discordant vascularized xenografts undergo delayed xenograft rejection (DXR), characterized by graft infiltration by macrophages (MO) and natural killer (NK) cells, endothelial cell activation, and widespread fibrin deposition. Given a lack of effect of T cell-directed therapies on development of DXR, we evaluated two novel agents, 15-deoxyspergualin (DSG) and leflunomide (LEF), with reported anti-B-cell and/or anti-MO actions. METHODS: DSG and LEF were administered to C-depleted, splenectomized rat recipients of guinea pig cardiac xenografts, and their effects on graft survival and production of anti-guinea pig antibodies were determined. Serial intragraft events were studied by immunohistology using monoclonal antibodies to rat leukocytes, cytokines, and novel proteins, including rat MO lectin, which in other systems is important to MO binding, activation, and target cell killing. RESULTS: Median graft survival was 62 hr in cobra venom factor (CVF)-treated controls versus 108 hr (DSG), 129 hr (LEF), and 120 hr (DSG and LEF; all groups P<0.01 vs. CVF alone). LEF and DSG each decreased (immunoglobulin M [IgM]) or abrogated (IgG) posttransplant production of anti-guinea pig antibodies. Immunohistologic studies showed that each agent also inhibited graft infiltration by NK and T cells, and expression of various cytokines, including the chemokine monocyte chemoattractant protein-1 (MCP-1), but did not affect the tempo or extent of MO infiltration. Consistent with this, the rapid induction of MO lectin postxenografting, and induction of MO lectin by rat MO exposed to guinea pig cells in vitro, were unaffected by therapy with DSG and/or LEF. CONCLUSIONS: LEF or DSG along with CVF can result in the longest prolongation of xenograft survival yet reported in this model, in conjunction with a dampening of host mononuclear cell responses, including suppression of B cell activation. However, the persistent influx of MO in this model, despite lack of C-, Fc receptor- or apparent chemokine-dependent mechanisms, suggests the presence of additional mechanisms for cell recruitment and activation. It was of importance that, in this regard, although MO depletion is technically difficult and can lead to undesired effects, the demonstration of rapid MO lectin induction postxenografting indicates opportunities for blockade of MO recruitment and functions during DXR by use of anti-MO lectin monoclonal antibodies or administration of competing sugars.

Animals↗
PubMed1997

The effect of 5,8,11,14-eicosatetraynoic acid on endothelial cell gene expression.

The endothelium plays a key role in inflammation, hemostasis and organ rejection. We report here that a synthetic polyunsaturated fatty acid, 5,8,11,14-eicosatetraynoic acid (ETYA), selectively inhibits the up-regulation of several genes on endothelial cells. ETYA suppresses endothelial cell activation by inhibiting the up-regulation of adhesion molecules like E-selectin. A runoff assay for E-selectin demonstrated that the suppression is at the level of transcription. The fact that ETYA inhibits E-selectin upon stimulation with a diverse group of stimuli like lipopolysaccharide, tumor necrosis factor-alpha or phorbol 12-myristate 13-acetate, suggests that ETYA does not exert its effect by modifying membrane-bound receptors. The messenger RNA for interleukin-8 and glyceraldehyde phosphate dehydrogenase are not affected. Pre-treatment of endothelial cells with ETYA also prevents the adherence of monocytes to tumor necrosis factor-alpha-stimulated cells.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1997

Effect of porcine endothelial tissue factor pathway inhibitor on human coagulation factors.

BACKGROUND: Delayed xenograft rejection (DXR) is characterized by inflammation and vascular thrombosis. Activation of coagulation may occur as a result of tissue factor (TF) expression on both activated donor endothelial cells (EC) and recipient infiltrating monocytes (Mo). In addition, natural anticoagulants associated with porcine endothelial cells may not function adequately across species. METHODS: In the present study, we examined the interaction of the TF pathway of coagulation with the natural anticoagulant TF pathway inhibitor, in xenogeneic leukocyte-EC cultures in vitro, and during rejection of discordant xenografts in vivo. RESULTS: Coculture of human Mo with pig aortic EC (PAEC) resulted in 1.7-fold and 2-fold higher induction of Mo TF and Mo intercellular adhesion molecule-1, respectively, when compared with coculture with human aortic endothelial cells (HAEC). In addition, TF-dependent and -independent activation of coagulation factor X was higher on PAEC than on HAEC. Low levels of mRNA for tissue factor pathway inhibitor (TFPI) and its variant, TFPI-2, in resting PAEC were up-regulated by stimulation with tumor necrosis factor alpha. Procoagulant activity of recombinant human TF complexed to activated factor VII was inhibited by PAEC and HAEC-associated TFPI by 22% and 56%, respectively. In contrast, human activated factor X (factor Xa) activity was inhibited by human, but not porcine, EC-associated TFPI, suggesting functional incompatibility of PAEC for human factor Xa. Endothelial TFPI was detected in pig control organs and after hyperacute rejection, but was lost from the vasculature during DXR. CONCLUSIONS: Lack of appropriate human factor Xa inhibition by porcine EC during hyperacute rejection and loss of porcine EC TFPI during DXR could promote the development of a procoagulant environment leading to xenograft rejection.

Amino Acid Sequence↗
PubMed1997

Human monocytes activate porcine endothelial cells, resulting in increased E-selectin, interleukin-8, monocyte chemotactic protein-1, and plasminogen activator inhibitor-type-1 expression.

Monocytes (Mo) are thought to be important effector cells in early xenograft rejection. Effects of Mo-endothelial cell (EC) interactions on EC activation in vitro were studied by coculturing human Mo or human monocytoid cell lines, U937 and THP-1, with porcine EC. Without preactivation, U937 cells and Mo induced mRNA for the EC-specific adhesion receptor, E-selectin, expressed only on activated cells, after 2 hr. Surface protein was maximal when equal numbers of EC and Mo were cocultured. Increased mRNA expression of the chemokines, interleukin-8 and monocyte chemotactic protein-1, and the antifibrinolytic protein plasminogen activator inhibitor type-1, confirmed EC activation. Like E-selectin, plasminogen activator inhibitor type-1 mRNA was rapidly induced and returned to baseline after 24 hr, whereas chemokine gene expression was slower and more prolonged. Interleukin-1 receptor antagonist failed to modulate induction of E-selectin. Soluble tumor necrosis factor (TNF) alpha receptor inhibited E-selectin induced by TNF alpha, but not by U937 cells, and mRNA and protein on EC in Mo-EC mixtures cocultured at 1:1 ratios were not significantly reduced. The TNF alpha inhibitor did reduce E-selectin expression (30-40%), as well as induced chemokine gene expression (80%), at higher Mo-EC ratios. Despite this, minimal TNF alpha was detectable in supernatants. These results, along with the transwell experiments that confirmed a requirement for Mo-EC contact, suggest that membrane-bound TNF alpha may be involved. Thus, Mo-EC interactions in the porcine to human combination activated several EC functions, suggesting that initial Mo contact with the vessel wall of a xenogeneic graft may promote leukocyte recruitment, inflammation, and maintenance of thrombus, resulting in eventual organ destruction.

Animals↗