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Biomedical subjects

F H Bach

Publications and source records attributed to F H Bach.

At least 109 records · Page 6Linked to original sources
PubMed1996

Genetic engineering of endothelial cells to ameliorate xenograft rejection.

The prospect of clinical xenotransplantation using pigs as donors (a discordant combination) holds out a potential solution to the shortage of human organs, as well as potentially widening the spectrum of patients who might benefit from a transplant procedure. While generalized clinical trials of pig to human organ transplantation (of immediately vascularized organs, such as the heart or kidney) will probably not occur in the very near future, there are reasons for optimism that this approach, deemed impossible a few decades ago, may find its way to the bedside. This optimism is based on two factors: First, a great increase in our understanding of the probable underlying causes of rejection and, second, the development of a number of important therapeutic approaches, including genetic manipulation of the donor animal, to tackle the manifold problems inherent in rejection of such a transplanted organ.

Animals↗
PubMed1996

Loss of rat glomerular ATP diphosphohydrolase activity during reperfusion injury is associated with oxidative stress reactions.

Endothelial cell ATP diphosphohydrolases or ATPDases degrade extracellular inflammatory mediators ATP and ADP, thus inhibiting the formation of platelet thrombi, but the modulation of these ecto-enzymes during vascular injury remains largely undetermined. Renal glomerular ATPDase levels were determined in the rat following ischemia-reperfusion or systemic complement activation, by direct biochemical methods and histochemistry. Ischemia followed by reperfusion times over 30 min were associated with loss of glomerular ATPDase activity. Cobra Venom Factor (CVF) inhibited ATPDase activity and potentiated the deleterious effects of reperfusion. Treatment with either soluble complement receptor type 1 (sCR1), an inhibitor of complement activation, or antioxidants prior to the ischemia-reperfusion was largely protective. Expression of rat glomerular ATPDase activity appears susceptible to the inflammatory injury associated with systemic complement activation and ischemia/reperfusion processes. Oxidative stress could, at least in part, result in the loss of ATPDase activity and thus thrombotic consequences of vascular injury.

Adenosine Diphosphate↗
PubMed1995

Inhibition of NF-kappa B by pyrrolidine dithiocarbamate blocks endothelial cell activation.

Endothelial cell activation is achieved by the rapid, protein synthesis-independent induction of a characteristic set of genes. Because of the abundance of binding sites for the transcription factor NF-kappa B in the regulatory region of the aforementioned genes, we hypothesized that this factor might play a key role. Reactive oxygen intermediates act as second messengers in the activation of NF-kappa B. We have used the antioxidant pyrrolidine dithiocarbamate to analyze the effect of NF-kappa B inhibition on TNF alpha-induced EC activation in vitro. We show that pyrrolidine dithiocarbamate strongly reduces the TNF alpha-mediated induction of E-selectin, VCAM-1, ICAM-1, PAI-1, tissue factor, IL-8 and I kappa B-alpha. We present evidence identifying NF-kappa B as a central of EC activation. Therefore, this factor may represent a prime target for therapeutic intervention in pathologic conditions associated with EC activation such as allo- and xenograft rejection, atherosclerosis, ischemic reperfusion injury and vasculitis.

Animals↗
PubMed1995

Regulation of the tissue factor promoter in endothelial cells. Binding of NF kappa B-, AP-1-, and Sp1-like transcription factors.

Tissue factor is up-regulated on endothelial cells and monocytes in response to cytokines and endotoxin and is the main trigger of the extrinsic pathway of the coagulation cascade. We have isolated the porcine tissue factor gene and studied the regulation of the promoter, which has not been investigated previously in endothelial cells. Comparison of the promoter sequences with the respective human and murine genes reveals short stretches of homology, which encompass potential binding sites for AP-1, NF kappa B, and Sp1 transcription factors. Using DNase I footprinting, we detect binding of nuclear factors to these promoter elements. Transfection experiments demonstrate that a 300-base pair fragment containing the conserved elements can mediate induced transcription and that the NF kappa B-like element is essential. In accordance, electrophoretic mobility shift assays show a strong increase in the binding of factors to the NF kappa B-like site following induction. We further provide evidence that RelA (p65), c-Rel, and possibly novel polypeptides bind to the tissue factor NF kappa B element. In addition, we show constitutive binding of members of the Fos/Jun and Sp1 families to the AP-1 and Sp1 sites, respectively. We propose a concerted action of AP-1-, NF kappa B-, and Sp1-like factors in transcription from the tissue factor promoter in endothelial cells.

Animals↗
PubMed1995

Intron-exon structure of the porcine I kappa B alpha-encoding gene.

Genomic clones of ECI-6 (endothelial cell inducible), the porcine I kappa B alpha gene that encodes a cytoplasmic inhibitor of the transcription factor NF-kappa B, were isolated, spanning the entire transcribed region plus 2.1 and 0.35 kb of 5'- and 3'-flanking sequences, respectively. The gene contains five introns ranging in size from 0.6 to 0.1 kb. Four of the introns are located in the coding regions for four of the five ankyrin-like repeats in the central part of the I kappa B alpha protein at similar positions. The fifth intron is located in the C-terminal region. Southern blot analysis indicates the presence of a single copy of ECI-6/I kappa B alpha in the porcine genome.

Amino Acid Sequence↗
PubMed1995

NKG2-C is a receptor on human natural killer cells that recognizes structures on K562 target cells.

NKG2-C is a member of the recently discovered NKG2 family of genes and proteins, which are preferentially expressed on human natural killer (NK) cells. These potential NK cell receptors belong to a larger class of type II transmembrane proteins with a C-type lectin domain. We show here that NKG2-C is expressed as a 36-kDa glycoprotein by translation in vitro, recombinant expression and immunoprecipitation from a human NK cell clone. Further, a recombinant soluble NKG2-C-receptor binds specifically to K562 cells, which are target cells for NK cell killing, and to RPMI 8866 cells, which are feeder cells for NK cells; several other hematopoietic cell lines tested do not show any binding. The binding structures on the surface of K562 cells disappear, concomitant with a loss in susceptibility to killing when the cells are induced to differentiate with phorbol ester and Ca2+ ionophore. Our data suggest the presence of specific target molecules for NKG2-C on K562 cells, since overall glycosylation, Lewis X and Lewis Y structures, as well as the mucin-like CD43 molecule, do not change following induction of the cells. We propose that NKG2-C mediates a specific interaction of NK cells and their target cells with functional importance for NK cell killing.

Animals↗
PubMed1995

Role of endothelial cells in transplantation.

Endothelial cell activation with accompanying vascular inflammatory changes is considered central to the experimental manifestations of both hyperacute and delayed xenograft rejection responses. Natural xenoreactive antibodies directed at alpha-galactosyl residues of xenogeneic glycoproteins and glycolipids, with associated complement activation via the classical pathway, are considered major immediate mediators of graft endothelial cell injury in the clinically relevant discordant swine to primate combinations. In delayed xenograft rejection processes, where recipients are treated prophylactically to ameliorate these initial events, activation of infiltrating mononuclear phagocytes and natural killer cells are associated with ongoing endothelial cell activation processes, procoagulant generation and vascular thrombosis. Allograft hyperacute rejection is observed when vascularised organs are transplanted to sensitized individuals with high levels of cytotoxic antibodies. Less dramatic forms of humoral allograft rejection (termed accelerated or vascular rejection) and the more common cell-mediated endothelialitis are associated with significant graft damage. Endothelial cell activation is also linked with graft preservation injury, forms of chronic rejection and delayed graft loss. Experimental work is currently being directed at the control of hyperacute rejection, the close understanding of endothelial cell thromboregulation in both transplanted xeno- and allografts and the development of novel therapeutic agents including gene therapy and the possible use of organs from transgenic animals.

Cell Transplantation↗
PubMed1994

Activation of intragraft endothelial and mononuclear cells during discordant xenograft rejection.

Most studies of discordant xenograft rejection have focused on the roles of recipient xenoreactive antibody and complement as mediators of hyperacute rejection; there are essentially no data from in vivo studies as to the contribution of endothelial cell responses to the pathobiology of xenograft rejection. We hypothesized that the mechanism by which xenoreactive natural antibodies and complement of the recipient are involved in rejection of a discordant, immediately vascularized xenograft involves donor organ endothelial cell activation, with the consequences of such activation contributing significantly to the rejection process. We performed a kinetic analysis of rejection of guinea pig hearts by untreated Lewis rats or recipients depleted of complement activity that underwent delayed xenograft rejection. We report that in both hyperacute rejection and delayed xenograft rejection there is widespread evidence of endothelial cell activation, including expression of P-selectin and E-selectin, upregulation of tissue factor, and downregulation of thrombomodulin and antithrombin III expression. Many of these changes occur very early posttransplantation in grafts that are not completely rejected until approximately 3 days. In delayed xenograft rejection, an intense cellular infiltrate is seen that results from progressive accumulation of activated macrophages and natural killer cells. T cell receptor alpha/beta+T cells are present only at relatively low levels. This cellular infiltrate is associated with dense expression of pro-inflammatory cytokines, including interferon gamma, interleukin 1, and tumor necrosis factor-alpha. We conclude that both endothelial cell activation and infiltration by activated macrophages and natural killer cells may play an important role in xenograft rejection. These newly described features of the xenogeneic rejection response may require targeting by future therapeutic regimens aimed at prolonging xenograft survival.

Animals↗
PubMed1994

NK-kappa B subunit-specific regulation of the I kappa B alpha promoter.

Stimulation of endothelial cells by cytokines and bacterial lipopolysaccharide leads to activation of the transcription factor NF-kappa B. NF-kappa B in turn regulates the expression of several genes involved in the inflammatory reaction, including cell adhesion molecules, interleukins, and transcription factors. One of these induced genes encodes an inhibitor of NF-kappa B, ECI-6/I kappa B alpha, that contains in its 5' regulatory region six consensus binding sites for NF-kappa B. We demonstrate here that these sites display striking differences in their ability in vitro to bind to various NF-kappa B subunits. In vivo, all six sites contribute, though to varying degrees, to transcription from the ECI-6/I kappa B alpha promoter, as demonstrated by deletion and mutation analysis. Among the NF-kappa B subunits tested p65, the p65/p50 heterodimer and, to a lesser extent, c-Rel, are able to activate transcription, whereas p50 or p50/Re1B were inactive. Since many genes regulated by NF-kappa B contain only one or two DNA-binding sites for this transcription factor, the presence of six functional NF-kappa B-binding sites in the ECI-6/I kappa B alpha promoter represents a unique feature of this gene.

Amino Acid Sequence↗
PubMed1994

In vivo IgM depletion by anti-mu monoclonal antibody therapy. The role of IgM in hyperacute vascular rejection of discordant xenografts.

Xenoreactive natural antibodies (XNA) and complement activation are thought to be the 2 main factors responsible for the hyperacute vascular rejection (HVR) of discordant xenografts. The aim of this work was to study the role of IgM XNA in the HVR of guinea pig to rat cardiac xenografts, a discordant model. Adult LOU/C rats were depleted of circulating IgM and therefore of IgM XNA using an anti-mu mAb (mouse anti-rat IgM mAb 7 [MARM-7]). Rats were injected with 10 mg and 5 mg of MARM-7 at days -3 and -1, respectively, and guinea pig cardiac xenografts were performed on day 0. Control animals were injected on the same days with 10 mg and 5 mg of anti-alpha mAb (MARA-1) or equivalent volumes of PBS. Xenografts were performed on day 0. Guinea pig cardiac xenograft survival time was significantly prolonged in IgM-depleted animals (62 min, P < 0.01) compared with controls using PBS (18 min) or MARA-1 mAb (12 min). This prolongation was not due to a decrease in the complement activity in IgM-depleted rats, since no significant variation of the C1q, C4, C3, and C5 complement hemolytic activity was observed between control and treated animals before HVR. Prolongation of the xenograft survival time in the MARM-7-treated group was correlated with an undetectable serum level of IgM and IgM XNA and a lack of IgM XNA deposits on the rejected xenograft vascular endothelium. Contrarily, both IgM-depleted and control animals showed C3 deposits on the rejected xenograft vascular endothelium and myocardium, as well as diffuse deposits of IgG2a XNA. Although HVR was not abrogated by the depletion of IgM XNA, our data indicate that IgM is implicated in the HVR and that the anti-mu approach is a potential therapeutic treatment for discordant xenografts. Finally, we suggest that other factors such as IgM-independent activation of complement might be one of the mechanisms responsible for the pathogenesis of HVR in the guinea pig to rat xenograft model.

Animals↗