Human xenoreactive natural antibodies of the IgM isotype activate pig endothelial cells

Abstract: Preformed, xenoreactive natural antibodies (XNA) and complement (C) are involved in the initiation of vascular rejection of organs transplanted between discordant species, presumably by stimulating donor organ endothelial cells (EC). Although C is known to play a role in the activation of EC, it has not been clear whether the antibodies serve only to anchor the initial components of C, and thus permit the C cascade to proceed, or whether the antibodies themselves deliver a signal to the EC. We have tested affinity-purified human IgM containing XNA (IgM-XNA) for its ability to stimulate in vitro the up-regulation of genes in pig EC. Northern blot analysis shows that IgM, which contains XNA, stimulates mRNA accumulation for certain genes (including IL-8, PAI-1, and ECI-7, a new gene that we have found is associated with EC activation), but not others known to be up-regulated in response to TNF, IL-1 or LPS. Our results show that XNA provide a signal to EC, and thus may themselves participate in activation of EC and consequent vascular rejection.     

REACTION OF COMPLEMENT WITH ENDOTHEUAL CELLS IN A MODEL OF XEN0TRANSPLANTATION

We review our studies on the role of complement (C) as mediator of xenograft hyperacute rejection using an in vitro model consisting of porcine endothelial cells as target and human serum as source of natural antibodies and C. Cytotoxicity of endothelial cells required IgM antibodies to porcine endothelial cells, and the classical pathway and membrane attack complex of C. These findings correlated with in vivo results of porcine organs transplanted into rhesus monkeys, which showed a) co-deposition of IgM, C3, C4 and C9, along blood vessels of rejecting organs, with trace deposits of factors B or P, and b) minimal deposition of IgM and C components in transplants with prolonged survival that were performed in rtiesus monkeys depleted of natural antibodies but with normal C levels. Human serum causes activation of porcine endothelial cells manifested by release of heparan sulfate proteogiycan. Heparan sulfate release was induced by C5a alone. A new approach to avert xenograft hyperacute rejection was tested. To inhibit cytotoxicity of porcine endothelial cells by human C, the membrane-associated C inhibitor decay-accelerating factor (DAF) of human origin was incorporated into endothelial cells. Human DAF was able to efficiently inhibit C-mediated killing of porcine endothelial cells, suggesting that the use of DAF and other C inhibitors could be used to interfere with C-mediated xenograft hyperacute rejection.     

Mutation analysis of Jewish hunter patients in Israel

We have performed molecular and mutation analyses on 14 unrelated Israeli Hunter families and have identified the IDS mutation in 8 of them. Three unrelated Ashkenazi patients had the same previously reported mutation (1246 C→T). Based on the haplotypes of the mutation-bearing chromosomes, we concluded that this is a recurrent mutation. In two patients, we identified a deletion spanning exons V–VII. Three novel mutations were observed in different patients: L410P, 717del4, and 244del3. In addition, the silent mutation (562 C→T) was observed in one patient. © 1994 Wiley-Liss, Inc.     

Protective genes expressed in endothelial cells of second hamster heart transplants to rats carrying an accommodated first graft

Abstract: Accommodation refers to survival of a xenograft despite the presence of anti-donor organ antibodies and complement. We have recently shown that accommodation of a hamster heart transplanted to a rat receiving short-term cobra venom factor (CVF) and continuing cyclosporine A (CyA) therapy is associated with i) the expression in the endothelial cells (EC) and smooth muscle cells of the graft of a number of "protective" genes, ii) a prominent intragraft Th2 cytokine profile, and iii) the relatively heavy deposition of IgG2c antibodies on the EC of the graft. In contrast, rejecting grafts do not express the protective genes, have a Th1 cytokine profile, and apparently have lesser amounts of IgG2c. These findings are consistent with host factors (Th2 cytokines and IgG2c) contributing to xenograft accommodation. To test whether these host factors may predispose to the development of accommodation, we placed a second hamster heart into each of 12 rats carrying a surviving first heart; recipients were, at the time, receiving only CyA. Whereas first grafts transplanted to rats receiving only CyA survive for 3 to 4 days, 11 out of 12 second transplants survived more than 20 days, and the other survived for 7 days. Nine of the twelve were not rejected: of these, four were removed between day 35 and 132 for study, and the remainder are still beating at 35 to 52 days. The surviving second hearts we studied had accommodated in that the picture on immunopathology was the same as for surviving first hearts. We suggest that the Th2 cytokines and perhaps the IgG2c response are factors in allowing prolonged survival of the second grafts and, further, that these factors contribute to the expression in the EC and smooth muscle cells of the surviving second hearts of the protective genes.     

Selective cytotoxicity associated with in vitro exposure of fresh rat renal fragments and continuous cell lines to atractyloside

The consumption of plants containing the diterpenoid atractyloside (ATR) causes selective proximal tubule injury, renal failure and death in humans. We have compared the effects of ATR in freshly isolated renal proximal tubules and glomeruli from rat and also in cell lines: NRK, derived from the proximal tubules, and MDBK and MDCK more closely representing the distal nephron. The effects of ATR (10– 500?μM) on proximal tubules and glomeruli were assessed by changes in lipid peroxidation, de novo protein synthesis and the leakage of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), glutamate dehydrogenase (GDH) and N-acetyl-β-D-glucosaminidase (NAG). The susceptibility of NRK, MDBK and MDCK cell lines to ATR was assessed by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, measuring mitochondrial reduction. Enzyme leakage was the most sensitive of the markers of cell injury in fresh fragments and ranked LDH>GDH>ALP>NAG in proximal tubules. As little as 20?μM ATR caused significant enzyme leakage from proximal tubules, but there were no increases in enzyme leakage from glomeruli at concentrations ?500?μM ATR. De novo protein synthesis was only inhibited 50% at ATR concentration >5?mM in the proximal tubules, but there were no effects in glomeruli. Malondialdehyde production was significantly elevated at 1?mM ATR for proximal tubules, and 500?μM for glomeruli. NRK cells were sensitive to ATR (IC₅₀, 120?μM), but MDBK or MDCK cells were unaffected by?1?mM of this diterpenoid. Both freshly isolated fragments and continuous cell lines representing the proximal tubules are more sensitive to ATR than either glomeruli or cells representing the distal nephron. These data also show that protein synthesis is a less specific and sensitive measure of ATR cytotoxicity than enzyme leakage in fragments. MTT reduction to formazan was the most sensitive in the NRK cell line. The low levels of lipid peroxidation products in proximal tubular fragments or sensitive renal cell lines at toxic levels of ATR suggest that oxidative injury is not a key mechanism.