Falsely incompatible B-cell flow cytometry crossmatch after pronase treatment: a case report.

This report presents a falsely incompatible B cell crossmatch by flow cytometry in a lung transplant recipient. The patient was a 35-year-old Caucasian male with end-stage lung disease secondary to cystic fibrosis whose pretransplantation serologic workup did not disclose the presence of anti-HLA class II antibodies by single antigen bead testing. Unexpectedly, crossmatch of recipient sera with pronase-treated donor lymphocytes resulted in antibody binding to B cells only. The positive reactivity was reproducible in pronase-treated autologous B cells. Recipient sera did not react with nontreated donor or autologous lymphocytes. Herein, we describe our approach to this unexpected crossmatch result and consider the implications of false-positive crossmatch results on transplantation.

A randomized, double-blind, placebo-controlled, multicenter study of rabbit ATG in the prophylaxis of acute rejection in lung transplantation.

ATG-Fresenius S (ATG-F) is a polyclonal anti-human-T-lymphocyte immunoglobulin preparation that has been clinically developed to prevent episodes of acute cellular rejection. This study evaluated the efficacy and safety of ATG-F at doses of 5 and 9 mg/kg versus placebo in adult recipients of a primary lung allograft. The primary efficacy composite end point was defined as death, graft loss, acute rejection and/or loss to follow-up within 12 months of transplantation. The interim analysis showed the ATG-F 5 mg/kg treatment to be inefficacious, and it would be impossible to enroll enough patients to power the study to show a difference between the 9 mg/kg arm and the placebo arm. Therefore, the main focus of the study shifted to the safety end points and a descriptive analysis of the primary end point. At 12 months posttransplant, the efficacy failure rate was not significantly different between the ATG-F 9 mg/kg group and the placebo group (40.2% vs. 36.7%, respectively). This large study did not demonstrate a significant reduction in acute cellular rejection, graft loss or death with single-dose induction therapy with ATG-F within the first year after lung transplantation.

Screening by tRNA primer extension analysis of porcine kidney mRNA libraries defines a novel endogenous porcine retroviral long terminal repeat.

BACKGROUND: We have taken advantage of the common requirement of all eukaryotic retroelements for a specific tRNA primer to initiate DNA synthesis and applied a previously described in vitro screening methodology to the analysis of in vivo porcine tissues for transcriptionally active retroviral sequences. METHODS: A series of 18-base pair (bp) 3' tRNA oligomers complementary to established primer binding sites for a variety of vertebrate retroviruses, retrotransposons, and retroposons were applied to primer extension analysis of kidney poly(A) mRNA. Primer extension products are predicted to represent "strong stop" signals characteristic of the initial stages of retroviral transcription. RESULTS: Several extension products were cloned, sequenced, and analyzed as probes for screening the porcine genome for potentially active retroviral sequences. We used this strategy to identify and clone a 655-bp 5' long terminal repeat of a porcine retrovirus with significant homology to the simian sarcoma virus. This transcriptionally active virus has an 82-bp U5 region, a conserved AATAAA polyadenylation sequence, a 39-bp repeat reminiscent of other retroviral enhancers, and a unique glycine primer binding site. CONCLUSION: Our results suggest that tRNA primer cloning can effectively identify novel retroviral elements.

The role of antibodies in acute vascular rejection of pig-to-baboon cardiac transplants.

Long-term success in xenotransplantation is currently hampered by acute vascular rejection. The inciting cause of acute vascular rejection is not yet known; however, a variety of observations suggest that the humoral immune response of the recipient against the donor may be involved in the pathogenesis of this process. Using a pig-to-baboon heterotopic cardiac transplant model, we examined the role of antibodies in the development of acute vascular rejection. After transplantation into baboons, hearts from transgenic pigs expressing human decay-accelerating factor and CD59 underwent acute vascular rejection leading to graft failure within 5 d; the histology was characterized by endothelial injury and fibrin thrombi. Hearts from the transgenic pigs transplanted into baboons whose circulating antibodies were depleted using antiimmunoglobulin columns (Therasorb, Unterschleisshein, Germany) did not undergo acute vascular rejection in five of six cases. Biopsies from the xenotransplants in Ig-depleted baboons revealed little or no IgM or IgG, and no histologic evidence of acute vascular rejection in the five cases. Complement activity in the baboons was within the normal range during the period of xenograft survival. In one case, acute vascular rejection of a xenotransplant occurred in a baboon in which the level of antidonor antibody rose after Ig depletion was discontinued. This study provides evidence that antibodies play a significant role in the pathogenesis of acute vascular rejection, and suggests that acute vascular rejection might be prevented or treated by therapies aimed at the humoral immune response to porcine antigens.

The role of natural anti-Gal alpha 1-3Gal antibodies in hyperacute rejection of pig-to-baboon cardiac xenotransplants.

Xenoreactive natural antibodies in humans and higher primates are directed predominantly at Gal alpha 1-3Gal. These antibodies are thought to initiate hyperacute rejection of porcine organ xenografts. The contribution of anti-Gal alpha 1-3Gal antibodies to the xenoractive natural antibody repertoire and to the initiation of hyperacute rejection was tested in a pig-to-baboon cardiac xenograft model. Anti-Gal alpha 1-3Gal antibodies were depleted from baboons by extracorporeal absorption of anti-Gal alpha 1-3Gal antibodies from plasma using columns with a matrix bearing Gal alpha 1-3Galb1-4GlcNAc. Specific removal of anti-Gal alpha 1-3Gal antibodies was achieved prior to transplantation as demonstrated by immunoassay. Porcine hearts were then transplanted into these baboons and the outcome of the transplants was analysed. Immunofluorescence revealed little deposition of baboon antibodies in the grafts. The porcine hearts did not undergo hyperacute rejection even though complement activity was approximately 90% of baseline at the time of transplantation. These findings demonstrate that anti-Gal alpha 1-3Gal antibodies constitute a major fraction of xenoreactive natural antibodies in primate blood and that these antibodies contribute significantly to the pathogenesis of hyperacute xenograft rejection.

A molecular epidemiological probe for pig microchimerism.

We have cloned and characterized a single-copy DNA sequence from the porcine alpha-1,3-galactosyltransferase gene that corresponds to a 547-base pair intron separating exons 3 and 4 of the protein coding domain. Polymerase chain reaction amplification of this sequence from flanking oligonucleotides generates a species-specific DNA probe (pgt34) capable of recognizing 50 pg chimeric template DNA at a pig to human cellular ratio of 1/10,000. Homologous DNA sequence is not identified in the macaque, baboon, or human genome by Southern hybridization. Analysis of a discordant model of pig to baboon xenotransplantation demonstrates peripheral blood microchimerism in the presence of a functioning pig kidney xenograft and persistence of microchimerism in lymphatic tissue after graft removal. This probe should be useful for tracking the fate of porcine cells in patients undergoing xenotransplantation of whole organs or free tissues such as pancreatic islet cells and should facilitate studies of microchimerism in experimental models of pig to monkey xenotransplantation.

Molecular strategies for clinical xenotransplantation in cardiothoracic surgery.

The current shortage of donors for allotransplantation has generated interest in the potential use of animal organs to meet increasing clinical transplant needs, ie, xenotransplantation. However, when phylogenetically distant species such as the pig are transplanted into unmodified primate hosts--discordant xenotransplantation--the grafts undergo a rapid rejection process characterized by edema, hemorrhage, and diffuse microvascular thrombosis. "Hyperacute rejection" as such is mediated by an IgM natural antibody directed against the galactose alpha(1,3) galactose epitope expressed on the endothelial cell surface of all mammals except old world monkeys, apes, and humans which collectively lack the galactosyltransferase enzyme necessary for antigen expression. Transplants between nonhuman primates and human recipients--concordant xenotransplantation--avoid hyperacute rejection, but nonetheless undergo "acute vascular rejection" and progressive microthrombotic injury. Acute vascular rejection is associated with endothelial cell "activation," loss of vascular integrity, and progressive thrombosis. Molecular strategies for avoiding xenograft rejection involve insertion of genes into the donor pig genome capable of modifying xenoreactive antigen expression and regulating antibody-mediated endothelial cell damage.

Mapping of the mouse Rxr loci encoding nuclear retinoid X receptors RXR alpha, RXR beta, and RXR gamma.

Recently, a novel subgroup of nuclear hormone receptors called RXRs implicated for retinoid-mediated gene regulation have been identified. RXRs appear to interact with many other nuclear hormone receptors and modulate their functions. We have mapped genetic loci Rxra, Rxrb, and Rxrg encoding three RXR subtypes, RXR alpha, RXR beta, and RXR gamma, respectively, using interspecific backcross mice. None of the Rxr loci cosegregated with each other or with the retinoic acid receptor loci (Rar) mapped previously. Rxra mapped to Chr 2 near the centromere, Rxrb mapped to the H-2 region of Chr 17, and Rxrg was tightly linked to the Pbx gene on distal Chr 1. These results underscore that RXR genes are dispersed in the genome.

Oral-facial clefts and associated malformations in the squirrel monkey (Saimiri sciureus).

Of the 414 squirrel monkey pregnancies recorded at this institution since 1977, seven (1.7%) have resulted in offspring with clefts of the lip and/or palate. Associated malformations include a ventricular septal defect, renal agenesis, anal atresia, axial skeletal anomalies, and craniorachischisis (anencephaly and spina bifida). Three of these infants are the result of consanguineous matings.