Minor H antigen matches and mismatches are equally distributed among recipients with or without complications after HLA identical sibling renal transplantation.

Studies of the effect of minor H antigen mismatching on the outcome of renal transplantation are scarce and concern mainly single center studies. The International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 16 laboratories of the IHIW, the role of 15 autosomal, 10 Y-chromosome encoded minor H antigens and 3 CD31 polymorphisms, was investigated in relation to the incidence of renal graft rejection and graft loss in 444 human leukocyte antigens (HLA)-identical sibling renal transplantations. Recipient and donor DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, C19Orf48, LB-ECGF-1, CTSH, LRH-1, LB-ADIR and HY. The correlation between minor H antigen mismatch and the primary outcome graft rejection or graft loss was statistically analyzed. The incidence of rejection was very low and no correlation was observed between one or more minor H antigen mismatch(es) and a rejection episode (n = 36), of which only eight resulted in graft loss. In summary, in our study cohort of 444 renal transplants, mismatching for neither autosomal nor HY minor H antigens correlate with rejection episodes or with graft loss.

Outcome of patients with preformed donor-specific antibodies following alemtuzumab induction and tacrolimus monotherapy.

It has been shown that low-level preformed donor-specific antibodies (DSAbs) detected by luminex beads in the setting of a negative CDC and flow cytometry crossmatch (CDC/FCXM) are associated with inferior allograft outcomes. The relevance of preformed DSAbs in patients receiving alemtuzumab induction and tacrolimus monotherapy has not been studied. Four hundred and eighty renal transplant recipients with a negative CDC/FCXM had their pretransplant sera retrospectively screened for DSAbs. 45/480 (9.4%) of patients were found to have preformed DSAbs. Females and patients receiving regrafts were more likely to have a DSAb (p = 0.008 and p < 0.0001, respectively). Patients with DSAbs had inferior allograft survival (p = 0.047), increased incidence of antibody-mediated rejection (p < 0.0001) and inferior allograft function at 6 months posttransplant (p = 0.017). Patients with HLA class I DSAb (alone or in combination with a Class II DSAb) with high mean fluorescence intensities (MFIs) were at highest risk. We conclude that patients with preformed DSAb are at high risk of adverse outcomes when receiving a minimal immunosuppressive regime incorporating alemtuzumab induction. Patients found to have a preformed DSAb despite a negative crossmatch might benefit from augmented immunosuppression.

Interpreting regulatory authority guidance on immunosuppressive therapy for renal transplantation: a response to the UK's National Institute for Clinical Excellence (NICE).

AIMS: A group of UK consultant transplant physicians and surgeons (the Consensus Group) met to consider the implications and interpretation of the National Institute for Clinical Excellence's (NICE) Technology Appraisal No. 85 on the use of immunosuppressive therapy for renal transplantation in adults. METHODS: This group considered what the implications of these guidelines might be for clinical practice and consensus was developed on those areas which were potentially open to different interpretations. A wider survey of nephrologists and transplant surgeons throughout the UK was also performed to gauge the impact of the NICE recommendations. RESULTS AND CONCLUSIONS: The outcome of the discussions of the Consensus Group are presented with particular reference to the recommendations of how to respond to calcineurin inhibitor (CNI) intolerance. The survey suggested that the publication of this NICE guidance has resulted in relatively few changes in prescribing practice: UK transplant centers continue to use a wide range of locally developed protocols for immunosuppressive therapy. These include the use of agents such as mycophenolate mofetil (MMF) and sirolimus, despite the fact that both drugs appeared to receive only conditional acceptance in the NICE Guidelines.

Proteolysis of the endothelial cell protein C receptor by neutrophil proteinase 3.

BACKGROUND: The endothelial cell protein C receptor (EPCR) presents protein C to the thrombin:thrombomodulin complex on the endothelium of large vessels, and enhances the generation of activated protein C (APC) and activation of protease-activated receptor-1. A previous report has demonstrated binding of soluble (s) EPCR to activated neutrophils via surface proteinase 3 (PR3). METHODS: We now report further characterization of this interaction. Activated neutrophils and purified PR3 both decrease endothelial cell (EC) surface EPCR, suggestive of its proteolysis. RESULTS: When added to purified recombinant sEPCR, PR3 produced multiple cleavages, with early products including 20 kDa N-terminal and C-terminal (after Lys(176)) fragments. The binding of active site blocked PR3 to sEPCR was studied by surface plasmon resonance. Estimates of the K(D) of 18.5-102 nM were obtained with heterogeneous binding, suggestive of more than a single interaction site. CONCLUSIONS: This work demonstrates PR3 binding to and proteolysis of EPCR and suggests a mechanism by which anticoagulant and cell protective pathways can be down-regulated during inflammation.

Splicing by overlap extension by PCR using asymmetric amplification: an improved technique for the generation of hybrid proteins of immunological interest.

Major histocompatibility complex (MHC) proteins play a central role in the immune recognition of antigen. The generation of hybrid MHC molecules has been of great value in elucidating the structure: function relationships of these key glycoproteins. In this report, the generation of cDNAs coding for seven such hybrid proteins is described. We have used the technique of splicing by overlap extension by the polymerase chain reaction (SOE by PCR) [Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. and Pease, L.R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61-68] to generate intermediate products of each of the components of the hybrid, tipped with a small sequence of the other, and then mixed these products in a second-stage PCR to produce the final spliced product. Where we were unable to generate final product, we introduced an additional step of asymmetric PCR synthesis to generate an excess of those strands which would anneal in the final PCR and found this to be effective. We noted a significant but manageable mutation rate, possibly contributed to by the tendency of DNA polymerase to add additional non-templated nucleotides [Hu, G. (1993) DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3' end of a DNA fragment. DNA Cell Biol. 12, 763-770]. To avoid this, we modified our protocol to include a stage of blunting our intermediate products with T4 DNA polymerase prior to mixing them in the final PCR. We present this system as an effective mechanism to splice DNA.

Transfected murine cells expressing HLA class II can be used to generate alloreactive human T cell clones.

Alloreactive human T cells are conventionally generated in vitro using peripheral blood mononuclear cells (PBMCs). The disadvantage of such an approach is that PBMCs express multiple HLA class II molecules and, as a consequence, it is difficult to generate T cells specific for an individual HLA alloantigen. This paper describes a technique in which T cell clones can be generated using stimulators which do express only one alloantigen. This has permitted the generation of HLA-DR-specific T cell clones and will be applied to produce T cell clones specific for other isotypes which cannot easily be obtained using other techniques. Murine DAP.3 cells were transfected with cDNAs encoding human class II molecules and used to stimulate primary alloresponses by purified human CD4+ T cells. The cloning of these T cells provided a good yield of cells allospecific for the class II molecule expressed by the transfected cells. A large percentage of the T cell clones were able to recognise human cells, suggesting that specificity for DR-bound peptides of mouse origin does not limit the applicability of this approach. Despite having been raised against mouse stimulators cells, the responses of the T cell clones to alloantigen-expressing human B cell lines were profoundly inhibited by anti-human LFA-3 monoclonal antibody. The possible mechanisms responsible for these results are discussed.

Function of porcine adhesion molecules in a human marrow microenvironment.

BACKGROUND: One way to circumvent the need for chronic immunosuppression in solid organ xenografting may be to induce donor-specific tolerance using bone marrow transplantation. If this approach is to succeed in the pig-to-human species combination, pig marrow must be capable of maturing into relevant tolerance-inducing cells and replenishing itself in host human marrow. One possible barrier is adhesion molecule incompatibility. We have studied the compatibility across the pig-human species barrier of two well-characterized ligands known to be important in hematopoiesis, CD44 and very late antigen (VLA)-4. METHODS: In vitro long-term bone marrow cultures were studied in which the effects of blocking antibodies were assessed by measuring cell numbers and colony-forming units. RESULTS: The blocking of CD44 had a comparable inhibitory effect on the hematopoiesis of human and pig marrow, even if the latter was maintained on a human stromal layer. Both cellular proliferation and colony-forming activity were inhibited by anti-CD44 monoclonal antibody. By contrast, a significant difference was observed in VLA-4 usage by hematopoietic cells of the two species. Blocking VLA-4 markedly inhibited human hematopoietic cellular proliferation but had no effect on pig hematopoiesis, on either porcine or human stroma. CONCLUSIONS: The data suggest that the incompatibility of either CD44 or VLA-4 is unlikely to limit the efficiency of porcine hematopoiesis in a human marrow environment. However, the difference in VLA-4 utilization between these species raises the possibility that other interactions may be important for effective porcine hematopoiesis and that their failure to function between species may contribute to the poor function of porcine hematopoietic cells in primate marrow microenvironments.

Cross-species compatibility of intercellular adhesion molecule-1 (CD54) with its ligands.

BACKGROUND: The molecular interactions of intercellular adhesion molecule-1 (ICAM-1; CD54) are potentially important in several situations in the context of pig-to-human xenotransplantation. If porcine bone marrow is to be used for the induction of xenograft tolerance in humans, the role that has been suggested for ICAM-1 in the interactions of haematopoietic stem cells makes its cross-species compatibility important. Similarly, the potential role of ICAM-1 interactions in graft rejection makes it an important molecule to study. METHODS: An in vitro static cell-to-cell adhesion study was used to look at the successful interaction of ICAM-1 with its ligands across the pig-human species barrier in both directions. A second in vitro system, the standard long-term bone marrow culture (LT-BMC), was used to study the functional role of ICAM-1 in haematopoiesis. RESULTS: Human ICAM-1 was able to adhere to ligands on porcine cells, including one or more ligand that contains CD18. Conversely, human CD18-containing ligands mediated adherence to porcine cells. Using the long-term bone marrow culture system, there was no evidence that blocking the interactions of ICAM-1 inhibited hematopoiesis, either in the human-human or pig-human combinations of precursor cells and marrow stroma. CONCLUSIONS: ICAM-1 is able to interact with at least some of its ligands across the species barrier, in both pig-human and human-pig combinations. However, the interactions of ICAM-1 do not appear to be central to hematopoiesis, at least in the model system used.

Cross-species interaction of porcine and human integrins with their respective ligands: implications for xenogeneic tolerance induction.

BACKGROUND: Organ transplantation is limited by the number of available donors. One possible solution would be the use of pigs as organ donors. However, current immunosuppressive protocols cannot prevent rejection of these organs. If donor-specific tolerance toward porcine antigens could be induced in recipients, subsequent implantation of porcine organs would be possible without further immunosuppression. Induction of tolerance can be achieved with a bone marrow transplant if donor antigen-presenting cells successfully differentiate in the recipient thymus to induce deletion of donor-reactive host cells. Migration of porcine progenitor cells to the host marrow and thymus and differentiation into tolerance-inducing antigen-presenting cells is likely to require successful interaction of porcine adhesion molecules with human ligands. In this study, we investigated whether very late antigen (VLA)4 and VLA-6 integrins, which play important roles in homing and differentiation of hematopoietic progenitor cells, function across the pig-to-human species barrier. METHODS: Static cell-to-cell and cell-to-extracellular matrix protein adhesion assays were used to examine the cross-species interaction of porcine adhesion molecules with human ligands. RESULTS: Our studies show that porcine cells adhere to various human endothelial cell monolayers and extracellular matrix proteins and demonstrate that porcine VLA-4 and VLA-6 appear to be fully cross-reactive to the human ligands vascular cell adhesion molecule-1 and laminin, respectively. CONCLUSIONS: It is likely that porcine hematopoietic progenitor cells will be able to successfully employ pVLA-4- and pVLA-6-human ligand interactions in a pig-to-human bone marrow transplantation model in order to induce donor-specific tolerance.

Presentation and recognition of major and minor histocompatibility antigens.

The structural basis of allorecognition is heterogeneous. For minor histocompatibility antigen-specific T cells and for a minority of anti-MHC T cells (indirect allorecognition), the allogeneic molecule acts as any other protein, and is processed and presented as a peptide in the context of self MHC. In circumstances where the MHC molecule is recognized unprocessed on the surface of the allogeneic cell, we have postulated that the factors important in recognition are determined by the relationship between the responder and stimulator MHC molecules. When the alloresponse is directed against an allogeneic molecule whose exposed surface closely resembles that of the responder homologue, the alloresponse can be regarded as resulting from mimicry of self MHC-restricted recognition of peptides which are differentially bound by responder and stimulator MHC molecules. When the alloantigen differs extensively in the MHC restriction-determining region of the molecule from the equivalent product in the responder, a chance high-affinity cross-reaction with the foreign MHC structure itself may be the most important mechanism.

Rejection mechanisms in transplantation.

While major improvements have been made in the prevention and treatment of hyperacute and acute transplant rejection, most grafts will succumb to chronic rejection: this reflects the extent of our knowledge of the mechanisms that drive these processes. Clinically, transplant rejection is classified according to timeframe and histology into hyperacute (minutes to hours), acute (days to months) and chronic rejection (months to years). Hyperacute and acute rejection are reasonably well understood and occur by immune mediated events whereas chronic rejection probably has immune and non-immune components. The trigger to cell-mediated rejection is allorecognition, where same-species, non-self antigens are detected by the host immune system. This occurs by two distinct mechanisms, called the direct and indirect pathways. The direct pathway results from the recognition of foreign major histocompatibility molecules, intact, on the surface of donor cells. Indirect allorecognition occurs when donor histocompatibility molecules are internalised, processed, and presented as peptides by host antigen presenting cells. Animal and human studies strongly suggest that acute rejection is predominantly triggered by the direct pathway although if the latter is blocked then the indirect pathway can suffice. Donor antigen presenting cells within the graft become depleted with time and the frequency of T cells reactive to the direct pathway diminishes irrespective of whether or not chronic rejection occurs. This implies that the direct pathway is unlikely to contribute to chronic rejection. Assays of T cell responses have, however, found an association between the indirect pathway and chronic rejection although it is unlikely that this is the whole story: there are numerous non-immunological risk factors for chronic rejection which probably interact with immune components causing gradual graft failure. Xenotransplantation, where tissue is transferred across species, causes rejection by processes analogous to those seen in allografts but they are faster and more vigorous. Novel approaches have overcome some early antibody mediated rejection events but then reveal a huge, intense, adaptive cellular response. We believe that by the careful study of the mechanisms of rejection, the problems of chronic rejection and xenograft rejection will be overcome, thus reversing the widening gap between organ demand and supply.

Thoracic outlet compression syndrome: the lack of reliability of its clinical assessment.

Four manoeuvres advocated for the assessment of thoracic outlet compression were performed on 64 randomly chosen volunteers. Although only 17% had any symptoms of thoracic outlet compression, 58% had a positive result in at least one of the manoeuvres. Only 2% were positive for more than two manoeuvres. We suggest this low specificity devalues these tests in clinical practice. We were unable to correlate our clinical results with the findings of digital photoplethysmography.