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Biomedical subjects

A N Warrens

Publications and source records attributed to A N Warrens.

At least 19 recordsLinked to original sources
PubMed2000

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.

Animals↗
PubMed1999

Efficacy of adhesive interactions in pig-to-human xenotransplantation.

Successful xenotransplantation depends on many factors, one being the interactions of cross-species adhesion molecule-ligand pairs. Depending on the approach used to facilitate xenotransplantation, these interactions can play differing roles. Here, André Simon, Anthony Warrens and Megan Sykes review the existing information on pig-to-human adhesive interactions and its implication for different approaches to pig-to-human xenotransplantation.

Animals↗
PubMed1999

Improving the therapeutic monitoring of cyclosporin A.

The narrow therapeutic window of cyclosporin A (CsA) and the variable pharmacokinetics of the traditional preparation, CsA-SIM (Sandimmune), have made it difficult to establish its optimal use. The introduction of a microemulsion preparation, CsA-ME (Neoral), with less variable pharmacokinetics, has made it possible to attempt to define its use more closely. One hundred and one renal allograft recipients were converted from CsA-SIM to CsA-ME. Absorption was monitored using a standard pharmacokinetic 'profile' measuring 'whole blood' CsA levels at several time points following drug administration. Areas under the resulting time-versus-CsA concentration curve (AUC) were calculated. Surprisingly, many patients showed very little fluctuation in 'whole blood' levels after administration of the conventional preparation: 31% had a difference between trough (to) and maximal levels of < 100 micrograms/L. On microemulsion cyclosporin, there was much better correlation between AUC and several parameters incorporating elements of trough and peak values. The best correlation was with t0 + (t1/4), where t1 represents the concentration at 1 h following administration, (r2 = 0.779 for microemulsion cyclosporin). The use of this parameter is a practical possibility in an out-patient setting. The very common, but under-recognised, pattern of almost flat absorption profiles in patients on conventional CsA suggests that the use of CsA in numerous clinical contexts should be reviewed, since CsA immunosuppression may previously have been inadequately monitored.

Absorption↗
PubMed1999

A case of acute renal failure and compartment syndrome after an alcoholic binge.

A 25 year old man presented with anuria and bilateral leg pain two days after an alcoholic binge. He subsequently developed rhabdomyolysis causing acute renal failure, with compartment syndrome of both lower legs. This required urgent dialysis and fasciotomy respectively within six hours of admission. He remained dialysis dependent for three weeks and only after four months was he able to weight bear on both legs. Alcohol is a leading cause of rhabdomyolysis. Early recognition and prompt treatment is essential to prevent serious complications.

Acute Kidney Injury↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

Studies on the interaction of T-cells with major histocompatibility complex class II antigens.

1. Major histocompatibility complex class II antigens have the central role in the immune response of 'presenting' antigenic peptide to CD4+ T-cells. This interaction with a T-cell's receptor may result in activation, but, if recognition occurs without collateral molecular interactions which cause 'co-stimulation', these T-cells will be tolerized. 2. In the light of current interest in muscle cell transplantation, a transformed myoblast, TE671, phenotypically comparable to untransformed cells, transfected to express class II, was studied as a stable model of antigen presentation by muscle cells. These cells failed to activate T-cells but induced tolerance. 3. The DR alpha chain is unusual being the only non-polymorphic classical class II polypeptide, raising the question of its functional contribution. To this end, several single polypeptide constructs were generated with contributions from different class II alpha-chains. On this basis, it was established that DR alpha makes significant contributions to peptide binding and that its alpha 2 domain is also important in T-cell recognition, possibly through CD4 binding. 4. One implication of the lack of polymorphism of DR alpha may be that it has a wider range of pairing partners, possibly including beta chains of different isotypes. To address this, it is planned to use transfectants expressing only a mixed isotype pair to generate T-cell clones in vitro. These reagents would be useful tools to detect whether such mixed pairs exist physiologically. In this paper, the development of a system is described which will allow this question to be addressed.

Antigen Presentation↗
PubMed1995

Definition of sites on HLA-DR1 involved in the T cell response to staphylococcal enterotoxins E and C2.

We have exploited the relative inefficiency of interaction between staphylococcal enterotoxins, SEE or SEC2, and H-2Ek compared to HLA-DR1 molecules to deduce which regions of the major histocompatibility complex (MHC) class II molecule are involved in the T cell response to these superantigens. Transfectants expressing hybrid DR/H-2E MHC class II molecules were used to present SEE to the T cell receptor V beta 8.1-expressing Jurkat cell line, and SEC2 to human peripheral blood T cells. For SEE, the critical region of the class II molecule for T cell reactivity and for binding was the beta 1 domain alpha-helix. The functional data were corroborated by measurements of direct binding. Sequence comparison between DR and H-2E raised the possibility that the glutamic acid at position 84 in the beta chain of H-2Ek, in place of glycine was responsible for the observed functional effects. This suggestion was supported by the finding that DQw2 (glutamine at 84) transfectants supported the SEE response much more efficiently than DQw6 that has glutamic acid at this position. In addition, amino acid substitutions at either position 36 or 39 in the DR alpha 1 domain abolished T cell reactivity without any obvious alteration in binding. For SEC2, use of transfectants expressing exon-shuffled alpha and beta chain genes showed that replacement of the alpha 1, alpha 2 and beta 1 domains with H-2E sequence inhibited the presentation of SEC2. Similarly, the substitutions at positions 36 and 39 in the alpha 1 domain abolished the T cell response to SEC2. Taken together, these data may be best explained by a model in which these two toxins have primary binding sites on the beta 1 domain (SEE) and the alpha 1 and alpha 2 domains (SEC2), but by virtue of a secondary binding site on the opposite surface of the class II molecule, cross-link two adjacent DR molecules. Such cross-linking may be important in the induction of T cell reactivity.

Amino Acid Sequence↗
PubMed1994

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.

Animals↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

Myoblasts fail to stimulate T cells but induce tolerance.

Recent interest in myoblast transfer and in the use of myoblasts as vehicles in gene therapy has made it important to understand the potential immunogenicity of allogeneic or neoantigen-expressing myoblasts. Given the problems of producing a pure population of myoblasts, in this study we used a tumour-derived muscle cell line (TE671), with phenotypic features of myoblasts, which we transfected to express HLA-DR1. However, this cell line was unable to stimulate either established HLA-DR1-specific allorective T cell clones or a primary alloresponse. Nor could it present haemagglutinin peptide HA 306-324 to DR1-restricted, HA 306-324-specific T cell clones or lines. Indeed, preincubation with DR1-expressing TE671 and HA 306-324 rendered such T cells tolerant as judged by their subsequent inability to proliferate in response to a DR1+ B cell line plus peptide HA 306-324. These results imply that myoblasts do not provide costimulatory signals, and are therefore unlikely to stimulate allospecific T cells following myoblasts transplantation or to initiate neoantigen-specific immune responses following in vivo transfection.

Antigen-Presenting Cells↗