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Anthony Dorling

Publications and source records attributed to Anthony Dorling.

17 recordsLinked to original sources
PubMed2006

NK-cell-dependent acute xenograft rejection in the mouse heart-to-rat model.

BACKGROUND: Acute humoral xenograft rejection is characterized by widespread intravascular thrombosis with a significant NK-cell and macrophage infiltrate. Although in vitro and ex vivo data have shown that NK cells are capable of killing xenogeneic tissue, the precise role they play in vivo is still not certain. Consequently, there are few tested strategies for dealing with NK-cell-mediated rejection, should this prove to be a problem. One reason for this has been the lack of a relevant rodent model in which rejection by these cells can be easily studied. METHODS: Prior to transplantation of mouse hearts, we depleted rat recipients of fibrinogen using a snake venom, ANCROD, from the Malayan pit viper. Graft survival was examined by manual palpation and the rejected hearts were examined by histology. Levels of circulating interferon gamma (IFN-gamma), used as a surrogate marker for NK-cell activation, were determined by an enzyme-linked immunosorbent assay. RESULTS: Depletion of fibrinogen to approximately 5% of normal allowed surgery without a significant increase in the technical failure rates and prolonged graft survival compared with that seen in unmanipulated rats. Rejected hearts showed no evidence of intravascular thrombosis but did show significant antibody and complement deposition. There was little T-cell infiltration and cyclosporin had no influence on survival. Instead, hearts were infiltrated with NK cells and macrophages and rejection was associated with significant IFN-gamma production. Depletion of NK cells with anti-asialo-GM-1 from ANCROD-treated recipients led to a further significant prolongation of graft survival. CONCLUSIONS: Inhibition of intravascular thrombosis by fibrinogen depletion, in the absence of any other manipulation, unmasks NK-cell-dependent acute xenograft rejection in the mouse-to-rat heart transplantation model. This relatively simple model is expected to be useful to investigate the mechanisms of NK-cell-mediated rejection and to provide insight into the types of graft manipulation that could modify this process.

Ancrod↗
PubMed2005

Generation of a polyclonal rabbit anti-mouse tissue factor antibody by nucleic acid immunisation.

Tissue factor (TF) the cellular receptor and cofactor for factor VII, initiates coagulation and has also been implicated in several coagulation-independent functions, including inflammation, angiogenesis and tumour metastasis. Investigations of TF expression in mouse models of these processes has been limited by the availability of antibodies that specifically recognise mouse TF. We have generated a rabbit polyclonal antibody to mTF by DNA immunisation. This has yielded an antiserum that recognises native mTF in immunohistochemical and flow cytometric analyses. Furthermore, the antiserum is inhibitory in coagulation assays. This antiserum will be a valuable investigative tool in the analysis of mTF expression.

Animals↗
PubMed2005

Achieving permanent survival of islet xenografts by independent manipulation of direct and indirect T-cell responses.

Recent success in pancreatic islet allotransplantation has raised expectations but has equally highlighted the acute shortage of donor tissue. The use of xenogeneic tissue would help to address this shortage; however, strong cellular immunity limits the application of this approach. T-cell responses to xenogeneic tissues involve recognition of intact species-mismatched major histocompatibility complex (MHC) molecules, the direct pathway, and xenogeneic proteins presented as peptides by responder-type MHC molecules, the indirect pathway. In this study, we exploited the species difference to selectively and sequentially inhibit direct and indirect xenoresponses after transplantation of porcine islets into mice. Selective inhibition of the direct response was achieved using porcine CTLA4-Ig, which binds preferentially to pig versus mouse B7 molecules. Selective inhibition of the indirect response was achieved using murine CTLA4-Ig, which binds preferentially to mouse B7 molecules. Administration of porcine CTLA4-Ig alone caused modest prolongation of islet survival. Injection of murine CTLA4-Ig alone had a minimal effect. However, the injection of the porcine fusion protein early and the murine homolog late after grafting led to permanent survival of the porcine islets, in the absence of any other immunosuppression. These results suggest that a similar approach could have clinical utility in porcine islet xenotransplantation.

Abatacept↗
PubMed2004

Developing a porcine transplantation model: efficient gene transfer into porcine vascular cells.

BACKGROUND: The pig is generally regarded as likely to be the preferred donor animal in xenotransplantation. Although many hurdles remain to be cleared, it would be useful to be able to manipulate porcine endothelium genetically, among other reasons, to test approaches in the modulation of inflammation. However, as a nondividing cell, it is less easy to manipulate. METHODS: The authors performed in vivo and in vitro gene transfection experiments using as an adjunct the DNA-binding agent 4',6'-diamidino-2-phenylindole (DAPI), which protects DNA from degradation. RESULTS: The introduction of DAPI into a liposomal transfection system was able to increase in vitro transfection efficiency of both endothelial and vascular smooth muscle cells from the pig, even in the presence of small amounts of serum. This last observation encouraged the authors to use this system in vivo in porcine carotid arteries. In this model, the authors were also able to demonstrate a high degree of transfection efficiency using DAPI, which seemed to work by protecting DNA from degradation. CONCLUSIONS: The authors believe this technique may allow them to address many biological questions relating to intervening in vascular disease, inflammation, and immune responses in the context of transplantation and beyond.

Animals↗
PubMed2004

Inhibition of intravascular thrombosis in murine endotoxemia by targeted expression of hirudin and tissue factor pathway inhibitor analogs to activated endothelium.

We have generated transgenic mice expressing the leech anticoagulant hirudin and human tissue factor pathway inhibitor tethered to the cell surface by fusion with fragments of human CD4 and P-selectin. Expression of the transgenes is under the control of the CD31 (platelet endothelial cell adhesion molecule [PECAM]) promoter, limiting expression to endothelial cells, monocytes, and platelets. In addition, the P-selectin sequence directs expression to secretory granules. Functional cell surface expression only occurs when the cells are activated. In a mouse model of systemic lipopolysaccharide (LPS)-induced endotoxemia, we show that expression of either anticoagulant on activated endothelium inhibits the widespread intravascular thrombosis, thrombocytopenia, and consumptive coagulopathy associated with endotoxemia. Importantly, non- LPS-treated transgenic mice had normal baseline bleeding times. We speculate that targeted delivery of anticoagulants to the endothelium may be a strategy worth pursuing in clinical sepsis to improve efficacy of systemic anticoagulation while minimizing potential hemorrhagic side effects.

Animals↗
PubMed2004

Modified dendritic cells coexpressing self and allogeneic major histocompatability complex molecules: an efficient way to induce indirect pathway regulation.

A feature of the tolerance that has been described in experimental models is that it can be transferred by CD4+ T cells to a naive recipient. Described is a novel approach to induce indirect pathway regulatory T cells in a rat model that exploits the natural processing and presentation of major histocompatability complex (MHC) molecules as peptide by the MHC class II molecules of the same cell. Dendritic cells (DC) coexpressing donor (AUG) and recipient (LEW) MHC molecules were rendered tolerogenic by treatment with dexamethasone. After injection into LEW animals followed by a single low dose of CTLA4-Ig, T cells were rendered unresponsive to indirectly presented AUG alloantigens, but retained direct pathway responsiveness to fully allogeneic AUG cells. The T cells from the DC-injected rats were unresponsive to (LEW x AUG)F1 stimulator cells, suggesting the presence of indirect pathway regulatory cells whose activity depended on the presence of LEW MHC molecules. Depletion of CD25+ cells from the responder population led to a marked increase in proliferation, and the T cells from the DC-injected rats inhibited the response of naive LEW T cells to (LEW x AUG)F1, but not to AUG, stimulator cells, further indicating indirect pathway-mediated regulation. Most importantly, pretreatment of LEW rats with the dexamethasone-treated DC led to the indefinite survival of AUG kidney grafts after a short course of cyclosporin to inhibit the early direct pathway response. Similarly treated AUG DC had no effect, confirming the privileged status of F1 cells in the induction of indirect pathway regulation.

Animals↗
PubMed2004

Complete inhibition of acute humoral rejection using regulated expression of membrane-tethered anticoagulants on xenograft endothelium.

Xenotransplantation promises an unlimited supply of organs for clinical transplantation. However, an aggressive humoral immune response continues to limit the survival of pig organs after transplantation into primates. Because intravascular thrombosis and systemic coagulopathy are prominent features of acute humoral xenograft rejection, we hypothesized that expression of anticoagulants on xenogeneic vascular endothelium might inhibit the process. Hearts from novel transgenic mice, expressing membrane-tethered fusion proteins based on human tissue factor pathway inhibitor and hirudin, respectively, were transplanted into rats. In contrast to control non-transgenic mouse hearts, which were all rejected within 3 days, 100% of the organs from both strains of transgenic mice were completely resistant to humoral rejection and survived for more than 100 days when T-cell-mediated rejection was inhibited by administration of ciclosporin A. These results demonstrate the critical role of coagulation in the pathophysiology of acute humoral rejection and the potential for inhibiting rejection by targeting the expression of anticoagulants to graft endothelial cells. This genetic strategy could be applied in a clinically relevant species such as the pig.

Animals↗
PubMed2003

Cross-species costimulation: relative contributions of CD80, CD86, and CD40.

BACKGROUND: The response of human CD4+ T cells against porcine cells is of comparable magnitude to that induced by human leukocyte antigen-mismatched allogeneic cells. This reflects productive interactions between key costimulatory molecules across the species barrier. Inhibition of these molecular interactions will be crucial in overcoming CD4+ T-cell-mediated rejection of xenografts. We have performed a detailed investigation to determine the expression profiles and relative contributions of the three key costimulatory molecules in the porcine-human xenogeneic response. Whereas only porcine CD86 is constitutively expressed on resting endothelial cells, both CD40 and CD80 are rapidly expressed after activation. All three costimulatory molecules are expressed by professional antigen-presenting cells. METHODS: We have isolated full-length cDNA clones for human and porcine CD80, CD86, and CD40. Human fibroblast cell lines (M1) coexpressing DR1 were transfected with these cDNAs and used in mixed lymphocyte reactions and flow cytometric studies in vitro. RESULTS: These data provide the first characterization of the expression profile and functional role of porcine CD80. Functional assays demonstrate that pCD40, pCD80, and pCD86 are independently capable of costimulating human CD4+ T cells, albeit with differing kinetics. Proliferative responses were of comparable magnitude to those obtained when costimulation was provided by human CD40, CD80, and CD86. CONCLUSIONS: These data have implications for therapy targeting the direct pathway of xenorecognition; costimulatory molecule blockade must be directed against both the B7/CD28 and CD40/CD40L pathways.

Animals↗
PubMed2003

Endothelial cell cytoprotection induced in vitro by allo- or xenoreactive antibodies is mediated by signaling through adenosine A2 receptors.

Endothelial cell (EC) expression of proteins such as hemoxygenase-1 or Bcl-xL is associated with enhanced survival of vascularized allo- or xenografts. These grafts are resistant to humoral rejection, a phenomenon called accommodation. In vitro, low concentrations of allo- and xenoreactive antibody (XNA) induce a cytoprotective phenotype in EC similar to that seen in accommodated grafts. In this study we examine whether adenosine plays a role in antibody-induced cytoprotection. Porcine EC were incubated with human anti-pig XNA or specific alloantibody. EC expressed Bcl-xL and were protected from TNF-mediated apoptosis. Bcl-xL expression was inhibited by an adenosine A2 receptor antagonist. Human anti-pig XNA were shown to bind and induce cyclic adenosine 3',5'-monophosphate (cAMP) generation through these receptors. This activity was abolished by depletion of anti-galalpha(1-3)gal-specific XNA. In contrast, alloantibody caused adenosine production. Protection from TNF-mediated apoptosis was also mediated through A2 receptor but involved additional non-cAMP-dependent signaling. This study indicates a molecular mechanism common to both antibody-mediated cytoprotection and ischemic preconditioning and suggests a potential therapeutic avenue based on adenosine for improving the outcome of transplanted grafts in those patients with pre-existing anti-graft antibody.

Adenosine↗
PubMed2002

Clinical xenotransplantation: pigs might fly?

Xenotransplantation has the potential to deliver an unlimited supply of organs for transplantation. However, this promise has yet to translate into clinical application, despite substantial research efforts in the last decade. Although increasing numbers of studies are being performed in relevant pre-clinical (pig-to-primate) transplantation models, so far these have highlighted the apparent elusiveness of long-term xenograft survival. Humoral rejection remains the main obstacle to success, but control of T cell-mediated rejection will be a problem in the future and there are major concerns about the possible transmission of porcine endogenous retroviruses (PERV) and other infectious agents. This article reviews recent advances in the understanding of acute vascular rejection (AVR), acute T cell-mediated rejection and PERV transmission and highlights some of the strategies that may prove successful in overcoming these problems. Although progress has been slow, the promise of an inexhaustible supply of organs is sufficient reason to continue research in these areas. Assuming the specific problem of AVR can be ameliorated by one of a number of strategies currently under investigation, there are grounds to believe that xenotransplantation will become a clinical reality. Pig xenografts, currently grounded, might eventually fly!

Animals↗