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Biomedical subjects

A Dorling

Publications and source records attributed to A Dorling.

At least 19 recordsLinked to original sources

The interface between coagulation and immunity.

Coagulation proteases are involved in generating fibrin after vascular injury (hemostasis) but they also have multiple other effects, many of which are mediated independently of fibrin generation, via interactions with specific cell membrane-expressed "protease activated receptors". In inflammation, this family of proteins has a complex influence, the facets of which are still incompletely understood, though a common feature in different models appears to be amplification of innate signals that are initially generated by pathogenic elements or, in the context of transplantation, ischemia or anti-graft antibodies, for instance. There is increasing evidence that these proteases may also have specific effects on cells involved in adaptive immunity and on cells that mediate chronic inflammation and fibrosis. Understanding whether these effects are relevant in the responses generated against transplanted organs is important, as it could lead ultimately to the development of novel ways to promote long-term graft survival.

Animals↗

Extraanatomic stents for transplant ureteric stenosis.

Surgical and standard endourology options are limited in transplant patients with severe ureteric stenosis, particularly when access to the transplant renal pelvis is limited. The use of a silicone-polytetrafluoroethelene (PTFE)-bonded extraanatomic urinary tract stent for urinary tract drainage is described in two patients. This technique of ureteric reconstruction in renal transplantation may be considered when standard approaches have failed. It appears to be safe when performed by radiologists and urologists with expertise in percutaneous renal access.

Adult↗

Postinjury vascular intimal hyperplasia in mice is completely inhibited by CD34+ bone marrow-derived progenitor cells expressing membrane-tethered anticoagulant fusion proteins.

BACKGROUND: Coagulation proteins promote neointimal hyperplasia and vascular remodelling after vessel injury, but the precise mechanisms by which they act in vivo remain undetermined. OBJECTIVES: This study, using an injury model in which the neointima is derived from bone marrow (BM)-derived cells, compared inhibition of tissue factor or thrombin on either BM-derived or existing vascular smooth muscle cells. METHODS: Two transgenic (Tg) mouse strains expressing membrane-tethered tissue factor pathway inhibitor (TFPI) or hirudin (Hir) fusion proteins driven by an alpha smooth muscle actin (SMA) promoter were generated (alpha-TFPI-Tg and alpha-Hir-Tg) and the phenotype after wire-induced endovascular injury was compared with that in wild-type (WT) controls. RESULTS: WT mice developed progressive neointimal expansion, whereas injury in either Tg was followed by repair back to a preinjured state. This was also seen when WT mice were reconstituted with BM from Tg mice but not when Tgs were reconstituted with WT BM, in which injury was followed by slowly progressive neointimal expansion. Injection of CD34+ cells from Tg mice into injured WT mice resulted in the accumulation of fusion protein-expressing cells from day 3 onwards and an absence of neointimal hyperplasia in those areas. CONCLUSIONS: Neointimal development after wire-induced endovascular injury in mice was completely inhibited when BM-derived cells infiltrating the damaged artery expressed membrane tethered anticoagulant fusion proteins under an alpha-SMA promoter. These findings enhance our understanding of the pathological role that coagulation proteins play in vascular inflammation.

Animals↗

Cure of disseminated cryptococcal infection in a renal allograft recipient after addition of gamma-interferon to anti-fungal therapy.

The immunosuppressive regimens that are used in solid organ transplantation are potent inhibitors of Th0 as well as Th1 and Th2 cell-mediated immune responses. This predisposes patients to disseminated cryptococcal infections. Mortality in such patients remains very high despite advances in anti-fungal chemotherapy. We describe a case of disseminated cryptococcal disease in a renal allograft recipient that failed to respond to prolonged treatment with several anti-fungal drugs. However, addition of the immuno-modulator, interferon-gamma, resulted in the formation of granulomas and the resolution of his disease within 4-6 weeks. As we cannot find a similar example of combination therapy for disseminated cryptococcal disease in the solid organ transplant literature, we propose that interferon-gamma could be used in synergy with anti-fungal drugs to cure disseminated cryptococcal infections in solid organ transplant patients.

Adult↗

Are anti-endothelial cell antibodies a pre-requisite for the acute vascular rejection of xenografts?

BACKGROUND: Vascular rejection occurring within the first few weeks after transplantation is still the major immunological barrier to the long term survival of xenografts. Currently there is no consensus about what to call this type of rejection (acute vascular rejection, delayed xenograft rejection or acute humoral xenograft rejection), nor about how to prevent or treat it. METHODS: A review of published evidence to define the heterogeneity of this phase of rejection and examine the role of antibodies, complement and graft-infiltrating inflammatory cells. RESULTS: i) antibodies are always involved in acute vascular rejection; ii) this antibody-mediated rejection may be complement-dependent or -independent; iii) inflammatory cells may mediate an antibody- and complement-independent phase of rejection in some small animal models (which, in its pure form cannot be called 'vascular rejection') iv) there remain significant questions about the relevance of 'accommodation' and the importance of coagulation abnormalities. CONCLUSIONS: Without doubt, future research would be helped by distinguishing between these different forms of delayed xenograft rejection, using terminology to reflect the involvement of specific pathophysiological mechanisms. An updated classification of the stages of xenograft rejection is proposed here.

Acute Disease↗

"Accomodated" pig endothelial cells promote nitric oxide-dependent Th-2 cytokine responses from human T cells.

BACKGROUND: Cardiac and renal allo- and xenografts can become naturally resistant to vascular rejection. Understanding this process of "accommodation" would enhance our understanding of vascular inflammatory responses and have implications for immune manipulation and tolerance induction. A feature of these grafts is infiltration by leukocytes secreting a Th-2 pattern of cytokines. METHODS: HLA-DR-1-transfected, immortalized porcine endothelial cells (IPEC) were incubated with polyclonal human immunoglobulin G (IgG) for 6 days before incubation with purified human CD4+ T cells. RESULTS: IgG-incubated IPEC stimulated a normal proliferative response from alloreactive T cells. However, interferon (IFN)-gamma levels were significantly reduced, whereas interleukin (IL)-5 and IL-10 were maintained at levels equivalent to those stimulated by control IPEC. Cognate interaction between T cells and IPEC was not required for this effect, because IgG-incubated, MHC-class II-negative IPEC caused reduced IFN-gamma secretion during a response to human Epstein-Barr virus-transformed B cells. Experiments with the nitric oxide (NO) donor, (z)-1-2-[2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), and the NO synthase inhibitor, NG-monomethyl-L-arginine.monoacetate (L-NMMA), showed that NO released by the IgG-incubated IPEC was actively involved in the development of this phenotype. CONCLUSIONS: These data suggest a novel, IgG-mediated, NO-dependent mechanism by which endothelial cells (EC) influence T cell responsiveness and that the Th-2 cytokine skewing seen in "accommodated" grafts may be a secondary phenomenon, resulting from the T-EC interactions.

Animals↗

Nitric oxide-mediated expression of Bcl-2 and Bcl-xl and protection from tumor necrosis factor-alpha-mediated apoptosis in porcine endothelial cells after exposure to low concentrations of xenoreactive natural antibody.

BACKGROUND: Cardiac and renal allo- and xenografts can acquire a natural resistance to vascular rejection. This "accommodation" involves endothelial cell (EC) expression of "survival genes" such as Bcl family members and hemoxygenase 1. Understanding what initiates this protective process would have profound implications; our hypothesis is that low concentrations of antigraft antibodies may mediate these changes. METHODS: In vitro cultured primary and immortalized porcine EC were incubated with polyclonal human IgG for 6 days and then examined for phenotype changes. RESULTS: The cells acquired resistance to tumor necrosis factor-alpha-mediated apoptosis (50-100% reduction at 6 hr) and up-regulated expression of Bcl-2 and Bcl-xl; sustained expression was accompanied by inducible nitric oxide (NO) synthase expression and by enhanced production of NO by EC. Two observations suggested that NO was actively involved in the process of Bcl-2 and Bcl-xl induction. First, (z)-1-2-[2-aminoethyl)-N- (2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, an NO donor, was able to induce similar changes in porcine EC to those induced by anti-pig antibodies. Second, an NO synthase inhibitor NG-monomethyl-L-arginine.monoacetate was able to specifically inhibit the anti-pig antibody-mediated expression of Bcl-2 or Bcl-xl. CONCLUSIONS: These data strongly support the hypothesis that Bcl-2 and Bcl-xl expression and protection from apoptosis in EC may result from antibody-mediated NO production through the neoexpression of inducible NO synthase.

Animals↗

Human thrombin and FXa mediate porcine endothelial cell activation; modulation by expression of TFPI-CD4 and hirudin-CD4 fusion proteins.

Aside from their critical role in thrombosis, activated coagulation factors also have inflammatory properties and these may be important during delayed xenograft rejection (DXR). This study assessed whether porcine EC could be activated by factor Xa (FXa) and thrombin (FIIa) and whether expression of tissue factor pathway inhibitor (TFPI)-CD4 and hirudin-CD4 fusion proteins could prevent such activation. Incubation of porcine EC with human FXa and FIIa induced cell surface expression of E-selectin, VCAM and tissue factor (TF) in a time-dependent and concentration-dependent manner. In contrast, porcine EC transfected with a human TFPI-CD4 fusion protein were selectively resistant to these pro-inflammatory effects of FXa but not FIIa. Likewise, the transfectants expressing the hirudin-CD4 fusion protein were selectively resistant to the pro-inflammatory effects of FIIa but not those of FXa. When combined, the FXa and FIIa had an additive effect on the activation of control EC. In contrast, coexpression of both hirudin-CD4 and TFPI-CD4 fusion proteins completely inhibited the upregulation of VCAM with the FXa/FIIa mix. These results indicate that expression of novel anticoagulant fusion proteins on the surface of porcine EC can protect against EC activation induced by human coagulation factors FXa and FIIa. In vivo, we anticipate that expression of these fusion proteins on the endothelium of transplanted xenografts, besides preventing intravascular thrombosis, will also protect against EC activation induced by trace amounts of FIIa and FXa, thereby further protecting the grafts from DXR.

Animals↗

Transplant accommodation in highly sensitized patients: a potential role for Bcl-xL and alloantibody.

Transplantation of renal allografts into recipients with circulating anti-HLA antibodies results in hyperacute rejection. In some cases, however, antibodies return without causing harm; this phenomenon has been termed 'accommodation'. We have investigated this process in human allotransplantation. We removed anti-HLA antibodies by immunoadsorption in seven highly sensitized dialysis patients who subsequently underwent renal transplantation. Immunohistochemistry of renal biopsies for IgG and antiapoptotic proteins was performed. We also developed a model of 'accommodation' using anti-HLA antibodies eluted from sensitized patients and incubated with human umbilical vein endothelial cells (HUVECs) at different concentrations. Their effect on HUVEC phenotype was then analysed. Anti-donor antibody returned in 4/7 patients, without evidence of hyperacute rejection. Three out of four of these 'accommodated' grafts showed specific endothelial up-regulation of Bcl-xL and 2/2 tested positive for endothelial IgG deposition. HUVECs incubated with subsaturating concentrations of anti-HLA antibody showed increased expression of Bcl-xL, were rendered refractory to endothelial cell activation and became resistant to complement-mediated lysis. In contrast, HUVECs incubated with saturating concentrations underwent activation and expressed low levels of Bcl-xL. In conclusion, endothelial Bcl-xL expression defines the accommodation process in human allografts and this phenotype may be initiated by exposure of endothelium to low concentrations of anti-donor HLA antibodies.

Adult↗

Porcine CTLA4-Ig lacks a MYPPPY motif, binds inefficiently to human B7 and specifically suppresses human CD4+ T cell responses costimulated by pig but not human B7.

The CTLA4 receptor (CD152) on activated T lymphocytes binds B7 molecules (CD80 and CD86) on APC and delivers a signal that inhibits T cell proliferation. Several regions involved in binding to B7 are known, but the relative importance of these is not clear. We have cloned porcine CTLA4 (pCTLA4). Although highly homologous to human CTLA4 (hCTLA4), the predicted protein sequence contains a leucine for methionine substitution at position 97 in the MYPPPY sequence. A fusion protein constructed from the extracellular regions of pCTLA4 and the constant regions of human IgG1 (pCTLA4-Ig) bound porcine CD86 with equivalent affinity to that of hCTLA4-Ig. However, pCTLA4-Ig bound poorly to human CD80 and CD86 expressed on transfectants and EBV-transformed human B cells. In functional assays with MHC class II-expressing porcine endothelial cells and human B cells, pCTLA4-Ig blocked human CD4+ T cell responses to pig but not human cells, whereas control hCTLA4-Ig inhibited responses to both. Comparison between mouse, human, and porcine CTLA4-Ig suggests that the selective binding of pCTLA4-Ig to porcine CD86 molecules is due to the L for M substitution at position 97. Our results indicate that pCTLA4-Ig may be a useful reagent to define the precise nature of the interaction between B7 and CTLA4. By failing to inhibit the delivery of costimulatory signals provided by human B7, it may also prove to be a relatively specific inhibitor of the direct human T cell response to immunogenic pig tissue.

Abatacept↗

Cloning of porcine intercellular adhesion molecule-1 and characterization of its induction on endothelial cells by cytokines.

BACKGROUND: The transplantation of pig organs into humans requires a detailed knowledge of similarities and differences between the two species in the molecular physiology of host defense mechanisms. We therefore set out to identify porcine intercellular adhesion molecule (ICAM)-1 and to characterize its expression by endothelial cells. METHODS: Porcine ICAM-1 cDNA was isolated from an endothelial cell cDNA library. An anti-pig ICAM-1 monoclonal antibody was generated and used to investigate the regulation by cytokines of ICAM-1 expression by porcine aortic endothelial cells (PAEC), using flow cytometry. RESULTS: We found that porcine ICAM-1 was similar in primary structure to human ICAM-1, with five Ig-like domains. COS-7 cells transfected with porcine ICAM-1 supported beta2 but not alpha4 integrin-dependent adhesion of human T lymphoblasts. There was a low-level surface expression of ICAM-1 on unstimulated PAEC and increased expression after stimulation with tumor necrosis factor (TNF)-alpha. However expression of ICAM-1 seemed to be significantly lower than that of vascular cell adhesion molecule-1, both on unstimulated and TNF-alpha-activated PAEC. Recombinant porcine interferon-gamma weakly stimulated ICAM-1 expression when incubated alone with PAEC but had an inhibitory effect on the increase in ICAM-1 due to TNF-alpha, both at 8 and 24 hr. CONCLUSIONS: Our observations confirm the existence of ICAM-1 in the pig and provide novel insights into how porcine and human endothelial cells differ in terms of adhesion molecule expression and cytokine responsiveness. Such differences are potentially important in interpreting models of inflammation in the pig and also in understanding the process of rejection of porcine xenografts.

Amino Acid Sequence↗

HLA-G inhibits the transendothelial migration of human NK cells.

The expression of the non-classical MHC class I molecule HLA-G is normally restricted to the placenta during pregnancy, where it is found on fetal endothelial cells and on invasive cytotrophoblast cells, specifically those at the maternal / fetal interface. Its precise physiological role has yet to be defined. HLA-G may have nonimmune functions relating to angiogenesis and placentation, but most evidence suggests that it protects fetal cells from lysis by maternal uterine NK cells, which are found in large numbers around invading trophoblast cells. This effect is due to specific interaction with inhibitory receptors expressed on NK cells. We have examined the hypothesis that another function of HLA-G is to inhibit NK cell migration. Using an in vitro transmigration assay system, we present data to support this hypothesis. NK cell migration across porcine endothelial cells transfected with HLA-G1 was specifically inhibited compared to migration across HLA-A2-transfected monolayers. HLA- G1 had no influence on the migration of a control T lymphocyte line. These results support the idea that in vivo, HLA-G may inhibit NK cell traffic across the placenta.

Cell Movement↗

Costimulatory blockade by the induction of an endogenous xenospecific antibody response.

Xenogeneic tissues induce vigorous T cell immunity, reflecting the ability of costimulatory molecules to function across species barriers. We describe a strategy to inhibit costimulation that exploits species differences using the model of porcine pancreatic islet transplantation into mice. Mice were immunized with chimeric peptides that contained a known T cell epitope and selected sequences of the porcine costimulatory molecule CD86. This resulted in anti-peptide antibody responses that recognized intact porcine CD86, blocked costimulation by porcine CD86 but not murine CD86 in vitro, and prolonged the survival of porcine islet grafts in vivo. This strategy of inducing endogenous donor-specific costimulatory blockade has potential clinical applicability.

Amino Acid Sequence↗

Regulated inhibition of coagulation by porcine endothelial cells expressing P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins.

BACKGROUND: Thrombotic vascular occlusion resulting in infarction occurs during hyperacute rejection of allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. A similar process is also found in disorders of diverse etiology including atherosclerosis, vasculitis, and disseminated intravascular coagulation. METHODS: We have previously constructed two membrane-tethered anticoagulant fusion proteins based on human tissue factor pathway inhibitor and the leech anticoagulant hirudin and demonstrated their functional efficacy in vitro. These constructs have now been modified by the addition of a P-selectin sequence to the cytoplasmic tail to localize them in Weibel-Palade bodies. They have been transfected into Weibel-Palade body-positive endothelial cells isolated from the inferior vena cava of normal pigs. RESULTS: In resting endothelial cells, fusion protein expression colocalized with P-selectin and was confined to Weibel-Palade bodies. These cells had a procoagulant phenotype in recalcified human plasma. However, after activation with phorbol ester the anticoagulant proteins were rapidly relocated to the cell surface where they specifically inhibited the clotting of human plasma. CONCLUSIONS: Novel anticoagulant molecules may prove useful therapeutic agents for gene therapy in thrombotic disease and postangioplasty or for transgenic expression in animals whose organs may be used for clinical xenotransplantation. Expression in vascular endothelial cells may be regulated by inclusion of P-selectin cytoplasmic sequence, to restrict cell surface expression to activated endothelium.

Animals↗