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A Azimzadeh

Publications and source records attributed to A Azimzadeh.

At least 19 recordsLinked to original sources
PubMed2003

Hyperacute lung rejection in the pig-to-human model. 2. Synergy between soluble and membrane complement inhibition.

BACKGROUND: The role of complement in hyperacute lung xenograft rejection has not been elucidated. The present study evaluates the effect of complement (C) C3/C5 convertase inhibition on hyperacute rejection of pig lung by human blood. METHODS: In an established ex-vivo model, lungs from pigs heterozygous for human decay accelerating factor (hDAF), non-transgenic littermate control pigs, or farm-bred pigs were perfused with fresh human blood that was either unmodified or treated with soluble complement receptor type 1 (sCR1: TP10, 100 microg/ml). RESULTS: Non-transgenic lungs from littermate controls had a median survival time of 35 min (range 5 to 210; P = 0.25 vs. farm-bred piglets: median 5 min, range 5 to 10). Lungs expressing hDAF survived for a median of 90 min (range 10 to 161; P = 0.5 and 0.01 vs. littermate and farm-bred controls, respectively), with sCR1, whereas hDAF (-) lungs failed by 35 min (range 6 to 307), hDAF (+) lungs survived for 330 min (range 39 to 577) [P = 0.002 vs. farm-bred; P = 0.08 vs. hDAF (-); P = 0.17 vs. sCR1/hDAF (-)]. The rise in pulmonary vascular resistance (PVR) at 5 min was blunted only by hDAF (+) with sCR1 (0.26 +/- 0.2 vs. 0.5 to 0.7 mmHg/ml/min for other groups). Plasma C3a and sC5b-9 and tissue deposition of C5b-9 were dramatically diminished using sCR1, and further decreased in association with hDAF. Histamine and thromboxane were produced rapidly in all groups. CONCLUSION: Complement plays an important role in lung HAR. However, even potent inhibition of C3/C5 convertase, both membrane bound in lung and by a soluble-phase inhibitor in the blood, does not prevent activation of inflammatory responses known to be particularly injurious to the lung. Our findings implicate a role for innate immune pathways resistant to efficient complement regulation. The role of anti-species antibody, coagulation pathway dysregulation, and additional environmental or genetic influences remain to be defined.

Acute Disease↗
PubMed2001

Effect of insulin on von Willebrand factor release in normal and diabetic subjects: in vivo and in vitro studies.

The aim of this study was to evaluate the effect of insulin on the release of vWf in vivo during an oral glucose tolerance test (OGTT) performed in normal, glucose-intolerant and diabetic subjects and in vitro on human endothelial cells. Twenty-eight subjects exhibiting risk factors for diabetes underwent an OGTT: 11 subjects proved to be normal, 7 were glucose-intolerant and 10 diabetic. In each group, the vWf and PAI-1 plasmatic levels were measured at t = 0, 30 min and 180 min after the beginning of the test. Human endothelial cells from non-diabetic and diabetic subjects were incubated in the presence of human insulin at various concentrations (0.25, 2.5, 25 and 250 mUI/ml). After 1, 4, and 24 hours of incubation, the release of vWf and endothelin 1 was measured in the cell supernatant and the intracellular amount of vWf in the cell lysate. During the OGTT, the vWf levels in plasma were not affected despite a 4.5-, 6-, and 2.5-fold increase in insulin levels in normal, glucose-intolerant and diabetic subjects, respectively. Although raised in all three groups, PAI-1 plasmatic levels remained constant during the test. After 24 hours of exposure to insulin (0.25 mU/ml), the release of vWf by endothelial cells reached 35.94 +/- 23.08 % of the basal value for non-diabetic subjects, and 27.57 +/- 10.05 % for diabetic patients. Similar results were observed in non-stimulated cells. Insulin had no influence on intracellular vWf content, which remained comparable to control values. Regardless of its concentration, insulin failed to stimulate the release of vWf by endothelial cells of non-diabetic and diabetic subjects, while its ability to stimulate the release of endothelin 1 was preserved. In conclusion, hyperinsulinemia had no adverse effect on circulating vWf in normal or diabetic subjects. Neither release nor intracellular vWf content in non-diabetic or diabetic endothelial cells was influenced by insulin in vitro.

Adult↗
PubMed2001

CD40-ligand in primate cardiac allograft and viral immunity.

Our laboratory has studied the role of CD40 ligand (CD40L, CD154) in the primate immune response to allogenic and infectious challenges. We find that intensive early blockade of CD40L reliably attenuates acute rejection of primate cardiac allografts. Monotherapy fails to prevent late graft loss, which often occurs in association with rising antidonor antibody titers and allograft vasculopathy, despite continuing anti-CD40L therapy. In contrast, the primary humoral response to T helper dependent influenza viral antigen is inhibited during anti-CD40L therapy, and responses to subsequent immunization are blunted after discontinuation of therapy. These results are encouraging with regard to the tolerogenic potential of costimulatory blockade for specific T helper dependent antigens. However, these findings also indicate that pathogenic allograft responses in primates are probably not entirely CD40L-dependent. As such, additional immunomodulatory strategies are needed to facilitate tolerance to a transplanted organ.

Abatacept↗
PubMed2001

Comparative phenotype and immunogenicity of freshly isolated and immortalized rat hepatocytes.

Immortalized hepatocytes are an attractive cell source for hepatocyte transplantation and gene transfer. We compared the phenotype and immunogenicity of freshly isolated (FIH) and immortalized (IMH) rat hepatocytes. Effect of culture and proinflammatory cytokines (TNF-alpha, IFN-gamma) was studied on phenotype. FIH were isolated by collagenase digestion. Two SV40 immortalized hepatocyte cell lines were tested (RH1 and P9). Immunophenotyping was performed by FACS analysis using anti-rat-specific antibodies. Immunogenicity was evaluated by a mixed lymphocyte hepatocyte reaction (MLHR). FIH suspension was an almost homogeneous parenchymal cell population with few (1-2%) CD8+ cells. FIH showed a positive staining for ICAM-1 (20-35%) and for Class I (RT1A, 30-60%) but no staining for Class II (RT1B). After 48 h of culture, the already ICAM-1-positive cells were more strongly stained and additionally 3.6% of the cells (possibly endothelial cells) were Class II positive. IMH showed a consistent expression of Class I (93-97%) and ICAM-1 (95-97%) but no expression of Class II. Culture of IMH for 48 h had no effect on Class II expression but increased ICAM-1 expression. Addition of TNF-alpha at 1000 UI/ml to cultures of FIH or IMH increased Class I and ICAM-1 expression whereas IFN-gamma (50 or 1000 UI/ml) had no evident effect. Hepatocyte immunogenicity, assessed in MLHR and appreciated by the stimulation index (SI) test/SI syngeneic control, was similar for IMH (RH1: 2.68+/-0.89; P9: 2.37+/-0.78) and FIH (2.52+/-0.18). In conclusion, despite some quantitative immunophenotypic differences, FIH and IMH induced the same proliferation rate of allogeneic T lymphocytes. Thus, immortalized hepatocytes may constitute an appropriate cellular model to study the prevention of hepatocyte rejection by gene transfer.

Animals↗
PubMed1999

Use of von Willebrand diseased kidney as donor in a pig-to-primate model of xenotransplantation.

BACKGROUND: The coagulation process in hyperacute and delayed xenograft rejection is essential and depends upon platelet adhesion and aggregation. The initial binding of platelets to the damaged endothelium is due to the interaction of the platelet receptor glycoprotein Ib with von Willebrand factor (vWF), which is present on activated endothelial cells and bound to the subendothelial matrix. We hypothesized that the use of organs from animals with homozygous von Willebrand disease (vWD), severely deficient in vWF, might prevent the thrombosis encountered in delayed xenograft rejection. METHODS: Ten baboons were treated by extracorporeal immunoadsorption of xenoreactive natural antibodies (XNA) through the donor pig liver to inhibit hyperacute rejection and received heterotopic vWD or control pig kidney xenografts. XNA levels, coagulation, and platelet activation markers were studied, and specimens of rejected kidneys were analyzed histologically. RESULTS: Although XNA depletion was comparable in both groups, neither kidney function nor survival times of control (n=5) or vWD (n=5) porcine kidneys showed any difference. Platelet and coagulation activation was evidenced in both groups after surgery and at rejection time. Immunohistochemical analysis revealed a weak endothelial vWF immunostaining in the rejected vWD kidneys, whereas it was undetectable in the nongrafted vWD kidneys, suggesting the deposition of baboon plasma vWF on the porcine vessels. CONCLUSIONS: The use of vWD organs did not improve the survival time of grafted kidneys in this xenotransplantation model. Further studies on the use of vWD organs, in association with other therapeutic approaches, such as complement inhibition, are nevertheless necessary to evaluate the usefulness of vWF deficiency as an adjunctive therapy to decrease the coagulation process during xenograft rejection.

Animals↗
PubMed1999

Characterization of a pig-to-goat orthotopic lung xenotransplantation model to study beyond hyperacute rejection.

BACKGROUND: A pig-to-goat orthotopic lung xenograft model was developed to test whether depletion of goat xenoreactive antibodies against pig red blood cells would prolong pig lung xenograft survival. METHODS: Adult goats with anti-pig xenoreactive antibodies underwent left pneumonectomy followed by orthotopic transplantation of pig left lung (group 1) or immunodepletion of their xenoreactive antibodies by extracorporeal right pig lung perfusion before transplantation without (group 2) or with (group 3) complete clampage of the right pulmonary artery. In group 4, goat left lungs were orthotopically transplanted into pigs and served as negative controls (pig serum does not have anti-goat xenoreactive antibodies). Each study group included 5 animals. Immunosuppression in surviving recipients included cyclosporine and azathioprine. RESULTS: Group 1 recipients died 7 +/- 3 hours after xenograft reimplantation of severe pulmonary hypertension and dysfunction and vasogenic shock, with little evidence of histologic xenograft injury. Group 2 xenografts had a stable circulatory and respiratory function on reperfusion and survived 9 +/- 4 days. Group 3 animals also tolerated complete occlusion of the right pulmonary artery, and xenografts assured the total respiratory support for 4 +/- 1 days. After immunodepletion, goat serum showed no detectable titers of xenoreactive antibodies, which began to reappear by postoperative day 2, where xenografts showed histologic stigmata of acute (humoral and cellular-mediated) rejection that evolved to a complete xenograft necrose at death. Group 4 xenografts showed scattered features of acute rejection 5 +/- 1 days after the operation. CONCLUSIONS: Pig left lung xenografts can provide prolonged and complete respiratory support after depletion of goat xenoreactive antibodies, but they ultimately necrose once recipient xenoreactive antibodies return to pretransplantation values.

Animals↗
PubMed1999

Protection against hyperacute xenograft rejection of transgenic rat hearts expressing human decay accelerating factor (DAF) transplanted into primates.

BACKGROUND: Production of transgenic pigs for multiple transgenes is part of a potential strategy to prevent immunological events involved in xenograft rejection. Use of a genetically engineerable rodent as a donor in primates could allow testing in vivo of the effects of different transgenes on controlling xenograft rejection. As a first step in the development of a donor containing multiple transgenes, transgenic rats for human decay-accelerating factor (DAF) were used as heart donors to test their resistance against complement (C)-mediated rejection by non-human primates. MATERIALS AND METHODS: Transgenic rats were generated by using a construct containing the human DAF cDNA under the transcriptional control of the endothelial cell (EC)-specific human ICAM-2 promoter. DAF expression was evaluated by immunohistology and by FACS analysis of purified ECs. Resistance of transgenic hearts against C-mediated damage was evaluated by ex vivo perfusion with human serum and by transplantation into cynomolgus monkeys. RESULTS: Immunohistological analysis of DAF expression in several organs from two transgenic lines showed uniform expression on the endothelium of all blood vessels. ECs purified from transgenic hearts showed 50% DAF expression compared to human ECs and >70% reduction of C-dependent cell lysis compared to control rat ECs. Hemizygous transgenic hearts perfused with human serum showed normal function for >60 min vs. 11. 2 +/- 1.7 min in controls. Hemi- or homozygous transgenic hearts transplanted into cynomolgus monkeys showed longer survival (15.2 +/- 7 min and >4.5 hr, respectively) than controls (5.5 +/- 1.4 min). In contrast to hyperacutely rejected control hearts, rejected homozygous DAF hearts showed signs of acute vascular rejection (AVR) characterized by edema, hemorrhage, and an intense PMN infiltration. CONCLUSIONS: We demonstrate that endothelial-specific DAF expression increased heart transplant survival in a rat-to-primate model of xenotransplantation. This will aid in the analysis of AVR and of new genes that may inhibit this form of rejection, thus helping to define strategies for the production of transgenic pigs.

Animals↗
PubMed1998

Evidence of human non-alpha-galactosyl antibodies involved in the hyperacute rejection of pig lungs and their removal by pig organ perfusion.

BACKGROUND: Human natural xenoantibodies represent a major hurdle to the clinical application of pig lungs in transplantation by initiating hyperacute rejection within minutes to hours. OBJECTIVE: The object was to compare pig organ perfusion and specific depletion of anti-alpha-galactosyl xenoantibodies for prevention of hyperacute rejection in the pig to human lung combination. METHODS: Large White pig (20-25 kg) left lungs were removed and continuously ventilated and reperfused ex vivo either with (1) whole human blood previously perfused in situ through pig right lung (group I), liver (group II), or spleen (group III) or with (2) human plasma in vitro immunoabsorbed on columns containing alpha-galactosyl disaccharide (Gal-alpha-(1-3)Gal-beta-(CH2)3NH2; B disaccharide) (group IV). Each study group included 6 animals. RESULTS: The in situ and in vitro preperfusions depleted anti-alpha-galactosyl xenoantibodies and all in situ perfused pig organs showed histologic signs of hyperacute rejection. After the ex vivo reperfusion, group I xenografts had a significantly (P < .001) longer functional and histologic survival than did xenografts in groups II, III, and IV. Human blood reperfusing group I xenografts had a significantly (P < 0.05) lower (1) decline of clotting factors and total circulating immunoglobulins, (2) total and membrane attack complex (C5b,6,7,8,9) complement activation, and (3) hemolysis. By Western blot analysis, the in situ lung preperfusion removed antibodies against non-alpha-galactosyl proteins of low molecular weight that were not eliminated by the alpha-galactosyl column. CONCLUSIONS: Results demonstrate that specific depletion of anti-alpha-galactosyl antibodies alone incompletely protects pig lungs from hyperacute rejection. It is speculated that the more complete prevention of this rejection afforded by pig lung preperfusion relates to the removal of other, non-alpha-galactosyl antibodies.

Acute Disease↗
PubMed1998

Removal of primate xenoreactive natural antibodies by extracorporeal perfusion of pig kidneys and livers.

Organ perfusion is one of the possible strategies to attenuate rejection of discordant xenografts by reducing the levels of the recipient's xenoreactive natural antibodies (XNA). Its efficacy in terms of XNA removal was studied in models of primate blood or plasma perfusion through porcine kidneys or livers, with special attention to haematological consequences and potential side-effects. We first perfused the blood of rhesus monkeys through pig kidneys and livers, and demonstrated that the perfusion of a pig liver resulted in higher XNA adsorption (72 +/- 13%) than the perfusion of a pig kidney (51 +/- 25%). However, when we normalized for the weight of the perfused organs and for levels of natural antibodies in individual monkeys, livers adsorbed less antibody (1.4 +/- 0.9 U antibody/g) than kidneys (7.2 +/- 7 U antibody/g). Histological signs of rejection were observed in perfused kidneys, but not in perfused livers. A major drawback of the perfusion of blood through livers was a considerable decrease in the primates' haemoglobin and platelet levels. To avoid this, we developed a plasma liver perfusion device. This method allowed a significant improvement in the haemodynamic state of primates and was particularly effective in preventing anaemia. Moreover, plasma liver perfusion was as effective as blood liver perfusion to remove natural antibodies and, resulted in a marked decrease in their functional activity as assessed by complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). The level of other plasma proteins was not significantly affected, apart from a dilution effect. After xenoperfusion a strong antibody response was evidenced by ELISA, CDC and ADCC between days 7 and 14 and then decreased progressively. We conclude that the separation of blood to allow the perfusion of plasma through a pig organ is safer than the perfusion of unseparated blood and is associated with efficient natural antibody removal. However, organ perfusion is limited by a rebound in antibody levels after a few days, and thus will have to be associated with anti-B cell immunosuppressive therapy for long-term or repeated applications.

Animals↗
PubMed1998

Antibodies to human adhesion molecules and von Willebrand factor: in vitro cross-species reactivity in the xenotransplantation setting.

Endothelial cell activation is thought to play an important role in xenograft rejection through cell retraction and expression of pro-coagulant and pro-inflammatory factors. Identification of antibodies recognizing porcine endothelial molecules would be useful to study and manipulate the inflammatory response to a xenograft. The aim of this study was to investigate the cross-reactivity of antibodies directed against human adhesion molecules and von Willebrand factor (vWF). Binding of monoclonal antibodies (mAbs) directed against human CD3 1, CD44, CD49, CD54, CD62E, CD102, and CD106 was evaluated on resting and activated endothelial cells from human and pig by flow cytometry. Among 30 antibodies tested, 4 were shown to react with pig cells. Two of them, directed against human CD62E (E-selectin) and rabbit CD106 (VCAM-1) reacted strongly with activated and/or resting pig cells, whereas two others, directed to human CD31 (PECAM) and CD44 (H-CAM), bound weakly to pig cells. In addition, we analyzed the cross-reactivity of five polyclonal or monoclonal antibodies to human or pig vWF with human, baboon, rhesus, pig, and rat vWF. Binding of antibodies was tested by ELISA by using platelet lysates as source of vWF from the different species. Four anti-human or porcine vWF antibodies exhibited a broad reactivity with vWF from all species, whereas one anti-human vWF antibody was specific for primate vWF. In this study, we identified a small number of cross-reacting antibodies that may prove useful to study in vitro and in vivo xenogeneic responses. However, the weak antibody cross-reactivity observed with most porcine molecules points out the necessity of producing species-specific antibodies to study the immune response to xenografts or for use as specific immunosuppressive therapeutic reagents.

Animals↗
PubMed1998

Metabolic capacities in cultured human hepatocytes obtained by a new isolating procedure from non-wedge small liver biopsies.

A new isolating procedure of human hepatocytes has been developed using two-step collagenase digestion by a non-perfusion procedure (NP) of non-wedge liver biopsies. 1. A yield of 2-7 x 10(6) hepatocytes/g liver, 52-95% viability and 13-75% attachment were obtained from liver biopsies weighing 6-60 g, comparable to that obtained when using the classical perfusion procedure (P) to isolate human hepatocytes from wedge liver samples of 50-150 g. 2. In culture, human hepatocytes obtained by NP remained attached to plastic for up to 5 days and displayed the usual morphological characteristics. Their metabolic capacities, assessed by liver-specific albumin and urea synthesis and by CYP-dependent and conjugation pathways, were equivalent to those of human hepatocytes obtained by P. In addition, they responded adequately to specific CYP inducers, demonstrating that they constitute a model in which human drug metabolism and toxicity studies can be performed.

Adult↗
PubMed1997

Comparative study of target antigens for primate xenoreactive natural antibodies in pig and rat endothelial cells.

BACKGROUND: A rat-to-primate cardiac xenograft model has been proposed as an alternative to the clinically relevant but more cumbersome pig-to-primate model for assessing the efficacy of strategies aimed at preventing xenograft hyperacute rejection. As in pig xenografts, the rejection of rat hearts was mediated by the binding of xenoreactive natural antibodies (XNA) and complement activation. The present study was conducted to identify target antigens recognized by cynomolgus and rhesus monkey IgM XNA on rat tissues and cells in comparison with pig cells. METHODS: The reactivity of rhesus or cynomolgus serum on pig and rat endothelial cells (ECs) was studied by flow cytometry, ELISA, and complement-dependent cytotoxicity, after removal of primate XNA by perfusion of pig livers, immunoadsorption on a Gal alpha(1,3)Gal affinity column, and enzymatic removal of alpha-galactosyl epitopes from the cell surface. Rat and pig EC extracts were also immunoprecipitated with primate serum and resolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The expression of the Gal alpha(1,3)Gal epitope was analyzed on rat tissues and ECs by immunohistochemistry, flow cytometry, and Western blot, using the isolectin B4 from Griffonia simplicifolia. RESULTS: Removal of primate XNA or of alphaGal epitopes resulted in a decrease in XNA binding to pig and rat cells, leaving a similar degree of residual reactivity in the two species. At least five proteins of 260, 210, 110, 56, and 50 kDa were immunoprecipitated on rat ECs, with molecular weight similar to several proteins identified on pig ECs. These results suggest that primate XNA recognize similar antigens on rat and pig ECs. Rat cells expressed lower levels of the Gal alpha(1,3)Gal epitope than pig cells. A large proportion, but not all, of primate XNA react with this epitope on pig and rat ECs. CONCLUSION: This study suggests that the rat is a valuable species for the evaluation of genetic engineering strategies on the vascular endothelium aimed at preventing hyperacute xenograft rejection.

Animals↗
PubMed1997

In vivo and in vitro optimization of depletion of IgM and IgG xenoantibodies by immunoadsorption using cell membrane proteins.

A system of immunoadsorption was developed for in vitro depletion of xenoreactive natural antibodies of classes IgG and IgM from monkey and human plasma. Porcine endothelial cell membrane proteins, platelet membrane proteins, and endothelial cells were used as affinity ligands, and cyanogen bromide-activated Sepharose 6 Fast Flow and Sepharose CL-4B gels were used for chromatography. Adsorption capacity was evaluated by means of ELISA, immunonephelometry, and cytotoxicity testing. Several consecutive adsorption-desorption cycles were performed. Different parameters influencing immunoadsorption were examined: ligand density on the column gel, adsorbent-plasma contact time, ratio of plasma volume to immunoadsorbent volume, desorption conditions, and temperature. After 2 adsorption-desorption cycles, 99% and 82 to 85% of IgG and IgM antipig antibodies were adsorbed, respectively. Furthermore, there was a 74 to 77% decrease in cytotoxicity. In vivo, we observed that after one adsorption-desorption cycle, 97% of antipig IgG antibodies and 96% of antipig IgM antibodies were adsorbed, and there was an 85% decrease in cytotoxicity. The immunoadsorption method studied and optimized in vitro and in vivo therefore efficiently depleted xenoantibodies and reduced the cytotoxicity. Thus, it can be used in xenotransplantation experiments without eliminating non-specific antibodies.

Adsorption↗
PubMed1996

Assessment of hyperacute rejection in a rat-to-primate cardiac xenograft model.

We studied a rat-to-cynomolgous monkey model for xenotransplantation of vascularized organs and found that a rat heart was rejected in 5.5 +/- 1.4 min (n = 10). This hyperacute rejection (HAR) was consistent with kinetic experiments in vitro that showed damage to rat endothelial cells (ECs) after 3 min of incubation with primate serum. Histopathology and ultrastructural analysis of rejected hearts showed marked EC damage and early adherence of platelets and polymorphonuclear leukocytes to the endothelium. Immunohistochemical analysis revealed deposition along endothelial surfaces of IgG, IgM, and complement (C) components of the classical but not the alternative pathway, suggesting that, as in the pig-to-primate model, HAR is mediated by the binding of recipient xenogeneic natural antibodies and C activation. The effect of C depletion on xenograft survival was evaluated in two recipients that were treated with cobra venom factor (CVF). CVF caused complete C inactivation, demonstrated by lack of serum hemolytic activity and C-dependent EC cytotoxicity at engraftment and until the animals died. The rat cardiac transplants survived for at least 9 hr and 77 hr. Histology showed massive interstitial hemorrhage, edema, and cellular infiltration with scanty fibrin deposits. These results in CVF-treated recipient monkeys indicate that C activation mediates the development of HAR in this rat-to-primate model. We suggest that the model may be of interest as an alternative to the more expensive and time-consuming pig-to-primate model for testing the efficacy of transgenic modification of donor organs to prolong xenograft survival and for studying mechanisms of discordant xenograft rejection.

Animals↗