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M Awwad

Publications and source records attributed to M Awwad.

At least 37 records · Page 2Linked to original sources
PubMed2000

High-dose porcine hematopoietic cell transplantation combined with CD40 ligand blockade in baboons prevents an induced anti-pig humoral response.

BACKGROUND: In pig-to-primate organ transplantation, hyperacute rejection can be prevented, but the organ is rejected within days by acute vascular rejection, in which induced high-affinity anti-Gal alpha1-3Gal (alphaGal) IgG and possibly antibodies directed against new porcine (non-alphaGal) antigenic determinants are considered to play a major role. We have explored the role of an anti-CD40L monoclonal antibody in modifying the humoral response to porcine hematopoietic cells in baboons pretreated with a nonmyeloablative regimen. METHODS: Porcine peripheral blood mobilized progenitor cells obtained by leukapheresis from both major histocompatibility complex-inbred miniature swine (n=7) and human decay-accelerating factor pigs (n=3) were transplanted into baboons. Group 1 baboons (n=3) underwent whole body (300 cGy) and thymic (700 cGy) irradiation, T cell depletion with ATG, complement depletion with cobra venom factor, short courses of cyclosporine, mycophenolate mofetil, porcine hematopoietic growth factors, and anti-alphaGal antibody depletion by immunoadsorption before transplantation of high doses (2-4 x 10(10)/cells/kg) of peripheral blood mobilized progenitor cells. In group 2 (n=5), cyclosporine was replaced by eight doses of anti-CD40L monoclonal antibodies over 14 days. The group 3 baboons (n=2) received the group 1 regimen plus 2 doses of anti-CD40L monoclonal antibodies (on days 0 and 2). RESULTS: In group 1, sensitization to alphaGal (with increases in IgM and IgG of 3- to 6-fold and 100-fold, respectively) and the development of antibodies to new non-alphaGal porcine antigens occurred within 20 days. In group 2, no sensitization to alphaGal or non-alphaGal determinants was seen, but alphaGal-reactive antibodies did return to their pre- peripheral blood mobilized progenitor cells transplant levels. In group 3, attenuated sensitization to alphaGal antigens was seen after cessation of cyclosporine and mycophenolate mofetil therapy at 30 days (IgM 4-fold, IgG 8-30-fold), but no antibodies developed against new porcine determinants. In no baboon did anti-CD40L monoclonal antibodies prevent sensitization to its own murine antigens. CONCLUSIONS: We believe these studies are the first to consistently demonstrate prevention of a secondary humoral response after cell or organ transplantation in a pig-to-primate model. The development of sensitization to the murine elements of the anti-CD40L monoclonal antibodies suggests that nonresponsiveness to cell membrane-bound antigen (e.g., alphaGal) is a specific phenomenon and not a general manifestation of immunological unresponsiveness. T cell costimulatory blockade may facilitate induction of mixed hematopoietic chimerism and, consequently, of tolerance to pig organs and tissues.

Animals↗
PubMed2000

Anti-Galalpha1-3Gal antibody levels in organ transplant recipients receiving immunosuppressive therapy.

The effect of long-term pharmacologic immunosuppression (PI) on anti-Galalpha1-3Gal (alphaGal) antibody (Ab) levels has not been determined previously in humans. In this study, we measured alpha Gal Ab levels by ELISA in 14 healthy volunteers (controls) and in 70 patients with grafts (kidney, heart, liver) who had received different combinations of PI (including cyclosporine, tacrolimus, azathioprine, mycophenolate mofetil, and steroids) for >3 months. There was great variation in Gal IgM (<80-fold) and IgG (<160-fold). There was no difference in Gal IgM or Gal IgG between any one group and any other. In kidney patients with either high (mean 68%) or low (mean 6%) panel-reactive alloantibodies, there was no difference in alpha Gal Ab level or serum cytotoxicity to pig cells. In vitro immunoadsorption of alphaGal Ab from the serum did not change panel-reactive alloantibody positivity. Therapy with OKT3, a mouse product that might stimulate alphaGal Ab production, led to no significant change in patient Ab levels. We conclude that long-term (>3 months) PI does not reduce Gal Ab levels sufficiently to be of clinical value in xenotransplantation.

Adult↗
PubMed2000

Development and analysis of transgenic mice expressing porcine hematopoietic cytokines: a model for achieving durable porcine hematopoietic chimerism across an extensive xenogeneic barrier.

The capacity of mixed hematopoietic chimerism to induce tolerance has not been demonstrated in discordant xenogeneic species combinations because of the difficulty in achieving lasting hematopoietic engraftment. In an effort to create a model of long-lasting disparate xenogeneic hematopoietic chimerism, we have developed transgenic (Tg) mice carrying porcine cytokines. Three lines of Tg mice were generated: one carrying porcine IL-3 and GM-CSF genes only (termed IL/GM) and the remaining two lines carrying in addition, the soluble SCF gene (termed IL/GM/sS) or membrane-bound SCF gene (termed IL/GM/mS). Sera from mice with IL/GM and IL/GM/sS transgenes markedly stimulated the proliferation of swine marrow cells in vitro. However, proliferation of swine marrow cells was not induced in cultures containing IL/GM/mS sera. Consistent with these observations, ELISA assays revealed detectable levels of porcine cytokines in the sera of IL/ GM and IL/GM/sS, but not in sera of IL/GM/mS Tg mice. Marrow stromal cells prepared from all three kinds of Tg mice, but not those from non-Tg littermates, were capable of supporting the growth of porcine hematopoietic cells in vitro. Immunodeficient Tg mice were generated by crossing Tg founders with C.B-17 SCID mice for five generations. All Tg immunodeficient mice showed improved porcine hematopoietic engraftment compared with non-Tg controls. These Tg mice provide a useful model system for studying porcine hematopoietic stem cells, and for evaluating the feasibility of donor-specific tolerance induction by mixed chimerism across highly disparate xenogeneic barriers.

Animals↗
PubMed2000

Plasma perfusion by apheresis through a Gal immunoaffinity column successfully depletes anti-Gal antibody: experience with 320 aphereses in baboons.

BACKGROUND: Anti-Galalpha1-3Gal (Gal) antibodies (Gal Ab) contribute to the rejection of porcine organs transplanted into primates. Extracorporeal immunoadsorption (EIA) has been developed to eliminate Gal Ab from the circulation. METHODS: Between 1995 and 1999 we performed 320 EIAs in baboons using a COBE-Spectra apheresis unit incorporating a synthetic Gal immunoaffinity column. Three plasma volumes were immunoadsorbed on each occasion. The 221 consecutive EIAs performed in 41 immunosuppressed baboons between January 1997 and April 1999 form the basis of this review. Of these 41 baboons, 29 underwent a series of three or four EIAs at daily intervals, seven had multiple series of three EIAs, and the remainder underwent single or double EIAs. Serum Gal Ab levels were monitored by ELISA before and at intervals after the course of EIA. RESULTS: There were two fatal complications, one from a respiratory mishap (unrelated to the EIA) and one from persistent hypotension unresponsive to therapeutic interventions. Seven procedures (3%) were terminated early owing to technical difficulties and/or persistent hypotension. Mean pre-EIA Gal Ab levels in naive baboons were 33.1 microg/ml (IgM) and 14.5 microg/ml (IgG). Immediately after three consecutive EIAs, IgM was depleted by a mean of 97.3% and IgG by 99.4%. By 18 to 24 h later, Gal Ab was returning but depletion remained at 80.1% (IgM) and 84.7% (IgG). The subsequent rate of return of Gal Ab depended on the immunomodulatory protocol used. CONCLUSIONS: (1) With appropriate monitoring, EIA is an acceptably safe procedure, even in small (<10 kg) baboons. (2) Three consecutive EIAs are effective in removing >97% of Gal Ab. (3) In the majority of cases, return of Gal Ab begins within 24 h, irrespective of the immunomodulatory protocol.

Animals↗
PubMed1999

Transfer of swine major histocompatibility complex class II genes into autologous bone marrow cells of baboons for the induction of tolerance across xenogeneic barriers.

BACKGROUND: The present study examined the potential role of gene therapy in the induction of tolerance to anti-porcine major histocompatibility complex (SLA) class II-mediated responses after porcine renal or skin xenografts. METHODS: Baboons were treated with a non-myeloablative or a myeloablative preparative regimen before bone marrow transplantation with autologous bone marrow cells retrovirally transduced to express both SLA class II DR and neomycin phosphotransferase (NeoR) genes, or the NeoR gene alone. Four months or more after bone marrow transplantation, the immunological response to a porcine kidney or skin xenograft was examined. Both the renal and skin xenografts were SLA DR-matched to the transgene, and recipients were conditioned by combinations of complement inhibitors, adsorption of natural antibodies, immunosuppressive therapy, and splenectomy. RESULTS: Although the long-term presence of the SLA transgene was detected in the peripheral blood and/or bone marrow cells of all baboons, the transcription of the transgene was transient. Autopsy tissues were available from one animal and demonstrated expression of the SLA DR transgene in lymphohematopoietic tissues. After kidney and skin transplantation, xenografts were rejected after 8-22 days. Long-term follow-up of control animals demonstrated that high levels of induced IgG antibodies to new non-alphaGal epitopes developed after organ rejection. In contrast, induced non-alphaGal IgG antibody responses were minimal in the SLA DR-transduced baboons. CONCLUSIONS: Transfer and expression of xenogeneic class II DR transgenes can be achieved in baboons. This therapy may prevent late T cell-dependent responses to porcine xenografts, which include induced non-alphaGal IgG antibody responses.

Animals↗
PubMed1999

Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption.

BACKGROUND: Xenotransplantation would provide a solution to the current shortage of organs for transplantation. Our group has been successful in inducing tolerance in mice and monkey models of allogeneic transplantation. The present study attempts to extend the same tolerance-inducing regimen to a pig-to-baboon organ transplantation model. METHODS: Nine baboons underwent a conditioning regimen (consisting of nonmyeloablative or myeloablative whole body and thymic irradiation, splenectomy, antithymocyte globulin, pharmacologic immunosuppression and porcine bone marrow transplantation [BMTx]), which has previously been demonstrated to induce donor-specific allograft tolerance in monkeys. In addition, immunoadsorption of anti-alphaGal antibody (Ab) was performed. Four of the nine baboons received pig kidney transplants (KTx), and one also underwent repeat transplantation with an SLA-matched kidney. Two received heterotopic pig heart transplants (HTx). Three baboons underwent conditioning without organ transplantation for long-term studies of natural Ab kinetics. RESULTS: In the three baboons that received the conditioning regimen without an organ transplant, immunoadsorption reduced Ab by approximately 90%, but recovery of Ab to pretreatment level or higher occurred within 7 days. In contrast, the level of Ab remained low after organ transplant. No Ab to pig antigens other than alphaGal was detected in any baboon before or after BMTx, KTx, or HTx. No graft succumbed to hyperacute rejection. KTx function began to deteriorate within 3-6 days, with oliguria and hematuria progressing to anuria, and the kidneys were excised after 3, 6, 9, 11, and 14 days, respectively. One HTx ceased functioning at 8 days; the second baboon died with a contracting HTx at 15 days. Features of coagulopathy and thrombocytopenia developed in all six transplanted baboons (high D-dimer, prolonged prothrombin time and partial thromboplastin time, and falling fibrinogen) resulting in serious bleeding complications in two baboons, one of which died on day 9. Donor organs showed progressive acute humoral rejection with deposits of IgM, IgG, and complement; a focal mononuclear cellular infiltrate was also observed. The ureter was the earliest structure of the KTx affected by rejection, with progression to necrosis. CONCLUSIONS: This conditioning regimen prevented hyperacute rejection but was ineffective in preventing the return of Ab, which was associated with the development of acute humoral rejection with features of coagulopathy. No baboon developed anti-pig Ab other than alphaGal Ab. Further modifications of the protocol directed toward suppression of production of Ab are required to successfully induce tolerance to pig organs in baboons.

Animals↗
PubMed1999

Different responses of human anti-HLA and anti-alphagal antibody to long-term intravenous immunoglobulin therapy.

Concentrated human immunoglobulin (IVIG) has been administered intravenously in the treatment of autoimmune disorders and to reduce anti-HLA antibodies in highly sensitized patients awaiting organ transplantation. It has also been shown, in experimental animals, to prevent the hyperacute rejection of discordant xenografts, possibly by anticomplement activity. The aim of the present study was to assess the effect of IVIG therapy on both acquired anti-HLA antibodies and natural antigalactose alpha1-3 galactose (alphaGal) antibodies in five patients awaiting heart transplantation. Five patients placed on mechanical circulatory support who had developed high HLA panel-reactive antibodies (PRA) or in whom the percentage of PRA was increasing rapidly were treated weekly with 500 mg/kg IVIG, which contained 1% of anti-alphaGal IgG. Levels of PRA, anti-alphaGal IgG and IgM, and serum cytotoxicity to pig cells were measured before, during, and after therapy. PRA percentages in the five patients were initially 85%, 53%, 23%, 19% and 19% (mean 39%). Mean PRA fell by 66% after 3 months of therapy (to a mean PRA of 14%), and by 96% after 6 months therapy (to a mean PRA of 2%). Anti-alphaGal antibody levels and serum cytotoxicity to pig aortic endothelial cells did not change significantly. These results confirm the effectiveness of IVIG therapy in reducing PRA in HLA highly sensitized patients. It is likely that IVIG does not contain the relevant anti-HLA antibody, resulting in an accelerated catabolism of native alloantibodies. However, as IVIG contains a normal level of anti-alphaGal IgG, catabolism of anti-alphaGal IgG is not modified, as it is being continuously replaced. To achieve a decrease in the anti-alphaGal IgG level it would be necessary to use IVIG depleted of this antibody.

Adult↗
PubMed1999

Immunohistologic evaluation of mechanisms mediating hyperacute lung rejection, and the effect of treatment with K76-COOH, FUT-175, and anti-Gal column immunoadsorption.

Although most investigators agree that lung dysfunction occurs rapidly in various pig-to-primate hyperacute lung rejection (HALR) models, the basic mechanisms mediating this phenomenon remain in question. Here we describe an immunohistochemical method for assessment of mechanisms driving HALR. Using an established model wherein piglet lungs are perfused ex vivo with human blood, six experimental groups (K76 COOH; FUT-175; K76 with FUT; anti-alpha-Gal column adsorption; column with FUT; and column with K76) and two control groups (unmodified human blood; autologous pig blood) were studied. Each lung was biopsied serially during perfusion, and assessed using an immunohistochemical technique, with vWF staining as an internal control to quantitate binding of human IgM, IgG, C3, C5b-9, properdin, and C1q. The effect of each treatment and subsequent lung perfusion on IgG and IgM anti-alpha-Gal titers(by ELISA) and on pig endothelial cell cytotoxicity were correlated with histologic findings. We found that [1] the classical complement activation pathway was activated, as has been shown for other pig organs in primate or human blood environments [2]; alternative complement pathway activation is also seen, which has not been described for other organs in pig-to-primate models, but only in the context of classical pathway activation; and [3] anti-Gal column absorption, pharmacologic inhibition of complement, or combination therapy each was associated with histologic evidence of partial protection, consistent with what would be predicted for each intervention. Further, immunohistologic differences correlated with physiologic outcomes [8] and with antibody assay results, and revealed that treatments used were incompletely effective. Our data suggest that more complete inhibition of antibody- and complement-driven pathways than was achieved in these experiments will be necessary to prevent the antibody and complement-mediated facets of hyperacute lung rejection. This immunohistologic technique may also help us identify additional pathogenic mechanisms important to eventual clinical application of pig-to-human lung xenografts.

Acute Disease↗
PubMed1998

A phase II study of BTI-322, a monoclonal anti-CD2 antibody, for treatment of steroid-resistant acute graft-versus-host disease.

BTI-322, a rat monoclonal IgG2b directed against the CD2 antigen on T cells and natural killer (NK) cells, blocks primary and memory alloantigen proliferative responses in vitro. We have evaluated the pharmacokinetics and safety of BTI-322 during treatment of 20 transplant recipients with steroid-refractory acute graft-versus-host disease (GVHD). Treatment consisted of BTI-322 by intravenous (IV) bolus or 30-minute infusion at approximately 0.1 mg/kg/d for 10 days in addition to continuing high-dose steroids and tacrolimus or cyclosporine. Pharmacokinetic sampling was performed in 10 patients; the t1/2 +/- SE was 9.1 +/- 1.3 hours, the Cmax was 2,549 +/- 291 ng/mL, the Vd was 3.97 +/- 0.95 L, and the Vd/kg was 0. 05 +/- 0.01 L/kg. Ten patients experienced transient dyspnea sometimes accompanied by nausea, vomiting, diarrhea, and tachycardia shortly after the initial bolus dose of drug, but serious drug-related adverse events were not seen during the remainder of the infusions. At the end of treatment (day 11), there were six patients with complete responses and five with a reduction in grade of GVHD for a total response rate of 55% (95% confidence interval [CI], 32% to 77%). Antibodies targeting CD2 may be active in the treatment of acute GVHD, and evaluation of a humanized form of BTI-322 is warranted.

Adult↗
PubMed1998

Anti-Gal(alpha)1-3Gal antibody response to porcine bone marrow in unmodified baboons and baboons conditioned for tolerance induction.

BACKGROUND: Mixed lymphohematopoietic chimerism can provide an effective means of inducing longterm immunological tolerance and has been documented in a monkey allograft model. A conditioning regimen including nonmyeloablative or myeloablative irradiation and splenectomy has been used to induce chimerism in a pig-to-primate transplantation model. Since the presence of anti-Gal(alpha)1-3Gal (alphaGal) natural antibodies leads to the hyperacute rejection of pig organs transplanted into primates, extracorporeal immunoaffinity adsorption (EIA) of anti-alphaGal antibodies is also included in the regimen. The effect of the tolerance induction protocol on the anti-alphaGal antibody response has been assessed. METHODS: Anti-alphaGal antibody was measured after the EIA of plasma through an alphaGal immunoaffinity column in baseline studies involving two unmodified baboons, one splenectomized baboon, and one baboon that received a challenge with porcine bone marrow (BM), and in three groups of baboons (n=2 in each group) that received different conditioning regimens for tolerance induction. Group 1 received a nonmyeloablative conditioning regimen without porcine BM transplantation. Group 2 received nonmyeloablative conditioning with pig BM transplantation and pig cytokine therapy. Group 3 received myeloablative conditioning, an autologous BM transplant (with BM depleted of CD2+ or CD2+/CD20+ cells), and pig BM transplantation. RESULTS: In the baseline studies, a single EIA of anti-alphaGal antibodies in an unmodified animal initially depleted anti-alphaGal antibody, followed by a mild rebound. Nonmyeloablative conditioning (group 1) in the absence of pig cell exposure reduced the rate of anti-alphaGal antibody return. Pig BM cells markedly stimulated anti-alphaGal antibody production in an unmodified baboon (alphaGal IgM and IgG levels increased 40- and 220-fold, respectively). This response was significantly reduced (to an only 2- to 5.5-fold increase of IgM and IgG) in baboons undergoing nonmyeloablative conditioning (group 2). A myeloablative conditioning regimen (group 3) prevented the antibody response to pig BM, with the reduction in response being greater in the baboon that received autologous BM depleted of both CD2+ and CD20+ cells. No new antibody directed against pig non-aGal antigens was detected in any baboon during the 1 month follow-up period. CONCLUSIONS: (i) EIA of anti-alphaGal antibody in unmodified baboons results in a transient depletion followed by a mild rebound of antibody; (ii) exposure to pig BM cells results in a substantial increase in anti-alphaGal antibody production; (iii) a nonmyeloablative conditioning regimen reduces the rate of antibody return and (iv) markedly reduces the response to pig BM cells; (v) the anti-alphaGal response is completely suppressed by a myeloablative regimen if CD2+ and CD20+ cells are eliminated from the autologous BM inoculum. Furthermore, (vi) challenge with pig BM cells appears to stimulate only an anti-alphaGal antibody response without the development of other (non-alphaGal) anti-pig antibodies. We conclude that regimens used for T-cell tolerance induction can be beneficial in reducing the anti-alphaGal antibody response to porcine BM.

Animals↗
PubMed1998

Removal of anti-porcine natural antibodies from human and nonhuman primate plasma in vitro and in vivo by a Galalpha1-3Galbeta1-4betaGlc-X immunoaffinity column.

BACKGROUND: Natural antibodies (NAbs) against a terminal alpha1-3 galactosyl (alphaGal) epitope have been identified as the major human anti-pig NAbs. METHODS AND RESULTS: We used two synthetic alphaGal trisaccharides--type 6 (alphaGal6) and type 2(alphaGal2)--linked to an inert matrix to remove NAbs from human plasma in vitro. Flow cytometry indicated that an average of 85% of the NAb binding activity was depleted by adsorption with alphaGal6. By measuring the binding of NAbs to pig peripheral blood mononuclear cells and bone marrow cells, we demonstrated that alphaGal6 was more effective than alphaGal2 in removing NAbs, and the combination of alphaGal6 + alphaGal2 did not further increase removal of NAbs. The specificity of the removal of NAbs (IgM and IgG) reactive with the alphaGal epitope by alphaGal6 matrix was shown by enzyme-linked immunosorbent assay. In vivo studies in nonhuman primates compared plasma perfusion through a alphaGal6 immunoaffinity column with hemoperfusion through a pig liver for changes in blood pressure, hematocrit, platelets, and NAb adsorption. CONCLUSIONS: Both methods reduced the level of anti-pig IgM and IgG xenoreactive antibodies to nearly background, but column perfusion caused less hypotension and reduction in platelets than liver perfusion. Four pig kidneys transplanted into monkeys after column perfusion did not undergo hyperacute rejection, remaining functional for 2-10 days, with a mean functional period of 7 days, demonstrating that a pig kidney can support renal function in a primate.

Animals↗