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T Kozlowski

Publications and source records attributed to T Kozlowski.

At least 19 recordsLinked to original sources
PubMed2006

Isolated donor specific alloantibody-mediated rejection after ABO compatible liver transplantation.

Antibody-mediated rejection (AMR) after liver transplantation is recognized in ABO incompatible and xeno-transplantation, but its role after ABO compatible liver transplantation is controversial. We report a case of ABO compatible liver transplantation that demonstrated clinical, serological and histological signs of AMR without evidence of concurrent acute cellular rejection. AMR with persistently high titers of circulating donor specific antibodies resulted in graft injury with initial centrilobular hepatocyte necrosis, fibroedematous portal expansion mimicking biliary tract outflow obstruction, ultimately resulting in extensive bridging fibrosis. Immunofluorescence microscopy demonstrated persistent, diffuse linear C4d deposits along sinusoids and central veins. Despite intense therapeutic intervention including plasmapheresis, IVIG and rituximab, AMR led to graft failure. We present evidence that an antibody-mediated alloresponse to an ABO compatible liver graft can cause significant graft injury independent of acute cellular rejection. AMR shows distinct histologic changes including a characteristic staining profile for C4d.

ABO Blood-Group System↗
PubMed2004

Precise measurement of the pi+-->pi0 e+nu branching ratio.

Using a large acceptance calorimeter and a stopped pion beam we have made a precise measurement of the rare pi(+)-->pi(0)e(+)nu (pi(beta)) decay branching ratio. We have evaluated the branching ratio by normalizing the number of observed pi(beta) decays to the number of observed pi(+)-->e(+)nu (pi(e2)) decays. We find the value of Gamma(pi(+)-->pi(0)e(+)nu)/Gamma(total)=[1.036+/-0.004(stat)+/-0.004(syst)+/-0.003(pi(e2))]x10(-8), where the first uncertainty is statistical, the second systematic, and the third is the pi(e2) branching ratio uncertainty. Our result agrees well with the standard model prediction.

Journal Article↗
PubMed2004

Precise measurement of the pion axial form factor in the pi+-->e+nugamma decay.

We have studied radiative pion decays pi(+)-->e(+)nugamma in three broad kinematic regions using the PIBETA detector and a stopped pion beam. Based on Dalitz distributions of 41 601 events we have evaluated absolute pi-->enugamma branching ratios in the three regions. Minimum chi(2) fits to the integral and differential (E(e(+)),E(gamma)) distributions result in the axial-to-vector weak form factor ratio of gamma identical with F(A)/F(V)=0.443(15), or F(A)=0.0115(4) with F(V)=0.0259. However, deviations from standard model predictions in the high-E(gamma)-low-E(e(+)) kinematic region indicate the need for further theoretical and experimental work.

Journal Article↗
PubMed2000

Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates.

BACKGROUND: Efforts to achieve tolerance to transplanted pig organs in nonhuman primates by the induction of a state of mixed hematopoietic chimerism have been associated with disorders of coagulation and thrombosis. Activation of recipient vascular endothelium and platelets by porcine hematopoietic cells and/or activation of donor organ vascular endothelium and/or molecular differences between the species may play roles. Irradiation or drug therapy could possibly potentiate endothelial cell activation and/or injury. METHODS: We have investigated parameters of coagulation and platelet activation in nonhuman primates after (1) a regimen aimed at inducing mixed hematopoietic chimerism and tolerance (TIR that included total body irradiation, T cell depletion, and splenectomy; (2) pig bone marrow or pig peripheral blood mobilized progenitor cell transplantation (PCTx); and/or (3) pig organ transplantation (POTx). Five experimental groups were studied. Baboons were the recipient subjects in all groups except Group 1. Gp 1 Cynomolgus monkeys (n=6) underwent TIR + allotransplantation of hematopoietic cells and a kidney or heart or TIR + concordant xenotransplantation (using baboons as donors) of cells and a kidney; Gp 2 Baboons (n=4) underwent TIR with or without (+/-) autologous hematopoietic cell infusion; Gp 3 (n=12) PCTx+/-TIR; Gp 4 (n=5) POTx+/-TIR; Gp 5 (n=4) TIR + PCTx + POTx. Platelet counts, with plasma prothrombin time, partial thromboplastin time, fibrinogen levels, fibrin split products and/or D-dimer were measured. RESULTS: In the absence of a discordant (porcine) cellular or organ transplant (Groups 1 and 2), TIR resulted in transient thrombocytopenia only, in keeping with bone marrow depression from irradiation. PCTx alone (Group 3) was associated with the rapid development of a thrombotic thrombocytopenic (TTP)-like microangiopathic state, that persisted longer when PCTx was combined with TIR. POTx (+/-TIR) (Group 4) was associated with a gradual fall (over several days) in platelet counts and fibrinogen with disseminated intravascular coagulation (DIC); after graft excision, the DIC generally resolved. When TIR, PCTx and POTx were combined (Group 5), an initial TTP-like state was superseded by a consumptive picture of DIC within the first week, necessitating graft removal. CONCLUSIONS: Both PCTx and POTx lead to profound alterations in hemostasis and coagulation parameters that must be overcome if discordant xenotransplantation of hematopoietic cells and organs is to be fully successful. Disordered thromboregulation could exacerbate vascular damage and potentiate activation of coagulation pathways after exposure to xenogeneic cells or a vascularized xenograft.

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↗
PubMed2000

Acute humoral xenograft rejection: destruction of the microvascular capillary endothelium in pig-to-nonhuman primate renal grafts.

The major cause of xenograft loss beyond hyperacute rejection is a form of injury, traditionally termed delayed xenograft rejection (DXR), whose pathogenesis is unknown. Here we analyze the immunologic and morphologic features of DXR that develops in pig kidney xenografts transplanted into nonhuman primates. Kidneys from miniature swine were transplanted into cynomolgus monkeys (n = 14) or baboons (n = 11) that received regimens aimed to induce mixed chimerism and tolerance. No kidney was rejected hyperacutely. Morphologic and immunohistochemical studies were performed on serial biopsies, and an effort was made to quantify the pathologic features seen. The early phase of DXR (Days 0-12) was characterized by focal deposition of IgM, IgG, C3, and scanty neutrophil and macrophage infiltrates. The first abnormality recognized was glomerular and peritubular capillary endothelial cell death as defined by in situ DNA nick-end labeling (TUNEL). Damaged endothelial cells underwent apoptosis and, later, frank necrosis. The progressive phase developed around Day 6 and was characterized by progressive deposition of IgM, IgG, C3, and prominent infiltration of cytotoxic T cells and macrophages, with a small number of NK cells. Thrombotic microangiopathy developed in the glomeruli and peritubular capillaries with TUNEL+ endothelial cells, platelet aggregation, and destruction of the capillary network. Only rare damaged arterial endothelial cells and tubular epithelial cells were observed, with rare endothelialitis and tubulitis. In the advanced phase of DXR, interstitial hemorrhage and infarction occurred. During the development of DXR, the number of TUNEL+ cells increased, and this correlated with progressive deposition of antibody. The degree of platelet aggregation correlated with the number of TUNEL+ damaged endothelial cells. We conclude that peritubular and glomerular capillary endothelia are the primary targets of renal DXR rather than tubular epithelial cells or arterial endothelium and that the earliest detectable change is endothelial cell death. DXR was characterized by progressive destruction of the microvasculature (glomeruli and peritubular capillaries) and formation of fibrin-platelet thrombi. Both cytotoxic cells and antibodies potentially mediate the endothelial damage in DXR; however, in this model, DXR is largely humorally mediated and is better termed "acute humoral xenograft rejection."

Acute Disease↗
PubMed2000

Decreased graft-versus-host disease after haplotype mismatched bone marrow allografts in miniature swine following interleukin-2 treatment.

Graft-versus-host disease (GVHD) is an important complication of bone marrow transplantation after transplants between HLA-mismatched donor/recipient pairs. In mice, giving IL-2 post transplant decreases GVHD in this setting. We studied high-dose IL-2 therapy in pigs. Transplants were carried out after conditioning with fractionated total body radiation and cyclophosphamide. Fourteen pigs received a fully mismatched bone marrow transplant (six with IL-2; eight without IL-2), and six received a single haplotype class II mismatched transplant (three with IL-2; three without IL-2). GVHD was evaluated by skin histology. All fully mismatched recipients had severe GVHD (grade 2-3) and died within 13 to 51 days whether or not they received IL-2. Pigs receiving a one haplotype class II mismatched transplant without IL-2 developed severe skin GVHD lasting for 8-45 days; all died within 57 days. Similar pigs receiving IL-2 post transplant had no or only mild skin GVHD for less than 15 days; two are long-term survivors. Bone Marrow Transplantation (2000) 25, 47-52.

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

Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcine-specific growth factors.

BACKGROUND: Mixed allogeneic hematopoietic chimerism has previously been reliably achieved and shown to induce tolerance to fully MHC-mismatched allografts in mice and monkeys. However, the establishment of hematopoietic chimerism has been difficult to achieve in the discordant pig-to-primate xenogeneic model. METHODS: To address this issue, two cynomolgus monkeys were conditioned by whole body irradiation (total dose 300 cGy) 6 and 5 days before the infusion of pig bone marrow (BM). Monkey anti-pig natural antibodies were immunoadsorbed by extracorporeal perfusion of monkey blood through a pig liver, immediately before the intravenous infusion of porcine BM (day 0). Cyclosporine was administered for 4 weeks and 15-deoxyspergualin for 2 weeks. One monkey received recombinant pig cytokines (stem cell factor and interleukin 3) for 2 weeks, whereas the other received only saline as a control. RESULTS: Both monkeys recovered from pancytopenia within 4 weeks of whole body irradiation. Anti-pig IgM and IgG antibodies were successfully depleted by the liver perfusion but returned to pretreatment levels within 12-14 days. Methylcellulose colony assays at days 180 and 300 revealed that about 2% of the myeloid progenitors in the BM of the cytokine-treated recipient were of pig origin, whereas no chimerism was detected in the BM of the untreated control monkey at similar times. The chimeric animal was less responsive by mixed lymphocyte reaction to pig-specific stimulators than the control monkey and significantly hyporesponsive when compared with a monkey that had rejected a porcine kidney transplant. CONCLUSION: To our knowledge, this is the first report of long-term survival of discordant xenogeneic BM in a primate recipient.

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

Effect of pig-specific cytokines on mobilization of hematopoietic progenitor cells in pigs and on pig bone marrow engraftment in baboons.

Mixed hematopoietic chimerism has been found to be a requirement for achieving specific immunologic hyporesponsiveness. Some of the requirements for in vitro and in vivo coexistence of discordant hematopoietic systems in the pig-to-baboon (or human) model have been investigated. We have tested the efficacy of pig-specific cytokines (PSC) (IL3, SCF, GM-CSF) in the mobilization of porcine bone marrow (BM) progenitors in vivo (i) in the pig and (ii) in baboons that underwent a conditioning regimen and porcine BM transplantation. In a preliminary in vitro study, porcine BM cells were incubated in various media to assess the effect of human plasma on pig progenitors in a colony-forming unit (CFU) assay. In in vivo studies, four pigs received PSC and one control pig did not. Six baboons underwent natural antibody removal, with subsequent pig BM transplantation. Four of these six underwent nonmyeloablative (n=2) or myeloablative (n=2) conditioning and all received PSC treatment. Two baboons did not receive PSC, one of which underwent a nonmyeloablative regimen. Sequential blood samples and BM biopsies in pigs and baboons were analyzed by CFU assay for the detection of porcine cells. Baboon samples were analyzed by polymerase chain reaction (PCR) to detect porcine DNA. In the case of the in vitro tests, colony forming by porcine progenitors was not inhibited by media containing human plasma and for the in vivo tests, PSC increased the number of progenitors in pig BM; mobilization of progenitors into the peripheral blood was observed. PSC-treated baboons which experienced transient depletion of leukocytes < 1,000/ml (as an effect of the conditioning regimen) had porcine BM cells detectable by PCR for as long as day 316 after BM transplantation. In conclusion we found that: (i) under the conditions of these studies, in vitro porcine progenitor cell growth was not inhibited by human plasma containing natural antibody and complement; (ii) PSC treatment led to an increased number of progenitors in pig BM and peripheral blood; (iii) the combination of an effective conditioning regimen and treatment with PSC was capable of inducing long-term survival of pig progenitors in baboons, although only a low level of engraftment was achieved.

Animals↗
PubMed1998

Disseminated intravascular coagulation in association with the delayed rejection of pig-to-baboon renal xenografts.

BACKGROUND: Intravascular fibrin deposition and platelet sequestration occur with porcine xenograft rejection by baboons. Disseminated intravascular coagulopathy may arise either as a direct consequence of the failure to fully deplete xenoreactive natural antibodies and block complement, or because of putative cross-species molecular incompatibilities in this discordant species combination. METHODS: Three baboons were conditioned with retrovirally transduced autologous bone marrow to induce tolerance to swine antigens. Xenoreactive natural antibodies and complement were depleted by plasmapheresis and the use of Gal alpha1-3Gal column adsorptions; baboons were then splenectomized and underwent renal xenografting from inbred, miniature pigs. Soluble complement receptor type-1 with protocol immunosuppression (mycophenolate mofetil, 15-deoxyspergualin, steroids, and cyclosporine) was administered. RESULTS: A bleeding diathesis was clinically evident from days 5 to 12 after transplantation in two baboons. Low levels of circulating C3a, C3d, and iC3b were measured despite the absence of functional circulating complement components. Profound thrombocytopenia with abnormalities in keeping with disseminated intravascular coagulopathy were observed. Prolongation of prothrombin and partial thromboplastin times was accompanied by evidence for tissue factor-mediated coagulation pathways, high levels of thrombin generation (prothrombin fragment F(1+2) production and thrombin-antithrombin complex formation), fibrinogen depletion, and production of high levels of the fibrin degradation product D-dimer. Importantly, these disturbances resolved rapidly after the excision of the rejected xenografts in two surviving animals. Histopathological examination of the rejected xenografts confirmed vascular injury, fibrin deposition, platelet deposition, and localized complement activation. CONCLUSIONS: Systemic coagulation disturbances are associated with delayed xenograft rejection.

Animals↗
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↗
PubMed1998

Depletion of anti-Gal(alpha)1-3Gal antibody in baboons by specific alpha-Gal immunoaffinity columns.

Ongoing studies at our center on facilitating transplantation of discordant xenogeneic organs are focused on tolerance induction. To abrogate hyperacute rejection, we have used adsorption methods to eliminate natural anti-Gal(alpha)1-3Gal (alphaGal) antibodies from the circulation of baboons. We have analyzed data concerning antibody removal in baboons that were 1) immunologically naive, 2) receiving conventional pharmacologic immunosuppressive therapy (IS), and 3) treated with a conditioning regimen for tolerance induction. We compared the efficiency of removing alphaGal antibody 1) by perfusion of whole blood through an alphaGal affinity column (CP; n=5) with 2) perfusion of plasma (separated from cellular components by apheresis) through an alphaGal column (CPA; n=39). Our studies demonstrate that 1) CP and CPA are equally effective in removing anti-alphaGal antibody, 2) CPA is the method of choice if multiple adsorptions are required, 3) CPA in naive animals transiently affects levels of total IgG and IgM, 4) four CPAs repeated at 2-4 day intervals in association with heavy IS reduce the pool of anti-alphaGal antibody and total Ig, and 5) splenectomy and/or IS delay the return of anti-alphaGal antibody.

Animals↗
PubMed1998

Pharmacologic immunosuppressive therapy and extracorporeal immunoadsorption in the suppression of anti-alphaGal antibody in the baboon.

The aim of this study was to deplete baboons of anti-(alpha)galactosyl (alphaGal] antibody and attempt to maintain depletion by pharmacologic immunosuppressive therapy (PI). In 12 experiments, involving nine baboons, repeated extracorporeal immunoadsorption (EIA) was carried out by plasma perfusion through immunoaffinity columns of synthetic alphaGal trisaccharide type 6. Five of the baboons were immunologically naive and four had undergone various procedures at least 6 months previously. All, however, had recovered lymphohematopoietic function and (with one exception) had levels of anti-alphaGal antibody within the normal range. Eleven protocols included continuous i.v. cyclosporine (to maintain whole blood levels of approximately 1,600 ng/ml). In addition, in ten protocols, the baboon received one or more of the following drugs: cyclophosphamide (1-20 mg/kg/day), mycophenolate mofetil (70-700 mg/ kg/day), brequinar sodium (1-12 mg/kg/day), prednisolone (1 mg/kg/day), melphalan (0.15-0.6 mg/kg/day), methylprednisolone (125 mg/day x3), and antilymphocyte globulin (ATG) (50 mg/kg/day x3). EIA was carried out on 1-9 occasions in each study and was temporarily successful in removing all antibody. When no PI was administered, antibody returned close to pre-EIA levels within 48 hr. Cyclosporine alone delayed the rate of antibody return only slightly. While EIA was continuing on a daily or alternate day schedule, antibody levels (both IgM and IgG) were maintained at 20-45% of pre-EIA levels. Once EIA was discontinued but PI maintained, IgM rose to 40-90% and IgG to 30-60% of pre-EIA levels. In vitro testing demonstrated significant cytotoxicity to pig cells at these antibody levels. We conclude that i) EIA utilizing columns of alphaGal trisaccharide is successful in temporarily depleting baboons of anti-alphaGal antibody, but ii) none of the PI regimens tested suppressed antibody production to levels which would be expected to prevent antibody-mediated rejection of pig xenografts. Additional strategies will therefore be required if xenotransplantation is to become a clinical reality.

ABO Blood-Group System↗
PubMed1997

Pig to monkey bone marrow and kidney xenotransplantation.

BACKGROUND: The intensity of discordant xenograft cellular rejection makes it unlikely that safe doses of immunosuppressive drugs will alone be sufficient to permit long-term survival. We have therefore concentrated our efforts on establishing tolerance to xenogeneic organs through lymphohematopoietic chimerism and the elimination of preformed natural antibodies (nAbs). METHODS: Here we report the most recent series of 11 technically successful porcine to nonhuman primate transplantation procedures. In eight experimental animals induction therapy consisted of (1) 3 x 100 cGy nonlethal whole body irradiation (day -6 and day -5) to all animals, (2) horse anti-human thymocyte globulin (day -2, day -1, and day 0) to seven of the animals, (3) 700 cGy thymic irradiation (day -1) to five of the animals, and (4) pig bone marrow infused on day 0 (2-9 x 10(8)/cells/kg). On day 0, just before the renal xenograft, the recipient was splenectomized, and antipig nAbs were removed by means of perfusion of the monkey's blood through either a pig liver (n = 6) or a Gal-alpha (1,3)-Gal adsorption column (n = 5). There control animals did not receive this pretransplantation induction therapy but did undergo hemoperfusion and posttransplantation immunosuppression identical to the experimental animals. All 11 recipients were treated after transplantation with cyclosporin A and 15-deoxyspergualin. Recombinant pig-specific growth factors (interleukin-3 and stem cell factor) were given to six experimental animals from day 0 until the termination of the experiment. RESULTS: Analysis of recipients' sera by means of flow cytometry indicated the effective removal of immunoglobulin M and immunoglobulin G nAbs by either liver perfusion or column adsorption. In the eight experimental animals, nAb titers remained low until death (up to 15 days), but in the three control animals nAb titers increased substantially with time. The longest surviving recipient maintained excellent kidney function with creatinine levels at 0.8 to 1.3 mg/dl throughout its course. Death occurred at day 15 from complications caused by a urinary leak and pancytopenia. Histologic examination of the xenograft revealed only focal tubular necrosis and cytoplasmic vacuolization, with trace amounts of fibrin and C3 in peritubular capillaries. In this animal a fraction of the peripheral blood cells (3%) at day 7 were of pig origin as detected by pig-specific monoclonal antibodies. In addition, colony-forming assays performed on a bone marrow biopsy specimen taken at day 14 indicated that approximately 30% of the relatively few myeloid progenitors detected were of swine origin. CONCLUSIONS: We have demonstrated that our protocol is effective in the prevention of hyperacute rejection and in the maintenance of excellent function of the renal xenograft for up to 15 days. These results also indicate that at least short-term engraftment of the xenogeneic donor bone marrow cells is possible to achieve in this discordant large animal combination. Longer survivals will be required to assess the possible effect of this engraftment on induction of tolerance.

Animals↗