The diagnostic utility of FIB-4 as a non-invasive tool for liver fibrosis scoring among NAFLD patients: a retrospective cross-sectional study.

OBJECTIVE: Liver biopsy is the gold standard method to evaluate patients with non-alcoholic fatty liver disease (NAFLD). However, due to its several limitations and complications, a reliable and non-invasive marker is required to assess liver fibrosis. In this study, we compared the performance of the FIB-4 index [based on age, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels and platelets count] with the Scheuer scoring system of liver biopsies to evaluate the diagnostic utility of FIB-4 among NAFLD patients with different liver fibrosis severities. PATIENTS AND METHODS: A cross-sectional study was conducted at An-Najah National University Hospital (NNUH) in Palestine. The FIB-4 index was calculated using laboratory data for 128 NAFLD patients who underwent liver biopsies between November 2014 and July 2022. The results of FIB-4 were compared with the Scheuer scoring system of liver biopsies (using F0, F1+F2, F3+F4) to determine the sensitivity and specificity of FIB-4 in detecting and staging liver fibrosis. RESULTS: Out of 128 patients involved in our study, 49 of them had advanced fibrosis according to liver biopsy (F3+F4), where their FIB-4 indices showed 87% sensitivity at 1.45 cut off point and 87% specificity at 3.25 cut off point. CONCLUSIONS: The FIB-4 index may be used as a screening tool in the primary care setting. To raise awareness of liver diseases, this non-invasive, inexpensive, simple, and quick marker could identify people in need of further liver fibrosis evaluation and diagnosis.

Renal and cardiac endothelial heterogeneity impact acute vascular rejection in pig-to-baboon xenotransplantation.

Xenograft outcomes are dictated by xenoantigen expression, for example, Gal alpha1, 3Gal (Gal), but might also depend on differing vascular responses. We investigated whether differential vascular gene expression in kidney and cardiac xenografts correlate with development of thrombotic microangiopathy (TM) and consumptive coagulation (CC). Immunosuppressed baboons underwent miniswine or hDAF pig kidney (n = 6) or heart (n = 7), or Gal-transferase gene-knockout (GalT-KO) (thymo)kidney transplantation (n = 14). Porcine cDNA miniarrays determined donor proinflammatory, apoptosis-related and vascular coagulant/fibrinolytic gene expression at defined time points; validated by mRNA, protein levels and immunopathology. hDAF-transgenic and GalT-KO xenografts, (particularly thymokidneys) exhibited prolonged survival. CC was seen with Gal-expressing porcine kidneys (3 of 6), only 1 of 7 baboons postcardiac xenotransplantation and was infrequent following GalT-KO grafts (1 of 14). Protective-type genes (heme oxygenase-I, superoxide dismutases and CD39) together with von Willebrand factor and P-selectin were upregulated in all renal grafts. Transcriptional responses in Gal-expressing xenografts were comparable to those seen in the infrequent GalT-KO rejection. In cardiac xenografts, fibrin deposition was associated with increased plasminogen activator inhibitor-1 expression establishing that gene expression profiles in renal and cardiac xenografts differ in a quantitative manner. These findings suggest that therapeutic targets may differ for renal and cardiac xenotransplants.

Preformed antibodies to alpha1,3-galactosyltransferase gene-knockout (GT-KO) pig cells in humans, baboons, and monkeys: implications for xenotransplantation.

OBJECTIVE: The aim of our study was to determine the prevalence and cytotoxicity of primate antibodies directed to antigens other than Galalpha1,3Gal (Gal), termed nonGal antigens. METHODS: Sera from human, baboon, and cynomolgus monkeys were tested by flow cytometry for IgM and IgG binding to both wild-type (WT) and GT-KO pig peripheral mononuclear cells (PBMC). Also, complement-dependent cytotoxicity assays were performed. RESULTS: All species demonstrated significantly higher antibody binding and cytotoxicity to WT cells compared to GT-KO cells (P < .01). Cynomolgus monkeys had significantly higher IgM binding to WT and GT-KO cells than did baboons or humans (P < .01). Furthermore, approximately 50% of both human and baboon sera proved to be lytic to GT-KO cells, compared to 76% of monkey sera (P < .01). CONCLUSIONS: We confirm the advantage of using GT-KO pig grafts over WT pig grafts. However, our results suggest that, compared to the cynomolgus monkey, the baboon may be a more suitable model to study antibody-mediated rejection of GT-KO pig grafts.

Initial experience with the human anti-human CD154 monoclonal antibody, ABI793, in pig-to-baboon xenotransplantation.

BACKGROUND: ABI793 (ABI) is a human monoclonal antibody (mAb) specific for human CD154. To assess the suitability of ABI for baboon transplantation studies, we carried out in vitro studies to determine ABI's reactivity with baboon cells expressing CD154, performed in vivo pharmacokinetic studies in two baboons, and tested the effect of ABI administration on elicited antibody production in two baboons undergoing either pig hematopoietic progenitor cell (PBPC) or heterotopic heart transplantation. METHODS: In vitro: Baboon peripheral blood mononuclear cells were activated in vitro to upregulate CD154, and binding of ABI to CD154 was measured by flow cytometry. In vivo: Serum levels of ABI were measured immediately before and 15 min after the intravenous administration of ABI (20 mg/kg) to two baboons over 28 days. Subsequently, ABI (25 mg/kg on days 0, 1, 4 and 7, and then 20 mg/kg every 5 days) was included in the immunosuppressive regimen in two pig-to-baboon transplants (PBPC or heart transplantation). RESULTS: In vitro: ABI was almost non-reactive to baboon T cells before stimulation, but bound to activated T cells. In vivo: In the pharmacokinetic study, trough levels of ABI (before the next dose) ranged between 190 and 580 microg/ml, and the estimated half-life was 10-15 days. There was no apparent toxicity. Following pig PBPC or heart transplantation, no elicited antibody was detected while ABI was being administered or during several weeks of follow-up. CONCLUSIONS: ABI functions in baboons, is well-tolerated, and prevents an elicited antibody response to pig antigens.

Bone marrow transplantation from alpha1,3-galactosyltransferase gene-knockout pigs in baboons.

BACKGROUND: Successful hematopoietic cell allotransplantation results in donor-specific tolerance, but this approach has been unsuccessful in the wild-type pig-to-baboon xenotransplantation model, as pig cells were lost from the circulation within 5 days. However, after cessation of immunosuppressive therapy on day 28, all baboons demonstrated non-specific unresponsiveness on mixed leukocyte reaction (MLR) for at least 30 days. We have now investigated the transplantation of bone marrow (BM) cells from miniature swine homozygous for alpha1,3-galactosyltransferase gene-knockout (GalT-KO). METHODS: Baboons (n = 3) were pre-treated with whole body and thymic irradiation, anti-thymocyte globulin, and splenectomy, and received immunosuppressive and supportive therapy for 28 days. BM was harvested from GalT-KO swine (n = 3). The baboons were monitored for the presence of pig cells by flow cytometry and colony-forming units (CFUs), and for cellular reactivity by MLR. RESULTS: A mean of 11 x 10(8) BM cells/kg was infused into each baboon. The mean absolute numbers and percentages of pig cells detected in the blood at 2 h and on days 1, 2 and 4, respectively, were 641/microl (9.5%), 132/microl (3.4%), 242/microl (3.9%), and 156/microl (2.9%). One baboon died (from accidental hemorrhage) on day 6, at which time chimerism was present in the blood (2.0%) and BM (6.4%); pig cell engraftment in the BM was confirmed by polymerase chain reaction (PCR) of CFUs. In the two other baboons, blood chimerism was lost after day 5 but returned at low levels (<1%) between days 9 to 16 and 7 to 17, respectively, indicating transient BM engraftment. Both surviving baboons showed non-specific unresponsiveness on MLR until they were euthanized on days 85 and 110, respectively. CONCLUSIONS: By using BM cells from GalT-KO pigs, chimerism was detected at levels comparable with previous studies when 30-fold more growth factor-mobilized peripheral blood progenitor cells had been transplanted. In addition, cellular hyporesponsiveness was prolonged. However, long-term engraftment and chimerism were not achieved.

In vitro and in vivo investigation of a novel monoclonal antibody to plasma cells (W5 mAb).

Natural antibodies (Abs), predominantly anti-Gal alpha 1-3Gal (Gal) Abs, in non-human primates and human beings present a major hurdle to successful pig-to-primate xenotransplantation. Attempts to inhibit anti-Gal Ab production in naïve baboons using non-specific immunosuppressive or B cell-specific reagents have failed. A new rat monoclonal antibody (W5 mAb) has been generated, which binds to all B cells, including memory cells, and to the majority of plasma cells, but not to T cells. It has been tested in vitro and in vivo. By immunoprecipitation, W5 mAb bound a human leukocyte antigen class II (HLA-DR) determinant. Sorting splenic or bone marrow W5+ cells resulted in a highly enriched anti-Gal Ab and total immunoglobulin (Ig)-secretory population. In vivo studies in baboons demonstrated that W5 mAb was safe but, despite the concomitant administration of an anti-CD154 mAb to inhibit sensitization, anti-rat Abs were detected within 10 days and inhibited the effect of the W5 mAb. High levels of W5 mAb were able to completely deplete B cells in the blood, but not in lymphoid tissues. Enzyme-linked spot-forming assay (ELISPOT) demonstrated that only 50 to 60% of secreting cells (SC) were depleted in the bone marrow. No reduction in the serum levels of anti-Gal Ab was observed. W5 mAb did not cause complete inhibition of anti-Gal Ab production, probably as a result of its inability to completely deplete B and plasma cells from all lymphoid compartments.

Differential expression of Galalpha1,3Gal epitopes on fetal and adult porcine hematopoietic cells.

Galalpha1-3Gal (Gal) is the major epitope on pig tissues bound by human natural antibodies. Xenogeneic hematopoietic cell transplantation is being investigated to induce immunological tolerance to xenografts. We have investigated the level of Gal expression on pig hematopoietic cells. Cells were collected from pig fetal liver and bone marrow (BM), and also from adult BM and peripheral blood, before and after treatment with pig-specific hematopoietic growth factors. Fluorescent activated cell sorting (FACS) analysis was performed with the M86 monoclonal antibody (specific for Gal), lineage markers, and biotinylated stem cell factor (SCF) to detect c-kit expression. In fetal pig BM and liver, there was no significant difference in Gal expression between monocytes/macrophages (myeloid cells) and lymphocytes. In adult hematopoietic cells from all sources, Gal-positive subpopulations in T cells showed weak expression of Gal, whereas B cells demonstrated higher expression, and myeloid cells showed highest expression. Adult BM and mobilized peripheral blood progenitor cells contained small populations with very low or negligible expression of Gal. A very small population of c-kit-positive cells, indicating progenitor cells, were Gal-negative. The small Gal-negative population that exists in progenitor cells might explain why some pig colony forming units (CFU) can be resistant to human serum.

Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade.

The aim of the present study was to assess the survival of adult porcine islets transplanted into baboons receiving either (I) conventional triple drug immunosuppressive therapy or (2) a non-myeloablative regimen and an anti-CD154 monoclonal antibody (mAb) aimed at tolerance-induction. Group 1 baboons (n = 3) were pancreatectomized prior to intraportal injection of 10,000 porcine islet equivalents (IE)/kg and immunosuppressed with anti-thymocyte globulin (ATG), cyclosporine and azathioprine. In Group 2 (n = 2), non-pancreatectomized baboons underwent induction therapy with whole body and thymic irradiation, and ATG. Extracorporeal immunoadsorption (EIA) of anti-Galalpha1,3Gal (Gal) antibody was carried out. Maintenance therapy was with cobra venom factor, cyclosporine. mycophenolate mofetil, methylprednisolone and anti-CD154 mAb. Porcine islets were injected intraportally (14,000 and 32,000 IE/kg, respectively) and high-dose pig mobilized peripheral blood progenitor cells (3 x 10(10) cells/kg) were infused into a systemic vein. Porcine islets were also implanted in the sternomastoid muscle to facilitate subsequent biopsies. In both groups. porcine C-peptide was measured, and histological examination of liver or sternomastoid muscle biopsies was performed at regular intervals. In Group 1, total pancreatectomy reduccd human C-peptide to < 0.1 ng/ml and induced insulin-requiring diabetes. The transplantation of porcine islets was followed by normalization of glycemia for 15-24 h. Porcine C-peptide was detected only transiently immediately after porcine islet injection (maximum 0.12 ng/ml). Histological examination of liver biopsies taken between days 2 and 19 did not reveal viable islets, but necrotic cell structures with mononuclear cell infiltrates were identified in portal venules. In Group 2, injection of porcine islets into non-pancreatectomized recipients induced a transient hypoglycemia (2-4 h) requiring concentrated intravenous dextrose administration. Porcine C-peptide was detectable for 5 and 3 days (maximum 2.8 and 1.0 ng/ml), respectively. Baboon #4 died on day 12 from small bowel intussusception. Liver and sternomastoid muscle biopsies showed well-preserved porcine islets, staining positive for insulin and glucacon, without signs of rejection. In baboon #5, viable islets were detected in the sternomastoid muscle biopsy on day 14, but not on day 28 or thereafter. A progressive mononuclear cell and macrophage infiltration was seen in the biopsies. In conclusion, conventional immunosuppression allowed survival of porcine islets in baboons for < 24 h. The non-myeloablative regimen prolonged survival of porcine islets for > 14 days. However, despite depletion of T cells, anti-Gal antibody and complement, and CD154-hlockade, porcine islets were rejected by day 28. These results suggest that powerful innate immune responses are involved in rejection of discordant xenogencic islets.

Pig hematopoietic cell chimerism in baboons conditioned with a nonmyeloablative regimen and CD154 blockade.

BACKGROUND: In an attempt to induce mixed hematopoietic chimerism and transplantation tolerance in the pig-to-primate model, we have infused high-dose porcine peripheral blood progenitor cells (PBPC) into baboons pretreated with a nonmyeloablative regimen and anti-CD154 monoclonal antibody (mAb). METHODS: Group 1 baboons (n=2) received a nonmyeloablative regimen including whole body irradiation, pharmacological immunosuppression, porcine hematopoietic growth factors, and immunoadsorption of anti-Galalpha1,3Gal (Gal) antibody before infusion of high doses of PBPC (2.7-4.6x10(10) cells/kg). In group 2 (n=5), cyclosporine was replaced by anti-CD154 mAb. Group 3 (n=3) received the group 1 regimen plus anti-CD154 mAb. RESULTS: In group 1, pig chimerism was detected in the blood by flow cytometry (FACS) for 5 days (with a maximum of 14%), and continuously up to 13 days by polymerase chain reaction (PCR). In group 2, pig chimerism was detectable for 5 days by FACS (maximum 33%) and continuously up to 28 days by PCR. In group 3, initial pig chimerism was detectable for 5 days by FACS (maximum 73%). Two of three baboons showed reappearance of pig cells on days 11 and 16, respectively. In one, in which no anti-Gal IgG could be detected for 30 days, pig cells were documented in the blood by FACS on days 16-22 (maximum 6% on day 19) and pig colony-forming cells were present in the blood on days 19-33, which we interpreted as evidence of engraftment. Microchimerism was continuous by PCR up to 33 days. CONCLUSIONS: These results suggest that there is no absolute barrier to pig hematopoietic cell engraftment in primates, and that this may be facilitated if the return of anti-Gal IgG can be prevented.

Depletion of anti-gal antibodies in baboons by intravenous therapy with bovine serum albumin conjugated to gal oligosaccharides.

BACKGROUND: Anti-Galalpha 1-3Gal (Gal) antibodies (Ab) play a key role in the rejection of pig cells or organs transplanted into primates. A course of extracorporeal immunoadsorption (EIA) of anti-Gal Ab using an immunoaffinity column of a Gal type 6 oligosaccharide depletes Ab successfully, but Ab returns during the next few days. Although therapy with an anti-CD154 monoclonal antibody (mAb) prevents an induced Ab response to Gal or non-Gal epitopes, T cell-independent natural anti-Gal IgM and IgG return to baseline (pretransplant) levels. We have investigated the capacity of continuous i.v. infusion of bovine serum albumin conjugated to Gal type 6 oligosaccharide (BSA-Gal) to deplete or maintain depletion of circulating anti-Gal Ab. METHODS: Porcine peripheral blood mobilized progenitor cells (PBPC) obtained by leukapheresis from MHC-inbred miniature swine (n=6) were transplanted into baboons. Group 1 baboons (n=4) underwent whole body (300 cGy) and thymic (700 cGy) irradiation, T cell depletion with antithymocyte globulin, complement depletion with cobra venom factor, short courses of anti-CD154 mAb therapy (20 mg/kg i.v. on alternate days), cyclosporine (CyA) (in two baboons only), mycophenolate mofetil, and porcine hematopoietic growth factors. Anti-Gal Ab depletion by EIA was carried out before transplantation of high doses (2-4x 1010 cells/kg) of PBPC. Group 2 baboons (n=3) received the group 1 regimen (including CyA) plus a continuous i.v. infusion of BSA-Gal. To prevent sensitization to BSA, anti-CD154 mAb therapy was continued until BSA-Gal administration was discontinued. RESULTS: In group 1, Gal-reactive Ab returned to pre-PBPC transplant levels within 15-21 days, but no induced Ab to Gal or non-Gal determinants developed while anti-CD154 mAb therapy was being administered. In group 2, anti-Gal Ab was either not measurable or minimally measurable while BSA-Gal was being administered. After discontinuation of BSA-Gal, Ab did not return to pre-PBPC transplant level for more than 40-60 days, and no sensitization developed even when all therapy was discontinued. In one baboon, however, Ab to Gal type 2, but not type 6, returned during BSA-Gal therapy. CONCLUSIONS: Prevention of the induced humoral response to Gal and non-Gal epitopes by anti-CD154 mAb therapy has been reported previously by our group, but our studies are the first to demonstrate a therapy that resulted in an absence of natural anti-Gal Ab for a prolonged period. The combination of BSA-Gal and T cell costimulatory blockade may facilitate survival of pig cells and organs transplanted into primates. The return in one baboon of Ab reactive with the Gal type 2 oligosaccharide, but not type 6, indicates some polymorphism of anti-Gal Ab and suggests that, to be effective in all cases, the infusion of a combination of type 6 and type 2 BSA-Gal may be required.

Pig kidney transplantation in baboons: anti-Gal(alpha)1-3Gal IgM alone is associated with acute humoral xenograft rejection and disseminated intravascular coagulation.

BACKGROUND: Kidneys harvested from miniature swine or pigs transgenic for human decay-accelerating factor (hDAF) were transplanted into baboons receiving an anti-CD154 monoclonal antibody (mAb) and either a whole body irradiation (WBI)- or cyclophosphamide (CPP)-based immunosuppressive regimen. METHODS: Group 1 baboons (n=3) underwent induction therapy with WBI and thymic irradiation, pretransplantation antithymocyte globulin, and immunoadsorption of anti-Gal(alpha)1-3Gal (Gal) antibody (Ab). After transplantation of a miniature swine kidney, maintenance therapy comprised cobra venom factor, mycophenolate mofetil, and an anti-CD154 mAb (for 14-28 days). In group 2 (n=2), WBI was replaced by CPP in the induction protocol. Group 3 (n=3) animals received the group 2 regimen, but underwent transplantation with hDAF pig kidneys. RESULTS: Group 1 and 2 animals developed features of disseminated intravascular coagulation (DIC), with reductions of fibrinogen and platelets and increases of prothrombin time, partial thromboplastin time, and fibrin split products. Graft survival was for 6-13 days. Histology showed mild acute humoral xenograft rejection (AHXR) of the kidneys, but severe rejection of the ureters. Group 3 animals developed features of DIC in two of three cases during the fourth week, with AHXR in the third case. Graft survival was for 28 (n=1) or 29 (n=2) days. Histology of day 15 biopsy specimens showed minimal focal mononuclear cellular infiltrates, with predominantly CD3+ cells. By days 28 and 29, kidneys showed mild-to-moderate features of AHXR. In all groups, the humoral response was manifest by reappearance of anti-Gal IgM below baseline level, with no or low return of anti-Gal IgG. All excised kidneys showed IgM deposition, but no complement and no or minimal IgG deposition. No baboon showed a rebound of anti-Gal Ab immediately after excision of the graft, and anti-Gal Ab increased over pretransplantation levels only when anti-CD154 mAb was discontinued. CONCLUSIONS: DIC was observed with WBI- or CPP-based therapy, and after miniature swine or hDAF kidney transplantation. AHXR+/-DIC was observed in all recipients even in the absence of complement and no or low levels of anti-Gal IgG, but was significantly delayed in the hDAF recipients. These results confirm our earlier observation that CD154 blockade prevents T cell-dependent sensitization in baboons to pig antigens, but that baseline natural anti-Gal Ab production is not inhibited. We suggest that IgM deposition, even in the absence of IgG and complement, leads to endothelial cell activation with the development of DIC, even when there are only minimal histologic changes of AHXR.

CD40-CD154 pathway blockade requires host macrophages to induce humoral unresponsiveness to pig hematopoietic cells in baboons.

The effect of CD154 blockade and macrophage depletion or inhibition on baboon humoral and cellular immune responses to pig antigens was studied in a pig-to-baboon peripheral blood mobilized progenitor cell (PBPC) transplantation model aimed at inducing tolerance. We infused pig PBPCs in baboons pretreated with a nonmyeloablative regimen along with murine anti-human CD154 monoclonal antibody (mAb) and macrophage-depleting or -inhibiting agents. Group 1 baboons (n=2) underwent a nonmyeloablative regimen and immunoadsorption of anti-Gal(alpha)1,3Gal (Gal) antibody (Ab) before intravenous infusion of high doses (1.3-4.6 x 10(10)cells/kg) of PBPCs. In group 2 (n=5), cyclosporine was replaced by 8 doses of anti-CD154 mAb over 14 days. Group 3 (n=3) received the group 2 regimen plus medronate liposomes (n=2) or commercially available human intravenous immunoglobulin G depleted of anti-Gal Ab (n=1) to deplete/inhibit recipient macrophages. Group 1 developed sensitization to Gal and also developed new Ab to non-Gal porcine antigens within 10 to 20 days. In group 2, no sensitization to Gal or non-Gal determinants was seen, but Gal-reactive antibodies did return to their preleukocyte transplantation levels. CD154 blockade, therefore, induced humoral unresponsiveness to pig cells. In group 3, sensitization to Gal was seen in all three baboons at 20 days, and Abs against new porcine determinants developed in one baboon. The depletion or inhibition of host macrophages, therefore, prevented the induction of humoral unresponsiveness by CD154 blockade. These results suggest that CD154 blockade induces humoral unresponsiveness by a mechanism that involves the indirect pathway of antigen presentation. In vitro investigation of baboon anti-pig mixed lymphocyte reaction confirmed that only the indirect pathway is efficiently blocked by anti-CD154 mAb. The mechanism in which blockade of the CD40-CD154 pathway induces its effect remains to be determined, but it could involve the generation of regulatory cells capable of suppressing the direct pathway.

Clearance of mobilized porcine peripheral blood progenitor cells is delayed by depletion of the phagocytic reticuloendothelial system in baboons.

INTRODUCTION: Attempts to achieve immunological tolerance to porcine tissues in nonhuman primates through establishment of mixed hematopoietic chimerism are hindered by the rapid clearance of mobilized porcine leukocytes, containing progenitor cells (pPBPCs), from the circulation. Eighteen hours after infusing 1-2 x 10(10) pPBPC/kg into baboons that had been depleted of circulating anti-alphaGal and complement, these cells are almost undetectable by flow cytometry. The aim of the present study was to identify mechanisms that contribute to rapid clearance of pPBPCs in the baboon. This was achieved by depleting, or blocking the Fc-receptors of, cells of the phagocytic reticuloendothelial system (RES) using medronate liposomes (MLs) or intravenous immunoglobulin (IVIg), respectively. METHODS: Baboons (preliminary studies, n=4) were used in a dose-finding and toxicity study to assess the effect of MLs on macrophage depletion in vivo. In another study, baboons (n=9) received a nonmyeloablative conditioning regimen (NMCR) aimed at inducing immunological tolerance, including splenectomy, whole body irradiation (300 cGy) or cyclophosphamide (80 mg/kg), thymic irradiation (700 cGy), T-cell depletion, complement depletion with cobra venom factor, mycophenolate mofetil, anti-CD154 monoclonal antibody, and multiple extracorporeal immunoadsorptions of anti-alphaGal antibodies. The baboons were divided into three groups: Group 1 (n=5) NMCR+pPBPC transplantation; Group 2 (n=2) NMCR+ML+pPBPC transplantation; and Group 3 (n=2) NMCR+IVIg+pPBPC transplantation. Detection of pig cells in the blood was assessed by fluorescence-activated cell sorter and polymerase chain reaction (PCR). PRELIMINARY STUDIES: ML effectively depleted macrophages from the circulation in a dose-dependent manner. Group 1: On average, 14% pig cells were detected 2 hr postinfusion of 1 x 10(10) pPBPC/kg. After 18 hr, there were generally less than 1.5% pig cells detectable. Group 2: Substantially higher levels of pig cell chimerism (55-78%) were detected 2 hr postinfusion, even when a smaller number (0.5-1 x 10(10)/kg) of pPBPCs had been infused, and these levels were better sustained 18 hr later (10-52%). Group 3: In one baboon, 4.4% pig cells were detected 2 hr after infusion of 1 x 10(10) pPBPC/kg. After 18 hr, however, 7.4% pig cells were detected. A second baboon died 2 hr after infusion of 4 x 10(10) pPBPC/kg, with a total white blood cell count of 90,000, of which 70% were pig cells. No differences in microchimerism could be detected between the groups as determined by PCR. CONCLUSIONS: This is the first study to report an efficient decrease of phagocytic function by depletion of macrophages with MLs in a large-animal model. Depletion of macrophages with MLs led to initial higher chimerism and prolonged the survival of circulating pig cells in baboons. Blockade of macrophage function with IVIg had a more modest effect. Cells of the RES, therefore, play a major role in clearing pPBPCs from the circulation in baboons. Depletion or blockade of the RES may contribute to achieving mixed hematopoietic chimerism and induction of tolerance to a discordant xenograft.

Mechanisms of thrombotic microangiopathy following xenogeneic hematopoietic progenitor cell transplantation.

INTRODUCTION: Attempts to induce tolerance though mixed hematopoietic chimerism in the discordant pig-to-baboon xenotransplantation model are sometimes complicated by a potentially fatal thrombotic microangiopathy in the recipient baboons. This state develops immediately after the infusion of porcine mobilized peripheral blood leukocytes, containing progenitor cells (PBPC). In our study, we examined the interaction of infused porcine PBPC with recipient platelets in vivo in baboons and investigated the underlying mechanisms using an in vitro model. METHODS: Two naïve baboons and six baboons preconditioned with irradiation and immunosuppression that received porcine PBPC were evaluated in vivo. The interaction of porcine and baboon PBPC with baboon platelets was investigated by an in vitro platelet aggregation assay. Fresh and cryopreserved PBPC were evaluated as well as PBPC obtained from growth-factor mobilized and unmobilized pigs. Furthermore, cellular subsets of PBPC were assessed for potential to induce platelet aggregation. Immunohistochemical staining was performed on platelet-leukocyte aggregates and potential inhibition of aggregation with anti-P-selectin and anti-CD154 mAbs, or eptifibatide (a GPIIb/IIIa receptor antagonist), was tested. RESULTS: All baboons that received porcine PBPC rapidly developed marked thrombocytopenia (<20,000/microl), elevated serum lactate dehydrogenase (>1,500U/liter), schistocytosis, and platelet aggregates on blood smear. Three baboons died (two untreated and one preconditioned), and substantive platelet aggregates containing porcine leukocytes were observed in the microvasculature of lungs and kidneys. In vitro, porcine, but not baboon, PBPC induced aggregation of baboon platelets in a dose-dependent manner. Immunohistological examination of these aggregates confirmed the incorporation of porcine leukocytes. Cryopreserved PBPC caused less aggregation than fresh PBPC, and growth-factor-mobilized PBPC induced less aggregation than unmobilized PBPC. Aggregation was fully abrogated by the addition of eptifibatide, and modulated by anti-P-selectin and anti-CD154 monoclonal antibodies that recognize adhesion receptors on activated platelets. Purified fractions (granulocytes, CD2+, and CD- cells) of porcine PBPC did not initiate aggregation, whereas addition of exogenous porcine PBPC membranes (erythrocytes, dead cells, and/or platelets) to the purified fractions exacerbated the aggregation response. CONCLUSIONS: These data indicate that porcine PBPC mediate aggregation of baboon platelets. This process likely contributes to the thrombotic microangiopathy observed after PBPC transplantation in the pig-to-baboon model. Eptifibatide can fully abrogate platelet aggregation induced by porcine PBPC in vitro. Purification of the progenitor cells from porcine PBPC and/or treatment of baboons with eptifibatide may be beneficial.

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.