Search

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. 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). 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 lg/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. [ABSTRACT FROM AUTHOR]

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 α1,3-galactosyltransferase gene-knockout (GalT-KO). 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. A mean of 11 × 108 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/μl (9.5%), 132/ll (3.4%), 242/ll (3.9%), and 156/ll (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. 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. [ABSTRACT FROM AUTHOR]

Thrombotic microangiopathy (TM) is a serious complication of bone marrow transplantation (BMT) that resembles thrombotic thrombocytopenic purpura (TTP). In attempting to achieve hematopoietic cell chimerism in the pig-to-baboon model, we have observed TM following infusion of high doses (>1010 cells/kg) of porcine peripheral blood mobilized progenitor cells (PBPC) into baboons. We performed investigations to analyze the pathobiology of this TM and to test therapeutic interventions to ameliorate it. PBPC were obtained by leukapheresis of cytokine-stimulated swine. The initial observations were made in two baboons that underwent a non-myeloablative regimen (NMR) prior to PBPC transplantation (TX) (group 1). We then studied three experimental groups. Group 2 (n = 2) received NMR without PBPC TX. Group 3 (n = 2) received PBPC TX alone. Group 4 (n = 6) received NMR + PBPC TX combined with prostacyclin, low-dose heparin, methylprednisolone, and cyclosporine was replaced by anti-CD40L mAb in five cases. Baboons in groups 1 and 3 developed severe thrombocytopenia (<10 000/mm3), intravascular hemolysis with schistocytosis (>10/high powered field (hpf)), increase in plasma lactate dehydrogenase (LDH) (2500–9000 U/l), transient neurologic changes, renal insufficiency, and purpura. Autopsy on two baboons confirmed extensive platelet thrombi in the microcirculation, and, similar to clinical BMT-associated TM/TTP, no unusually large vWF multimers or changes in vWF protease activity were observed in the plasma of baboons with TM. In group 2, self-limited thrombocytopenia occurred for 10–15 days following NMR. Group 4 baboons developed thrombocytopenia (<20 000/mm3) rarely requiring platelet transfusion, minimal schistocytosis (<3/hpf), minor increase in LDH (<1000 U/l), with no clinical sequelae. We conclude that high-dose porcine PBPC infusion into baboons induces a microangiopathic state with vWF biochemical parameters... [ABSTRACT FROM AUTHOR]

Abstract: Background: Anti-Galα1–3Gal (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 immuno-suppressed 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 hypo-tension. Mean pre-EIA Gal Ab levels in naïve baboons were 33.1 µg/ml (IgM) and 14.5 µg/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 immuno-modulatory protocol. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We have examined the cell surface phenotype of porcine natural killer (NK) cells and compared them with classic porcine cytotoxic T lymphocytes (CTL). NK cells were susceptible to treatment with monoclonal antibodies to CD2 (PTI 1), CD8 (PT8) and Ia plus complement (C), as well as with antiserum to asialo-GM 1 (ASGM 1) plus C. In addition, monoclonal antibodies to leucocyte function antigen I (LFA-1) but not to CD8 blocked NK-mediated lysis in the absence of C. This is in contrast to porcine CTL, which were eliminated by antibodies to CD2, CD8 and Ia plus C, but not by antis ASGMI plus C, and which were blocked by anti-CD8 in the absence of C. Therefore, although porcine NK cells arc similar to porcine CTL, they are distinguished by several important differences. [ABSTRACT FROM AUTHOR]

Heterologous anti-idiotypic reagents have been prepared against a BALB/c anti-swine MHC (SLAd) monoclonal antibody (74-11-10) in order to test for possible interspecies idiotypic cross-reactions of anti-MHC antibodies. Using these reagents to examine xenoantisera produced in BALH/c mice immunized with swine SLAdd peripheral blood lymphocytes, all animals tested were found to express detectable levels of the 74-11-10 idiotype (Id). The Id could also be detected in one out of five BALB/c mice immunized with swine SLAcc PBL. Swine anti-SLAdd alloantibodies were also tested, but failed to show detectable levels of the 74-11-10 Id. The in vivo administration of anti-idiotypic reagents to BALI/c mice induced detectable levels of 74-11-10 Id positive antibodies that bound specifically to SLAdd PHL. Similar treatment of SLAgg swine (recombinant swine expressing the class I MHC molecules of c) with anti-idiotypic antibodies failed to induce anti-SLAd antibody activity. These results thus indicate that 74-11-10 represents a shared idiotype of BA LB/c anti-SLAdd antibodies. The presence of 74-11-10 Id in one mouse immunized with SLA∞ PBL suggests that the failure of pigs to express the 74-11-10 Id following treatment with anti-idiotypic antibodies may be the result of tolerance rather than absence of the relevant variable region gene(s). [ABSTRACT FROM AUTHOR]

MHC class I and II-defined, partially inbred miniature swine have recently become available as a large animal model in transplantation immunology. To investigate cutaneous immunocompetence in this model, cutaneous antigen presenting cell (APC) function was assessed. For morphologic analysis, punch biopsies were examined by electron microscopy. By this technique, epidermal Langerhans cells bearing typical Birbeck granules could be detected. For functional studies, epidermal cell (EC) suspensions were prepared from split thickness skin specimens. Using FACS analysis, freshly prepared epidermal cell suspensions contained 1.8 4.7% MHC class II-positive cells. These EC potently stimulated allogeneic nylon wool-enriched peripheral blood T cells in the primary mixed EC-lymphocyte reaction. For in vivo assessment of cutaneous APC function, EC suspensions enriched for or depleted of class II-positive EC were generated by panning of class II-positive EC using mouse anti-MHC class II antibodies and anti-mouse IgG-coated petri dishes. EC were then coupled to the hapten trinitrophenol (TNP) and injected s.c. into autologous or MHC-mismatched pigs twice at a one week interval. One week later, pigs were challenged by s.c.-injection of 0.5-1 X 10[SUP7] TNP-coupled or uncoupled EC. Autologous unseparated EC as well as EC enriched for MHC class II-positive cells were able to sensitize naive animals against TNP, whereas neither TNP-coupled EC depleted of class II-positive APC, MHC-mismatched EC coupled to TNP, nor uncoupled EC induced immunity to TNP. Our data indicate that inbred miniature swine possess competent cutaneous APC which are able to induce cutaneous immunity in a manner similar to Langerhans cells in murine or human skin. [ABSTRACT FROM AUTHOR]

After mice of several inbred strains were injected with Listeria monocytogenes, two parameters of resistance, the 50% lethal dose and the suppression of bacterial proliferation in spleen, were determined. The strains of mice tested could be segregated into two groups: the resistant C57BLjlOSn mice and the sensitive AIJ and DBAj2J mice. Congenic resistant strains of mice were used because they would express the H-2 haplotype of the sensitive strains (H-2a or H-2d) on the background of a resistant strain, C57BL/10Sn. Both the B10A/SgSn (H-2a) and the B10.D2/Sn (H-2d) mice were as resistant as mice from their background strain and were significantly more resistant than the strains that donated their H-2 locus (A/J or DBA/2J). Therefore, the resistance of mice to Listeria, although genetically controlled, is not controlled by gene (s) linked to the H-2 haplotype. On the other hand, the level of specific immunity to listeria antigens (as indicated by the footpad reaction) was higher in the C57BL/10Sn (H-2b) mice than in either the A/J and B10.A/SgSn (H-2a) mice or the DBA/2J and B10.D2/Sn (H-2d) mice. This observation suggests an H-2 linkage of specific immunity to Listeria. [ABSTRACT FROM PUBLISHER]

Murine peritoneal cells, both induced and noninduced, were examined for Ia antigens by a variety of techniques. Complement-mediated cytotoxicity and indirect immunofluorescence, analyzed by both visual microscopy and the fluorescence-activated cell sorter, detected Ia antigens on the surface of an average of 8% to 15% of cells with the morphologic and functional characteristics of macrophages. Internal radioisotope labeling studies showed that these antigens were actually synthesized by the macrophages. The antigens were borne on molecules which consisted of two components with apparent molecular weights of roughly 33,000 and 25,000 daltons. At least some of those molecules existed as a two-chain structure of 58,000 daltons linked by disulfide bonds. Although macrophage Ia antigens appeared to be structurally similar to the Ia antigens found on spleen cells, the radioisotope labeling studies indicated that the quantity of labeled Ia-bearing molecules isolated from peritoneal macrophages was at most 1/15 that found for B lymphocytes. In addition. anti-Ia antisera failed to inhibit the binding of heat-aggregated immunoglobulin to the Fc receptor of macrophages. Thus, Ia antigens appear to be present at very low levels in macrophage populations, similar to the low levels of Ia antigens found in T-lymphocyte populations. These studies suggest that Ia antigens exist on only a subpopulation of peritoneal macrophages. Alternatively, all cells in the population might bear small amounts of Ia antigens with only a fraction having sufficient numbers of molecules to be detected by the assay systems used. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about localized myocardial infarction following pig-to-baboon heart transplantation.

Success in xenotransplantation relies on engineering of the pig genome to express human transgenes, such as CD55/CD39, that can control coagulation and inflammation to prolong the graft survival of a 1,3-galactosyltransferase depleted (Gal-/-) pig organs in nonhuman primates and then able to bypass the hyperacute rejection. The aim of our work was to produce Gal-/- piglets overexpressing CD55/CD39. In experiment (Exp.) 1 exploiting 2 ubiquitous expression vectors (pCAGGS-CD55 and pCAGGS-CD39), we transfected immortalized porcine kidney cells (PK15) with CD55 and CD39 using Nucleofector (Amaxa Biosystems, Cologne, Germany) and selected 5 cell colonies each (PK15-CD55 and PK15-CD39) that were expanded and analyzed by RT-PCR, immunohistochemistry (IHC) and Western blot (WB). The monoclonal antibodies IA10 for hCD55 and BU61 for hCD39 were used. Transgenic transcription was confirmed by Northern blot (NB) using digoxigenin-labeled probes. In Exp. 2, a neonatal pig Gal-/- fibroblast line was co-transfected by Nucleofector using 2 ubiquitous expression vectors (hEF-CD55 and pCAGGS-CD39) for the expression of CD55 and CD39. Colonies were analyzed by RT-PCR and IHC only, because of the limited number of cells available. Cells from one colony with a high level of CD55/CD39 expression according to IHC were used for nuclear transfer into enucleated oocytes. Day 5 compact morula/blastocyst (n= 144) were transplanted in 2 synchronized sows. Porcine aortic endothelial cells (PAEC) and fibroblasts derived from 2 stillborn piglets were analysed with IHC, NB, and WB. The expression level of transgenes from both experiments was compared with human umbilical vein endothelial cells (HUVEC) by fluorescence-activated cell sorting (FACS), using IA10, BRIC110, IH4, 2G2, and MEM-118 antibodies for hCD55 and TU66 for hCD39. In Exp. 1, RT-PCR showed CD55 mRNA expression in 3 out of 5 (2, 15, 24) PK15-CD55 colonies. A high level of CD55 expression was confirmed only in colony 24 by IHC, NB, WB, and FACS. Low expression level in colony 2 revealed by FACS was not detected by IHC, indicating that FACS analysis is more accurate to quantify the level of expression. All PK15-CD39 colonies were positive according to RT-PCR and IHC. Only one colony PK15-CD39 was further analyzed by NB and WB and confirmed positive. In Exp. 2, IHC, NB, WB, and FACS analyses of fibroblasts and PAEC derived from both cloned piglets confirmed the high level of CD39 expression detected by IHC in donor cells used for nuclear transfer. However, strong CD55 expression detected by IHC was not confirmed by NB analyses and, by FACS, was lower than in HUVEC cells. In conclusion, we produced cloned CD55-CD39 transgenic Gal-/- piglets with a high level of CD39 expression but the expression level of CD55 was lower than in HUVEC cells. We found that although IHC is the method of choice in preliminary screening, it is not sufficiently quantitative when only a few cells for each clone are available. Thus, IHC needs to be complemented with additional methods (e.g. WB, FACS, real-time RT-PCR) to obtain complete evaluation of the expression pattern of transgenes before nuclear transfer experiments. This study was supported by EU grant no. LSHB-CT-2006-037377 and Fondazione Banca Popolare di Cremona. [ABSTRACT FROM AUTHOR]