106 Background: In the pig to baboon xenograft model, the performed IgM xenoantibodies are presumed to be one of the most important factor involved in hyperacute vascular rejection (HAVR). Methods: We therefore developed a rat monoclonal antibody which recognizes human as well as baboon IgM (LO-BM2) in order to deplete "in vivo" the total seric IgM. As assessed by ELISA, two to four consecutive iv injections of LO-BM2 at 15-20 mg/kg totally eliminated the baboon circulating IgM. Results: As control, we transplanted a pig renal xenograft to three baboons without any immunosuppressive therapy and a fourth baboon underwent a splenectomy three days prior to the renal xenograft. All the four animals hyperacutely rejected their xenograft between the first hour after unclamping and the first postoperative day (POD). At histology, signs of HAVR were evidenced. An experimental group of five baboons was then designed to assess the effect of a selective depletion of circulating IgM on the renal xenograft survival. During the preoperative LO-BM2 treatment, the total concentration of seric IgM was assessed daily by ELISA in order to perform the renal xenograft in absence of circulating anti-pig IgM. The first baboon was splenectomized on day -3 and received four injections of LO-BM2 (20 mg/kg/day) prior to grafting. During the following 4 POD, the xenokidney functioned well (creatinine 0.8; 1.0; 1.2 and 2.0mg/dl) and the renal function eventually acutely deteriorated on POD 6 (creatinine 6.4 mg/kg). At this time, the kidney was completely rejected, cyanotic and swollen and the pathology mainly showed signs of vascular rejection. The second animal also underwent a splenectomy on day -3 and a depletion of the circulating IgM by LO-BM2. During three days, the creatinine remained normal and the kidney was acutely rejected on POD4. A third animal was similarly treated by three LO-BM2 injections but without splenectomy. This baboon maintained a normal renal function during 3 days (0.9; 0.8; 0.9mg/kg) and eventually acutely rejected the xenograft on POD4. In these three animals, the circulating seric IgM were undetectable by ELISA up to the rejection time. By Flow Cytometry on porcine aortic endothelial cells specific anti-pig xenoantibodies were detected concomitantly or just after the renal function deterioration. Two additional animals were similarly treated by LO-BM2 prior to transplant but in addition received, in the first case, five injections of DSG at 9mg/kg (from day-2) and, in the second case, 6 days of RS at 250 mg/kg (from day 0). After elimination of the circulating IgM, both animals maintained a normal renal function for 3 and 4 POD but eventually rejected acutely the graft on POD4 and 6, respectively. The lack of prolonged effect of LO-BM2 over five or six days is related to a baboon anti-rat sensitization. Conclusions: These experiments show for the first time in a pig to baboon model that a pig kidney xenograft can survive up to six days without immunosuppression besides the complete elimination of the circulating IgM. The rejection occurred simultaneously to the reappearance of a low level of circulating IgM and was of the vascular type. These results also suggest that neither the alternate pathway of the complement nor the preformed IgG are able in this model to produce alone a HAVR.
It is believed that IgM xenoreactive natural antibodies (XNA) and activation of complement are the two main effectors involved in the hyperacute rejection (HAR) of discordant xenografts, such as pig-to-primate kidney, liver or heart transplants. We have hypothesized that long-term depletion of circulating IgM XNA might be able to overcome HAR and induce the "accommodation" of pig-to-primate vascular discordant xenografts. Several techniques have been described to eliminate circulating XNA in primates but, up to now, none has been able to totally deplete these antibodies for a sufficiently long period of time in order to test the hypothesis of discordant xenograft "accommodation". Previous reports from our laboratory have shown that, in rodents, B-cell immunosuppression could be achieved by neonatal administration of anti-mu antibodies. Recently we have shown that administration of an anti-mu mAb, in adult rats, was able to totally deplete circulating IgM and IgM XNA, without immune complex disease. Furthermore, we have used different methods such as splenectomy, plasma exchange and an anti-B cell immunosuppressive agent mycophenylate mophetil (RS61443, Syntex, Palo Alto, USA) to pre-deplete circulating IgM before administration of anti-mu mAb (MARM-7) and showed that the effectiveness of anti-mu mAb to deplete circulating IgM was increased by 100-fold. Depletion of circulating IgM in adult rats by anti-mu mAb (MARM-7) was used as an experimental model to study the role of IgM XNA in the pathogenesis of HAR in guinea pig-to-rat cardiac xenografts. Our data show that IgM XNA play a major role in HAR, even if in this discordant combination direct activation of complement, probably through the alternative pathway, seems to be the main effector involved in HAR. We have analyzed the mechanisms of anti-mu depletion of circulating IgM in adult animals and shown that, besides anti-mu/IgM immune complex formation, depletion of circulating IgM results from the very significant inhibition of B-cell differentiation and secretion of IgM following in vivo crosslinking and internalization of surface IgM on B cells. As well, we provide evidence demonstrating that anti-mu mAb blocks B cells at an early stage of maturation, probably in the bone marrow. Furthermore, we have developed several rat anti-human and anti-baboon IgM mAb and tested their ability to deplete circulating IgM and IgM XNA in baboons, after splenectomy or splenectomy and plasma exchange.(ABSTRACT TRUNCATED AT 400 WORDS)
1. Louvain rats (IgK-1a) were immunized with horse IgG(T). To generate mAb to IgG(T), popliteal lymph node cells taken from the immunized animals were fused to a non-secreting LOU/C immunocytoma (IR983F). The hybridomas were cultured in HAT-containing medium and cloned under limiting dilution conditions. Supernatants from the growing hybrids were screened by ELISA using plates coated with horse IgG(T) or IgGa+b+c. 2. The anti-IgG(T) mAb obtained was named LO-HoGT-1 (LOU anti-horse IgG(T)). It is an IgG2a rat antibody whose light chain allotype is IgK-1a, and with an affinity constant of 2.9 x 10(10) M-1. 3. Ascites was induced in LOU (IgK-1b) rats by injecting the hybridoma cells and incomplete Freund's adjuvant ip. To obtain purified mAb, ascitic fluid was applied to a Sepharose anti-rat LOU IgK-1a chain column. 4. The purified mAb was then coupled to Sepharose. Immunoelectrophoretically pure IgG(T) was obtained by passage of horse serum through this column. The entire procedure took less than 30 min and resulted in a highly purified IgG(T).
Cyclosporin A (CsA) and IMM‐125, a hydroxyethyl derivative of D‐serine CsA, are cyclic undecapeptides of molecular weight 1201.8 and 1261.8, respectively. The main metabolites still possessing the undecapeptide structure were found to be compounds resulting from the biotransformation of amino acids 4, 9 and 1. Under the influence of the hepatic cytochrome P‐450‐dependent monooxygenase system, CsA and IMM‐125 amino acid 1 are metabolized to a mono‐hydroxylated compound (metabolite M‐17) and to a dihydrodiol. A metabolite M18 was found to be the result of a non‐enzymic intramolecular formation of a tetrahydrofuran derivative from metabolite M17. Since the existence of a CsA dihydrodiol was reported and since epoxides are considered as the dihydrodiol precursors, the aim of the present work was to prove that the same non‐enzymic intramolecular formation of a tetrahydrofuran ring could occur by nucleophilic attack of the amino‐acid 1 β ‐hydroxy group at the ɛ ‐position of the freshly formed epoxide by reaction of IMM‐125 with m‐chloro‐perbenzoic acid and cyclosporin A with selenium oxide. The immunosuppressive activity of the compounds, as measured by the mixed lymphocyte reaction and by the luciferase activity of a Jurkat‐T‐cell line stably transfected with the NF‐AT/luc reporter plasmid, was found negligible for IMM‐125 compared to the parent drug as well as for the cyclosporin A derivative. Structures of the IMM‐125 and CsA derivatives were elucidated by electrospray mass‒spectrometry and NMR spectroscopy.
Different tacrolimus epoxides and dihydrodiol epoxides arising from the chemical oxidation of the parent drug are described. Open-chain tautomeric forms involving the lactone function were identified for the tacrolimus epoxides. Moreover, the identification by electrospray and electrospray linked scan mass spectrometry of an SDZ-RAD C16-C27 O-demethyl 17, 18-19, 20-21, 22 tris-epoxide new metabolite isolated from pig liver microsomes is reported. The in vitro immunosuppressive activity, using mixed lymphocyte reactions of the two macrolide reported oxidation compounds are discussed.
SummaryBetween 1980 and 1985, 11 children (8 boys, 3 girls aged between 18 months and 16 years) with poor prognosis ALL (Acute Lymphoblastic Leukaemia) were treated with ablative therapy and allogeneic bone marrow transplantation. Six patients (55 %) are alive at the time of evaluation , one died in aplasia, two died of interstitial pneumonia and two died of bone marrow relapse.The median time to recover a neutrophil count of 0.5 x 109/1 was 19 days. The total number of lymphocytes reached normal values after 6 months. The percentage of OKT3 cells (mature T lymphocytes) was slightly depressed during the first 3 months. For at least 9 months, the number of OKT4 (helper lymphocytes) was low with a normal number of OKT8 (suppressor lymphocytes) resulting in an OKT4/OKT8 inverted ratio.As far as B lymphocytes function was concerned, there was a moderate decrease of the immunoglobulin levels. Normal values were obtained after 3 months for IgG, 6 months for IgA and IgM.All the immunological parameters tested returned to normal within one year after the bone marrow transplantation.
P1089 Aims: In the pig-to-baboon model, acute vascular rejection (AVR) remains the main hurdle for successful long-term xenograft survival. The production of galactosyl knock-out pigs could solve concomitantly the problem of hyperacute and acute vascular rejections. This work studies in vitro the cell-mediated cytotoxicity of natural killers (NK) and T cells after priming of baboon peripheral blood lymphocytes (PBLs) with pig antigens to evaluate whether cytotoxicity is galactosyl-dependent. Methods: Peripheral Blood Lymphocytes (PBLs) from naive and primed baboons were used as effectors cells on porcine aortic endothelial cells (PAECs) to assess cytotoxicity. Untreated and galactosidase-digested PAECs were used as targets cells to assess the role of galactosyl residues on cell-mediated cytotoxicity. Two rat-anti baboon monoclonal antibodies were tested to inhibit either T + NK cells (LO-CD2b) or NK cells alone (LO-CD94). Results: When using PBLs from naive animals, spontaneous lysis of PAECs occurred (38.7 ± 3.0 %) but was completely inhibited by both LOCD2-b or LO-CD94. In comparison, lysis of PAECs was significantly higher (71.4 ± 3.9 %) when baboon PBLs were firstly primed in vivo with pig xenoantigens (PAECs intravenously injected). In that case, LO-CD2b inhibited completely the lysis whereas LO-CD94 inhibited it only partially (23.5 ± 4.1 %). Reduction of galactosyl residues by enzymatic digestion (α-galactosidase) showed that spontaneous lysis of PAECs completely disappeared with naïve baboon PBLs but not with primed baboon PBLs, suggesting that anti-pig T cell response is not galactosyl-dependent. Conclusions: Galactosyl knockout pigs could solve hyperacute rejection and also prevent the activation of NK cells even after xenogeneic priming. T cells will then be the next hurdle for the success of xenografting.
