P. A. Lear

Comparison of microemulsion and conventional formulations of cyclosporine A in preventing acute rejection in de novo kidney transplant patients. The U.K. Neoral Renal Study Group.

Background. The microemulsion preconcentrate formulation of cyclosporine A (CsA) (Neoral) exhibits more uniform pharmacokinetics than the conventional formulation (Sandimmun; SIM). This randomized, open-label, U.K. multicenter study compared the efficacy, safety, and tolerability of Neoral and SIM in preventing acute rejection in de novo renal transplant recipients. Methods. Adult cadaveric kidney recipients (n=293) received Neoral or SIM twice daily for 12 months. Initially identical Neoral and SIM doses were titrated, maintaining trough CsA levels within locally defined therapeutic limits. Results. In the year after transplantation, acute rejection occurred in 34% of the Neoral and 47% of the SIM recipients (P=0.037). In the intent-to-treat population, fewer treatment failures (defined as acute rejection, graft loss, withdrawal, or death) occurred in the Neoral (45%) than the SIM recipients (58%) (P=0.015) and therapeutic CsA levels (≥250 μg/L) were reached faster with Neoral than SIM (P=0.0017). Antibody treatment of refractory rejection was used slightly less in the Neoral group (Neoral: 10%; SIM: 12%). One-year patient and graft survival rates (excluding deaths with functioning grafts) were 95% and 88%, respectively, for Neoral and 96% and 89% for SIM. Both formulations were well tolerated. No differences were observed between therapies in the nature, frequency, or severity of adverse events. Neoral use was not associated with increased nephrotoxicity or excessive immunosuppression. Conclusions. Neoral reduced the incidence of acute rejection compared with SIM, without significant increases in adverse events. This was achieved without altering existing SIM protocols and was attributed to improved absorption of CsA from Neoral and less variability in whole blood CsA concentrations.

The effect of rejection and graft-versus-host disease on small intestinal microflora and bacterial translocation after rat small bowel transplantation

Bacterial translocation and the development of sepsis after small bowel transplantation may be promoted by immunological damage to the intestinal mucosa or by quantitative and qualitative changes in intestinal microflora. This study assessed the effects of rejection, graft-versus-host disease (GVHD) and immunosuppression on intestinal microflora and bacterial translocation after heterotopic rat small bowel transplantation. Isografts, allografts with and without CsA immunosuppression, and the semi-allogeneic parent to the F1 hybrid GVHD model were studied. Intestinal microflora in graft and host loops and bacterial translocation to host organs and the graft mesenteric lymph node were determined. Bacterial colonies were counted and individual colonies identified using API 20E nutrient and fermentation indicator techniques. Colony counts in isografts and allografts were significantly higher than in the native intestine, whereas there was a massive overgrowth in the native intestine in the GVHD group. The species profile for the host and graft loops was similar in animals that had received isografts, allografted animals receiving CsA, and animals undergoing GVHD. However, there was a large increase in Staphylococcus epidermidis in animals with rejection. Bacterial translocation was not detected in isografted animals, but was observed in all other animal groups, with S. epidermidis being the most prevalent organism. These findings demonstrate that rejection and GVHD are associated with shifts in intestinal microflora toward potentially pathogenic organisms and that bacterial translocation into recipient tissues poses a major threat for the development of sepsis.

Donor cell infiltration of recipient tissue as an indicator of small bowel allograft rejection in the rat

This study assessed whether screening of host tissues for graft cells could be used as an effective monitor of rejection following small bowel transplantation. Allogeneic rat small bowel transplantation was performed with or without cyclosporin (CyA) immunosuppression and cellular infiltration of host tissues assessed by immunohistological staining. Without immunosuppression, grafts were completely rejected within 1 week. CyA treatment for 7 days preserved the graft for 28 days although there was histological evidence of mild rejection in some of the animals studied. Continuous CyA treatment preserved the graft for up to 56 days. The peripheral lymph nodes and spleens of untreated animals were transiently infiltrated by low numbers of donor cells that disappeared by day 6. There was a marked donor cell infiltration of the lymph nodes and spleens of 7-day, CyA-treated animals that was maintained during the administration of immunosuppressive therapy but that declined thereafter. Continuous CyA treatment sustained donor cell infiltration up to day 56. These findings suggest the presence of donor cells in recipient lymph nodes and spleen to be indicative of effective control of rejection and their disappearance to be predictive of developing rejection responses. Examination of recipient peripheral tissues for donor cells may provide an improved technique for monitoring clinical small bowel transplantation.

Reduction of graft-versus-host reactivity after small bowel transplantation: ex vivo treatment of intestinal allografts with an anti-T cell immunotoxin

A specific T lymphocyte immunotoxin was used to pre‐treat small bowel grafts in an attempt to prevent graft‐versus‐host (GVH) reactivity and GVH disease in a rat transplant model. The immunotoxin used was a conjugate of the anti‐CD5 MoAb MRC OX‐19 with ricin A chain. The grafts were perfused ex vivo with a standard solution of immunotoxin followed by incubation at 4°C for 1 h before transplantation. In a semi‐allogeneic strain combination (parent to F1 hybrid offspring) graft treatment with immunotoxin led to a prolongation of recipient survival compared with groups receiving similar transplants without immunotoxin treatment. An additive effect on survival was observed when the host was treated with cyclosporin. The effect of immunotoxin was greater than that of mensenteric lymphadenectomy in increasing host survival. The effect of graft treatment with the immunoloxin on cellular migration from graft to host lymphoid tissues was assessed in fully allogeneic transplantation (PVG to DA). Host lymphoid tissues were subjected to immunohistochemical analysis using a MoAb specific for donor class I MHC antigens. Graft treatment with the immunotoxin led to a significant decrease in the number of graft cells found in host lymphoid tissues 7 days after transplantation. However, this effect was less marked than that achieved by graft mesenteric lymphadenectomy. With our current protocol graft treatment with a specific T cell immunotoxin can significantly reduce but not abolish GVH reactivity in rat small bowel transplantation.

Ex vivo perfusion of intestinal allografts with anti-T cell monoclonal antibody/ricin a chain conjugates for the suppression of graft-versus-host disease

The removal of T lymphocytes from intestinal allografts prior to transplantation would prevent graft-versus-host disease and might also weaken the unusually severe rejection response mounted by graft recipients. Ex vivo perfusion by monoclonal antibody-toxin conjugates represents a potentially ideal approach to achieve this goal. Monoclonal antibody-toxin conjugates were prepared by coupling the mouse anti rat CD5 antibody MRC OX19 to the A chain of ricin. The resultant conjugate contained 1 or 2 ricin A chain molecules per molecule of immunoglobulin and had high selective toxicity for rat T lymphocytes. Following ex vivo perfusion of the small intestine, the mesenteric lymph nodes and Peyers patches were removed and the level of penetration of the MRC OX19 antibody was assayed by immunohistological techniques. In lymph nodes, there was ready access of the antibody to the medulla, and to a lesser extent to the B cell areas of the cortex. However, only the T cells in the peripheral regions of the paracortex were stained, suggesting that the paracortex was resistant to penetration by blood-borne antibody, and was being stained only by diffusion from the lymph node medulla. Some penetration of the antibody also occurred in the immediate vicinity of the postcapillary venules. In the Peyers patches, staining was seen in the germinal centers, but not at all in the T cell areas. The addition of agents such as histamine to the perfusate to increase vascular permeability did not alter this picture. These studies suggest the presence of a potentially interesting resistance to penetration of the paracortex of the lymph node by blood-borne substances. Nevertheless, sufficient penetration occurred to influence favorably the course of GVD disease following ex vivo perfusion prior to transplantation of the donor intestine with the MRC OX19-ricin A chain conjugate.

Effect of small bowel transplantation, denervation and ischaemia on rat intestinal microflora

The effects of denervation and warm ischaemia on quantitative and qualitative changes in small intestinal microflora following rat heterotopic small-bowel isotransplantation were assessed. Animals with Thiry-Vella fistula, but without transplants, acted as controls. Thirty and 40-fold increases in bacterial colony counts were seen in the isografts compared to controls at 2 and 7 days, respectively (P< 0.05). Aerobic faecal organisms predominated at 2 and 7 days, but an overgrowth of Flavobacterium meningosepticum occurred at 28 days in the transplanted and host bowels. The effect of warm ischaemia on intestinal microflora was assessed by the application of a microvascular clamp to the superior mesenteric artery for 90 min. The effect of denervation was assessed following microsurgical division of all nervous tissue around the superior mesenteric artery. After 7 days, lengths of jejunum and ileum were removed and intraluminal microflora assessed. The number of bacterial colonies isolated from the ileum in the warm ischaemia group was six times greater than the number in the control group, whereas no significant changes were seen in the upper bowel. In contrast, denervation led to a slight, but consistent, decrease in colony counts. These findings suggest that the increase in bacterial numbers in an isografted small bowel primarily results from warm ischaemia rather than from mesenteric denervation, and that physical aspects of the procedure may affect the development of sepsis following smallbowel transplantation.