Antithymocyte globulin and cyclosporin A are synergistic in an experimental transplantation tolerance model in the rat.

The aim of the present investigation was to study the possible synergistic effect between cyclosporin A (CsA) and antithymocyte globulin (ATG), using the potent immunostimulator, Linomide. DA rats were transplanted with a PVG/c heart to the neck vessels, and the recipients were treated for 10 days with oral CsA (10mg/kg), oral Linomide (160mg/kg) and/or ATG, which was given as a single dose of either 0.02 ml, 0.1 ml or 0.2 ml prior to transplantation. Rats given a combination of ATG and Linomide or CsA and Linomide were used as controls. Synergism between CsA and ATG was tested using the two immunosuppressive drugs given in combination in order to override the challenge of Linomide. CsA or ATG treatment alone resulted in rats with long-term surviving grafts. Addition of Linomide to CsA-treated recipients was followed by early graft rejection. Similarily, Linomide triggered rejection in rats given a low dose of ATG and in recipients given a high dose of ATG if Linomide treatment was prolonged to 21 days. The combination of ATG, CsA and 21 days of Linomide resulted in a significantly prolonged graft survival compared with either ATG + Linomide or CsA + Linomide. These findings demonstrate the synergistic capacity of ATG and CsA in combined immunosuppressive therapy.

A morphological sequential study of mouse-to-rat cardiac xenografts.

Long-term survival of a concordant xenograft can be achieved by using cyclosporine (CyA) and deoxyspergualin (DSG) for immunosuppression. We have demonstrated in a mouse-to-rat heterotopic heart transplantation model that DSG treatment can be stopped after 4 weeks with the grafts remaining beating. In this investigation we have sequentially characterized the morphological changes and infiltrating cells in the transplanted hearts. Graft recipients were killed 9 days, 28 days and 56 days after transplantation. At days 9 and 28, the grafts exhibited a well-preserved morphology, with infiltrating cells restricted only to the periphery. These cells stained positive for rat MHC class II antigens, the ED1-macrophage marker and the CD4 antigen, and were thus considered to be macrophages. In comparison, grafts harvested at day 56 had signs of interstitial fibrosis and some arteries showed pronounced intimal thickening. There was a moderate infiltrate of cells both in the peripheral and central parts of the graft, consisting mainly of MHC class II+/ CD4+/ED1+ macrophages. Very few T cells and NK cells were noticed. Termination of DSG after 28 days does not trigger a humoral rejection. However, the grafts exhibit morphological changes equivalent to those seen in chronic allograft rejection. In addition, the characteristics of the infiltrating cells conformed with cellular infiltrates associated with chronic allograft rejection. Hence, this model could in the future prove to be useful for studies of mechanisms involved in chronic xenograft rejection.

Successful retransplantation of mouse-to-rat cardiac xenografts under immunosuppressive monotherapy with cyclosporine.

The present study was undertaken to investigate whether retransplantation with a second xenograft, from the same species as the primary graft, is possible to achieve using only moderate immunosuppression. Heterotopic mouse-to-rat cardiac transplantations were performed, and the recipients were treated with 15-deoxyspergualin (DSG) and cyclosporine (CsA) at high doses for days -1 to 4 and at moderate doses for days 5 to 28. From day 29 and onward, the immunosuppressive protocol consisted of daily oral administration of CsA 10 mg/kg as monotherapy. Animals that had beating grafts when DSG treatment was stopped were retransplanted 56-154 days after the primary transplantation, either with a vascularized graft (heart) or with nonvascularized graft (pancreatic islets), under continued therapy with CsA. Six of 10 secondary cardiac xenografts functioned for more than 50 days and were harvested beating after 60-100 days. In contrast, nonimmunosuppressed or DSG-treated rats are known to reject a second cardiac mouse graft hyperacutely. The unresponsiveness was confined to cardiac tissue, as the pancreatic islets, transplanted under the kidney capsule, were totally rejected after 14 days. Long-term functioning cardiac xenografts, primary and secondary, had a well-preserved morphology, and infiltrating mononuclear cells were found just in the periphery of the grafts. A majority of these cells were macrophages expressing the ED1, but not the ED2, antigen. No deposition of IgG or complement was seen in any of the graft vessels, whereas a slight deposition of IgM was observed in some vessels of both primary and secondary grafts. In conclusion, we have demonstrated that unresponsiveness can be induced by effective immunosuppression of the recipient at the time of the initial transplantation, so that retransplantation with a second xenograft can be performed successfully under single-drug immunosuppressive therapy with CsA.

Alterations in mucosal immune cell distribution in septic rats.

A reduced cell-mediated immunity in the intestinal mucosa might promote gut barrier failure and bacterial translocation in shock. This study was performed to investigate changes from the normal distribution of cellular components of the immune system within the intestine during sepsis. Intra-abdominal sepsis was induced by cecal ligation and puncture. After 24 h, the animals were killed and specimens were taken from the small bowel. Immunohistochemical stainings were performed on frozen sections using monoclonal antibodies reactive with MHC class II positive cells (OX6), the alpha/beta antigen receptor on T lymphocytes (R73), CD4+ T lymphocytes (W3/25), CD8+ T lymphocytes (OX8), and macrophages (ED1). There was a significant reduction in the number of pan T lymphocytes as well as both CD4+ and CD8+ subsets in the mucosa of the septic rats as compared with sham operated rats (p < .01). In contrast, the populations of MHC class II positive cells and macrophages increased in sepsis (p < .01). We conclude that 24 h after the induction of peritonitis, there is an altered pattern of immunocompetent cells within the intestinal mucosa.

Moderate additive immunosuppressive effect of thalidomide combined with cyclosporin A in rat cardiac transplantation.

Thalidomide is an immunomodulating agent shown to prolong graft survival in experimental skin, renal, cardiac and bone marrow transplantation. The main purpose of the present study was to investigate the possible additive effect of combining thalidomide with cyclosporin A (CyA). Members of our group have previously created a basis for such studies by demonstrating the ability of Linomide to abolish the effect of CyA. The additional effect of combined treatment with a second drug is thereby more readily evaluated, compared with using subtherapeutic dose levels to induce early rejection. Cardiac grafting was performed in three rat strain combinations (BN to WF, DA to Lew, and BN to Lew). Rats were given no treatment, or thalidomide, CyA and/or Linomide in single, double or triple drug therapy. Except for a consistent beneficial effect of CyA as single drug treatment, graft survival varied depending on the rat strain combination used. In the DA to Lew combination, the expected effects of Linomide were seen, and thalidomide was shown to prolong graft survival significantly (P = 0.004) when added to CyA and Linomide. However, there was no effect of thalidomide when given alone. In WF recipients of BN hearts, thalidomide tended to prolong graft survival (P = 0.07), and surprisingly Linomide manifested a marked immunosuppressive effect (P = 0.0002) and did not counteract the effect of CyA. When transplanting BN grafts to Lew recipients, Linomide reduced significantly but did not abolish completely the effect of CyA. Neither Linomide nor thalidomide had any beneficial impact on graft survival on their own. To sum up, thalidomide was shown to have a minimal or moderate immunosuppressive effect additive to that of CyA. The effects of the two immunomodulating drugs, thalidomide and Linomide, varied depending on the rat strain combination used, and were similar with respect to prolongation of graft survival when used as single drug treatment in BN to WF grafting, a fact which may indicate them to have a similar mechanism of action, both having been shown to exert similar effects on levels of tumour necrosis factor alpha.

Anti-carbohydrate antibodies associated with hyperacute rejection in a vascularized mouse heart-to-rat xenotransplantation model.

Specificity of immune reactions has always been sought, because it facilitates intervention with unwanted mechanisms. Specific carbohydrate antigens have been proposed to be targets of antibodies in early immune responses in pig-to-man xenografts. This work was undertaken to determine carbohydrate structure for antibody response in the experimental xenograft model mouse-to-rat. Glycolipids were prepared from nine different mouse organs and separated for carbohydrate size on thin layer plates. Sera taken from normal untreated rats showed only weak or absent IgM antibody-binding to the separated mouse glycolipids. This is in accordance with the observation that mouse heart grafts are not hyperacutely rejected by the rat. However, sera taken from mouse heart xenografted rats show clear IgG and IgM antibody binding to neutral glycolipids migrating in the five-sugar region of the thin-layer plate. These rats have previously been reported to hyperacutely reject a second xenograft. Glycolipids with this particular mobility and immunostaining properties are the dominant ones in the mouse caval vein preparation, which probably represents a rather pure vascular structure. The target antigen structure was identified, by mass spectrometry and proton nuclear magnetic resonance spectroscopy, to be the Forssman pentaglycosylceramide. A commercial monoclonal antibody directed toward the Forssman antigen bound the same biochemical structure as the antibodies derived from the mouse heart-xenografted rats. Most of the IgM activity, but very little of the IgG activity was adsorbed using the Forssman terminal disaccharide solid phase.

Is kidney transplantation in sensitized recipients justified?

The objective of the study was to determine if it is justified to use the scarce resources of cadaveric kidneys on HLA-sensitized patients, by reviewing the initial and long-term outcome of cadaveric renal transplantation at Uppsala University Hospital, Sweden. Between January 1988 and December 1994, 402 renal transplantations were performed. The patients were divided into one group of sensitized recipients (peak panel antibody reactivity > or = 25%; n = 84) and a second of non-sensitized recipients (panel reactive antibodies < 25%; n = 318). The groups were comparable in terms of recipient and donor age, gender, HLA-A, -B and -DR mismatches and numbers of diabetics. None of the sensitized patients received a six-antigen-matched kidney. For the non-sensitized group, life table analysis showed a 1-year actuarial graft survival (GS) of 91.8% and a 4-year GS of 84.4%. The corresponding GSs for the sensitized group were 79.9% and 68.7%, respectively (P < 0.01). The statistical significance vanished if patients with primary non-function were excluded. When excluding donors above 55 years of age, kidneys with cold ischemia time above 20 h, and two-antigen (HLA-DR) mismatches, there was no detectable difference between the non-sensitized and sensitized groups at 1-year or 4-year GS. Although there is a statistical significance in GS between non-sensitized and sensitized recipients of a kidney transplant, this does not differ from other risk groups such as diabetics, rheumatoid disease sufferers or elderly recipients. We therefore conclude that the sensitized patient should be accepted on the waiting list for a kidney transplant and that it is worthwhile to do the utmost to transplant this category of patients. Our data indicate that kidney GS in sensitized recipients is more affected by negative risk factors such as older donors, long cold ischemia time and two-antigen HLA-DR mismatch, than the non-sensitized recipient. To improve the outcome, those negative factors should be avoided or reduced.

Simultaneous occurrence of rejection and graft-versus-host reaction after allogeneic small bowel transplantation.

Small bowel transplantation may be complicated not only by rejection but also by graft-versus-host reaction (GVHR). So far, little is known about the association between these two immunological reactions, partly because of the lack of standardized, reproducible experimental models for such studies. In this work, a rat model in which GVHR and rejection occur simultaneously was established. When transplanting small bowel grafts from BN donors to Lewis recipients and thereafter treating the grafts locally with the immunomodulating substance LS-2616, a clinically visible GVHR occurred on day 6, at the same time the first signs of rejection became visible. The GVHR was confirmed by immunohistochemical staining for MHC class II-positive cells in liver and ear skin biopsy specimens. An obvious quantitative difference in the number of positive cells in both organs was observed when local treatment was compared with oral LS-2616 treatment or with findings in organs from untreated animals. We conclude that GVHR and rejection are not mutually exclusive and thus may occur simultaneously, and that this pharmacological model might facilitate further studies of the impact of GVHR on graft rejection and recipient survival.

Role of antibody synthesis and complement activation in concordant xenograft retransplantation.

A mouse-to-rat heart retransplantation model was used to study the effects of complement depletion and antibody production with regard to graft survival and anti-donor antibody specificity. Retransplantation was performed 3 weeks after the first transplantation in the presence of absence of 15-deoxyspergualin (DSG) immunosuppression. Untreated animals rejected their first graft after 3 days and retransplantation resulted in a hyperacute rejection within 2 min. A low titer of preformed anti-mouse lymphocytotoxic antibodies of the IgM subclass was found in serum collected from the unoperated rat. The rejection gave rise to a synthesis of IgG antidonor antibodies reacting with both graft endothelium and sarcolemma. Immunofluorescent staining of the rejected first heart graft showed moderate IgM and IgG antibody deposits on the graft vascular endothelium, while only IgG was found in the second graft. There was no C3 deposition found in the first mouse graft, as was the case in the second mouse graft. Anti-mouse antibodies cross-reacted with hamster antigens and a hyperacute rejection of a hamster heart graft occurred in a mouse-sensitized rat. Immunofluorescent staining revealed that the antibodies did not bind to hamster heart endothelium, as was expected, but, instead, to graft sarcolemma. DSG treatment prolonged the survival of the first graft by a median of 8 days. Continuous treatment until retransplantation resulted in a prolongation to 30 (20-127) min of the survival of the second graft and no increase in antibody titers against mouse antigens was observed. However, immunofluorescent staining revealed a weak binding of anti-mouse antibodies of the IgM subclass in the rejected mouse heart graft. Additional complement depletion with cobra venom factor in DSG-treated animals resulted in a prolongation of the median graft survival to 48 hr (6-96). No sign or minimal signs of antibody deposition were found in these grafts, but histology revealed massive mononuclear infiltration. In conclusion, xenograft transplantation in a concordant situation results in a shift of antidonor antibody Ig synthesis from IgM to IgG. If daily DSG treatment is administered from the day of transplantation, this reduces the synthesis of antidonor antibodies, and if complement is also depleted, the survival of the second graft is prolonged. The significance of the mononuclear infiltration remains to be established.