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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.

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.

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. Charcterization of anti-forssman antibodies.

The levels of human natural anti-porcine antibodies were followed in six HLA-immunized patients treated with plasmapheresis (PF) in combination with either 15-deoxyspergualin (DSG) or cyclophosphamide (CYP). PF was carried out three times a week for 4 weeks, and DSG was administered in a dose of 5 mg/kg following every third PF. CYP was given in a daily dose of 1–1.5 mg/kg. Four patients were randomized to DSG treatment and two were given CYP. Following the last (4th) DSG infusion, all patients continued on CYP treatment. Levels of antibodies toward porcine thyroglobulin were measured in an ELISA assay; thyroglobulin carries the major target for human anti-porcine natural antibodies, Gal-α(1,3)-Gal determinant. In all patients there was a drop in antibody levels. However, the antibodies never disappeared. The drop was more pronounced and more rapid in the DSG-treated group, but following the end of the PF series, antibody levels in both treatment groups rapidly returned to baseline or above baseline. In two patients in the DSG group, treatment had to be stopped because of side effects. No adverse reactions were noted in the CYP group. It is concluded that plasmapheresis in conjunction with DSG seems more effective than CYP treatment in reducing the levels of anti-porcine Gal-α(1,3)-Gal antibody. However, the dose of DSG should be reduced to lower the risk of side effects.

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.

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.

New immunosuppressants: testing and development in animal models and theclinic: with special reference to DSG.

In two kidney transplant patients, one of whom had panel-reactive antibodies (PRA) before transplantation, a pretransplant negative donor-recipient crossmatch became positive within the 1st week after transplantation. Simultaneously, good graft function deteriorated to a state of anuria. One patient graft biopsy showed a vascular rejection, whilst the other patient biopsy was unrevealing. Both patients were treated with plasmapheresis and a new immunosuppressive drug, 15-deoxyspergualin (DSG). Plasmapheresis was performed for 6 and 9 days, respectively, and DSG was given for 5 days in a dosage of 6 mg/kg body weight per day. One of the patients received methylprednisolone i.v. in addition. During treatment the cross-match became negative and has since remained that way. In both patients the graft function was restored. No adverse effects were seen from the treatment, except for a slight leukocytopenia and thrombocytopenia.

A simple technique for grafting a mouse heart heterotopically to rat neck vessels in experimental xenotransplantation is described. In this series, graft survival was tested with the following modes of immunomodulation: ciclosporin (Cy), athymic and nude rats, splenectomy, total lymphoid irradiation (TLI), 15-deoxyspergualin in two different doses and various combinations of these. The mean graft survival time, with Cy or athymic and nude rats as recipients, was 3 days as also seen in animals without treatment. With either irradiation, splenectomy or 15-deoxyspergualin, 10 mg/kg daily, the graft survival time was prolonged to 7.8, 4.7 and 6.8 days, respectively. In combination, TLI + splenectomy gave a graft survival of 9.7 days and Cy + splenectomy 7.2 days. With 15-deoxyspergualin at 10 mg/kg/day plus splenectomy, done 1 week prior to transplantation, graft survival was extended to 17.8 days.

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.

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.

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.

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.

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 longterm 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 ≥ 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.

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.

Increases in the volume of organ transplantation and in the supply of new organs have been accompanied by a dramatic surge of interest in chemical immunosuppression. In the 1950s, the selection of immunosuppressive drugs was limited to azathioprine and steroids, polyclonal antilymphocyte and anti-thymocyte globulins being added in the 1960s. The 1970s witnessed a real breakthrough with the introduction of the first second-generation selective immunosuppressant, cyclosporine. During the 1980s, the focus of interest shifted to biological agents, mainly monoclonal antibodies, though only one of these, anti-CD3 (OKT3), gained wide clinical use. Finally, in the early 1990s emerged a broad range of third-generation immunosuppressants, most of them with highly specific sites of action. At least half a dozen of these are currently undergoing clinical trials.

The immunostimulator LS-2616 abolishes the effect of cyclosporin A in a rat cardiac transplantation model. The present paper compares the characteristics of rejection obtained under different immunosuppressive regimens with and without additional LS-2616 application in the same model. Cyclosporin A (CyA, 10 mg/kg daily), prednisolone (15 mg/kg daily), or 15-deoxyspergualin (2, 5, or 10 mg/kg daily), all given from the day of transplantation until day 9, protected the grafts during the treatment period. The addition of LS-2616 (160 mg/kg, day -1 until stop) resulted in a total abrogation of the immunosuppressive effect of CyA and prednisolone. However, LS-2616 could only partially or not at all reverse the effect of 15-deoxyspergualine. These results show a certain drug selectivity of LS-2616 in promoting rejection of immunosuppressed allografts. Further studies with LS-2616 may be of benefit in evaluating the mode of action of different immunosuppressive compounds and, thus, contribute to finding more effective antirejection therapies.

The immunostimulator LS-2616 abolishes the effect of cyclosporin A in a rat cardiac transplantation model. The present paper compares the characteristics of rejection obtained under different immunosuppressive regimens with and without additional LS-2616 application in the same model. Cyclosporin A (CyA, 10 mg/kg daily), prednisolone (15 mg/kg daily), or 15-deoxyspergualin (2, 5, or 10 mg/kg daily), all given from the day of transplantation until day 9, protected the grafts during the treatment period. The addition of LS-2616 (160 mg/kg, day -1 until stop) resulted in a total abrogation of the immunosuppressive effect of CyA and prednisolone. However, LS-2616 could only partially or not at all reverse the effect of 15-deoxyspergualine. These results show a certain drug selectivity of LS-2616 in promoting rejection of immunosuppressed allografts. Further studies with LS-2616 may be of benefit in evaluating the mode of action of different immunosuppressive compounds and, thus, contribute to finding more effective antirejection therapies.

A retrospective study of 37 patients with abdominal aortic aneurysms treated at the small hospital of Bollnäs over a 16-year period is described. No surgery was performed on these cases during the first eight years. During the last eight years, six patients with ruptured aneurysms were operated on acutely and eight patients with intact aneurysms were treated by elective surgery. Peri-operative mortality was 50% in the group with ruptured aneurysms and 0% in the group with intact aneurysms. It is concluded that the facilities of a small hospital are adequate to enable trained vascular surgeons to treat abdominal aortic aneurysms successfully.