Göran Gannedahl

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.

15-deoxyspergualin effects in rat heart allograft transplantation--relation to dose, timing, and cyclosporine

15-deoxyspergualin effects in rat heart allograft transplantation--relation to dose, timing, and cyclosporine

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 α/β 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.

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 simulta neously 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 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.

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.

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 (10 mg/kg), oral Linomide® (160 mg/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.