The role of speckle tracking imaging in the noninvasive detection of acute rejection after heterotopic cardiac transplantation in rats.

OBJECTIVE: Acute cardiac allograft rejection continues to be the cause of graft loss and contributes to the morbidity and mortality after cardiac transplantation. Repetitive endomyocardial biopsies are necessary to monitor the effects of immunosuppressants after cardiac transplantation. In this study, we investigate whether speckle tracking imaging (STI) is a valuable method in assessing acute cardiac rejection. METHODS AND RESULTS: Hearts from Brown Norway rats or Lewis rats were transplanted into other Brown Norway rats. Isografts and groups of allografts, either untreated or treated with cyclosporine A (CsA) at a low dose (3 mg x kg(-1) x d(-1)) or a high dose (10 mg x kg(-1) x d(-1)), were compared 7 days after transplantation. Echocardiography-derived left ventricular post wall thickness was increased only in untreated allografts.The left ventricular ejection fraction was significantly lower in the allografts compared with the isografts, but allografts treated without or with low-dose CsA showed similar results. The radial velocity and systolic radial strain rate showed a lower value in untreated allografts than other grafts, but there is no significant difference between allografts treated with high- or low-dose CsA and isografts. The circumferential strain and circumferential strain rate were comparable among the 4 groups. However, the radial strain exhibited a clear gradient in these groups (2.8 +/- 1.3 in untreated allografts, 5.2 +/- 10.9 in allografts treated with low-dose CsA, 6.3 +/- 1.8 in allografts treated with high-dose CsA, and 12.7 +/- 7.9 in isografts, P < 0.001). CONCLUSIONS: STI is able to offer a noninvasive method for detecting transplant allograft rejection.

Donor T-cell development in host thymus after heterotopic limb transplantation in mice.

We developed a mouse model of heterotopic limb transplantation in which we took advantage of Thy1.1 and Thy1.2 congenic strains to track and characterize donor T cells, to determine the role of recipient's thymus in mixed T-cell chimerism induction as well as transplant immunocompetence. The vascularized Thy1.1 limb graft composed of femur, muscle, and skin (VBT) survived long-term in more than 87.5% of Thy1.2 recipients. Percentages of donor-type Thy1.1 T cells increased from day 30 to 90 in thymus and spleen of recipients. Most peripheral donor T cells displayed a naïve phenotype and a few others were regulatory T cells. Thymectomy prevented peripheral T-cell chimerism. Congenic VBT in immunodeficient RAG mice restored their ability to reject skin allografts. These observations suggest that donor T cells differentiated in host thymus might contribute to maintenance of mixed chimerism after transplantation of tissue composite grafts that include vascularized bone.

Embryonic stem cell immunogenicity increases upon differentiation after transplantation into ischemic myocardium.

BACKGROUND: We investigated whether differentiation of embryonic stem cells (ESCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection. METHODS AND RESULTS: In one series, 129/SvJ-derived mouse ESCs (ES-D3 line) were transplanted by direct myocardial injection (1 x 10(6) cells) into murine hearts of both allogeneic (BALB/c, n=20) and syngeneic (129/SvJ, n=12) recipients after left anterior artery ligation. Hearts were procured at 1, 2, 4, and 8 weeks after ESC transplantation and analyzed by immunohistochemistry to assess immune cell infiltration (CD3, CD4, CD8, B220, CD11c, Mac-1, and Gr-1) and ESC differentiation (hematoxylin and eosin). In a second series (allogeneic n=5, sham n=3), ESC transplantation was performed similarly; however after 2 weeks, left anterior descending artery-ligated and ESC-injected hearts were heterotopically transplanted into naive BALB/c recipients. After an additional 2 weeks, donor hearts were procured and analyzed by immunohistochemistry. In the first series, the size of all ESC grafts remained stable and there was no evidence of ESC differentiation 2 weeks after transplantation; however, after 4 weeks, both allogeneic and syngeneic ESC grafts showed the presence of teratoma. By 8 weeks, surviving ESCs could be detected in the syngeneic but not in the allogeneic group. Mild inflammatory cellular infiltrates were found in allogeneic recipients at 1 and 2 weeks after transplantation, progressing into vigorous infiltration at 4 and 8 weeks. The second series demonstrated similar vigorous infiltration of immune cells as early as 2 weeks after heterotopic transplantation. CONCLUSIONS: In vivo differentiated ESCs elicit an accelerated immune response as compared with undifferentiated ESCs. These data imply that clinical transplantation of allogeneic ESCs or ESC derivatives for treatment of cardiac failure might require immunosuppressive therapy.

Embryonic pig pancreatic tissue transplantation for the treatment of diabetes.

BACKGROUND: Transplantation of embryonic pig pancreatic tissue as a source of insulin has been suggested for the cure of diabetes. However, previous limited clinical trials failed in their attempts to treat diabetic patients by transplantation of advanced gestational age porcine embryonic pancreas. In the present study we examined growth potential, functionality, and immunogenicity of pig embryonic pancreatic tissue harvested at different gestational ages. METHODS AND FINDINGS: Implantation of embryonic pig pancreatic tissues of different gestational ages in SCID mice reveals that embryonic day 42 (E42) pig pancreas can enable a massive growth of pig islets for prolonged periods and restore normoglycemia in diabetic mice. Furthermore, both direct and indirect T cell rejection responses to the xenogeneic tissue demonstrated that E42 tissue, in comparison to E56 or later embryonic tissues, exhibits markedly reduced immunogenicity. Finally, fully immunocompetent diabetic mice grafted with the E42 pig pancreatic tissue and treated with an immunosuppression protocol comprising CTLA4-Ig and anti-CD40 ligand (anti-CD40L) attained normal blood glucose levels, eliminating the need for insulin. CONCLUSIONS: These results emphasize the importance of selecting embryonic tissue of the correct gestational age for optimal growth and function and for reduced immunogenicity, and provide a proof of principle for the therapeutic potential of E42 embryonic pig pancreatic tissue transplantation in diabetes.

An experimental technique to assess the immunologic consequences of segmental small bowel transplantation.

BACKGROUND: The amount of native small bowel required for adequate nutrition is variable, but lies between 10% and 20% of full length. Currently, for patients requiring small bowel transplantation (SBT), standard practice is to transplant the entire small bowel if space permits. Few experimental studies have addressed the effect of the length of small bowel transplanted on immune responses and in those that have, the amount of mesenteric lymph node (MLN) transplanted has always been a potential confounding factor, as have differences between jejunum and ileum. METHODS: Full-length and segmental heterotopic rat SBT was performed between PVG donor and DA recipients. To transplant reduced length small bowel grafts but to exclude immunologic differences between jejunum and ileum, equal lengths of bowel were resected from proximal and distal ends in the donor. A proportional amount of MLN was carefully dissected using a microvascular technique and then excised. Serial serum samples from the transplant recipients were tested for anti-PVG (rejection) and anti-DA (graft-versus-host) antibodies using a two-color flow cytometric technique, described previously, with the aim of looking for differences in immunologic responses to full and segmental grafts. RESULTS: We have established a model of segmental SBT that includes a proportional amount of MLN and is free from differences between jejunum and ileum. Preliminary data have demonstrated the development of circulating anti-host and anti-graft antibodies with time for both full-length and segmental SBT.

Reciprocal role of cyclins and cyclin kinase inhibitor p21WAF1/CIP1 on lymphocyte proliferation, allo-immune activation and inflammation.

BACKGROUND: Immune activation that results due to the aberrant proliferation of lymphocytes leads to inflammation and graft rejection in organ transplant recipients. We hypothesize that the cell cycle control and inflammation are parallel events, inhibition of cellular proliferation by cyclin kinase inhibitor specifically p21 will limit inflammation and prevent allograft rejection. METHODS: We performed in vitro and in vivo studies using lymphocytes, and rat heart transplant model to understand the role of cyclins and p21 on mitogen and allo-induced lymphocyte activation and inflammation. Lymphocyte proliferation was studied by 3H-thymidine uptake assay and mRNA expression was studied RT-PCR. RESULTS: Activation of allo- and mitogen stimulated lymphocytes resulted in increased expression of cyclins, IL-2 and pro-inflammatory cytokines, which was inhibited by cyclosporine. The over-expression of p21 prolonged graft survival in a completely mismatched rat heart transplant model resulted by inhibiting circulating and intra-graft expression of proinflammatory cytokines. CONCLUSION: Cyclins play a significant role in transplant-induced immune activation and p21 over-expression has potential to inhibit T cell activation and inflammation. The results from this study will permit the design of alternate strategies by controlling cell cycle progression to achieve immunosuppression in transplantation.

Intercellular adhesion molecule-1 induction: a sensitive and quantitative marker for cardiac allograft rejection.

OBJECTIVES: Rats with abdominal heterotopic heart transplants were studied to determine whether cardiac allograft rejection could be assessed by immunoscintigraphy targeting intercellular adhesion molecule-1 (ICAM-1), which was induced on allografted organ cells in association with rejection. BACKGROUND: It is important to detect early rejection before development of myocyte necrosis. Although a variety of methods for the detection of cardiac rejection have been investigated, histologic inspection of biopsied samples is still used routinely for clinical diagnosis of rejection. METHODS: DA rat (RT-1a) hearts were transplanted into PVG rats (RT-1c). Immunohistologic examination of the allografts demonstrated that ICAM-1 induction on vascular endothelial cells was observed as early as 4 days after transplantation in this combination. Thirty-nine allografted rats and seven isografted rats were studied. One day after injection of 100 microCi of 111Inlabeled anti-ICAM-1 monoclonal antibody (1A29), planar images were obtained. RESULTS: Rejecting allografts showed increased radiotracer uptake and could be identified on the images as early as 5 days after transplantation. In contrast, nonrejecting cardiac allografts and isografts did not show specific uptake. Mildly rejecting allografts, with mononuclear cell infiltration but without significant myocyte necrosis, could be scintigraphically identified, and the level of radiotracer uptake reflected the histologic severity of rejection. Accumulation of 111In-labeled monoclonal antibody of isotype-matched irrelevant specificity was not detected in the rejecting allografts. CONCLUSIONS: These data indicate that ICAM-1 induction can be assessed quantitatively by radioimmunoscintigraphy. Radioimmunoscitigraphy is a sensitive method for early detection and assessment of cardiac allograft rejection.

Immunologic benefits of longer graft in rat allogenic small bowel transplantation.

BACKGROUND: The effect of graft length on rejection reaction in small bowel transplantation (SBT), which is poorly understood, is tested using rat allogenic SBT models with a short course of tacrolimus. MATERIALS AND METHODS: Inbred Brown Norway rats (major histocompatibility complex: RT1) and Lewis rats (RT1) were used as donors and recipients, respectively. The intestinal tract of the recipient was partially or totally replaced by segmental (15 cm) or whole (70 cm) donor intestine, using two different SBT models. With tacrolimus treatment (0.64 mg/kg per day, 0-13 postoperative days, intramuscularly), recipients' body weights and their survival were evaluated. To compare the extent of peripheral chimerism, donor passenger leukocytes were followed using flow cytometry with a donor-specific monoclonal antibody, OX-27. For the periodical histologic analysis, heterotopic SBT and protocol biopsies of the graft were also performed with short or long intestinal grafts. RESULTS: In a classical Monchik and Russell orthotopic SBT model, whole SBT recipients survived more than 60 days. However, all of the allogenic segmental SBT recipients died within 14 days without histologic sign of graft rejection. In the modified orthotopic SBT model using a cuff technique without systemic clamping, all recipients with segmental allograft survived longer than 29 days. However, recipients with whole graft tended to survive longer than those with segmental graft. The suffering period, lasting from the onset of rejection to death, was significantly shorter in the segmental group than in the whole group. Flow cytometric analysis showed that recipients with whole intestinal grafts had significantly higher ratio of donor passenger leukocytes in peripheral blood. Histologic studies of the protocol biopsies showed that the shorter graft tended to be more severely rejected than the longer graft. CONCLUSIONS: We have demonstrated experimentally that long intestinal grafts have immunologic advantage over short grafts.

Heterotopic transplantation of glycerin-preserved trachea: effect of respiratory epithelium desquamation on acute rejection.

An effective preservation method and decreased rejection are essential for tracheal transplantation in the reconstruction of large airway defects. Our objective in the present study was to evaluate the antigenic properties of glycerin-preserved tracheal segments. Sixty-one tracheal segments (2.4 to 3.1 cm) were divided into three groups: autograft (N = 21), fresh allograft (N = 18) and glycerin-preserved allograft (N = 22). Two segments from different groups were implanted into the greater omentum of dogs (N = 31). After 28 days, the segments were harvested and analyzed for mononuclear infiltration score and for the presence of respiratory epithelium. The fresh allograft group presented the highest score for mononuclear infiltration (1.78 +/- 0.43, P < or = 0.001) when compared to the autograft and glycerin-preserved allograft groups. In contrast to the regenerated epithelium observed in autograft segments, all fresh allografts and glycerin-preserved allografts had desquamation of the respiratory mucosa. The low antigenicity observed in glycerin segments was probably the result of denudation of the respiratory epithelium and perhaps due to the decrease of major histocompatibility complex class II antigens.

Antigenicity of fresh and cryopreserved rat valve allografts.

Aortic valve allografts have demonstrated excellent clinical performance, but the importance of antigenic differences between donor and recipient is largely unknown. To determine the antigenicity of aortic valve grafts, rat aortic valves with a short portion of thoracic aorta were transplanted into the abdominal aorta of recipient rats. Valves were used immediately after harvest (fresh) or following cryopreservation. Three weeks after this procedure, the recipient rats received a skin graft from a rat of a strain syngeneic to that of the aortic valve donor. Additional groups of rats were subjected to sham operation (sham) followed three weeks later by skin grafting. Recipient rats were of the Lewis strain. Donor rats were of the Lewis, F344 (weakly allogeneic, RT1-compatible, non-RT1-incompatible), LBN F1 (moderately allogeneic, one-haplotype-identical and one-haplotype-incompatible at both the RT1 and non-RT1 loci), or BN (strongly allogeneic, RT1 and non-RT1-incompatible) strain. Time to skin graft rejection was measured. Among rats receiving the F344 grafts, the time to skin graft rejection (mean +/- SD) was sham: 9.1 +/- 1.0 days, fresh: 7.1 +/- 1.2 days, cryopreserved: 6.9 +/- 0.7 days. Among rats receiving the LBN F1 grafts, the corresponding times were sham: 7.8 +/- 0.8 days, fresh: 5.6 +/- 0.5 days, cryopreserved: 5.4 +/- 0.5 days. Among rats receiving the BN grafts, the corresponding times were sham: 7.1 +/- 0.3, fresh: 4.5 +/- 1.0 days, and cryopreserved: 4.3 +/- 0.7 days. Significant differences (P less than 0.05) existed between sham and fresh and between sham and cryopreserved, but not between fresh and cryopreserved. Significant differences (P less than 0.05) also existed between each histocompatibility grouping. It is concluded that aortic valve allografts in rats are antigenic and produce recipient sensitization. Cryopreservation does not diminish this sensitization. The degree of antigenicity is related to the degree of histoincompatibility between donor and recipient. Both RT1 and non-RT1 antigens appear to play a role in this process.

The necessity of differential immunosuppression for prevention of immune rejection by FK506 in rat islet allografts transplanted into the liver or beneath the kidney capsule.

The purpose of the present study was to achieve prevention of immune rejection in rat islet allografts by FK506. WKA/Qdj (RT1u) islets were transplanted either into the liver via the portal vein (p.v.) or beneath the kidney capsule (k.c.) of streptozotocin (60 mg/kg) induced diabetic Lewis (RT1(1)) rats. Fresh or cultured (24 degrees C, 1 week) islets were used as donors. A miniosmotic pump (0.2 ml, Alzet 2001) containing 5 mg FK was implanted s.c. at the time of transplantation for continuous delivery of FK506 for 7 days after transplantation. The mean survival time (MST) of the fresh p.v. grafts with a pump was offater than 61.4 +/- 37.2 days (mean +/- SD, n = 17) (control 5.5 +/- 0.6, n = 4). Ten out of 17 were normoglycemic for more than 90 days after transplantation. When low-temperature cultured islets were used and FK506 was delivered for 7 days, all the rats were normoglycemic for more than 90 days after transplantation. The MST of the fresh or cultured k.c. grafts with a pump was 22.0 +/- 14.2 or 24.7 +/- 5.0 days, respectively. Long-term administration of FK506 by repeated implantations (5 times; days 0, 7, 14, 21, and 28) of pumps containing 5 mg FK506 produced marked prolongation of the fresh or cultured k.c. graft survival with an MST of greater than 58.7 +/- 22.1 or greater than 56.9 +/- 18.0 days, respectively. These findings clearly demonstrate that the prevention of immune rejection in the islet allografts transplanted into the liver was achieved by short-term post-transplant administration of FK506 and low-temperature culture of donor islets, and also show that long-term continuous administration of FK506 was needed for the prolongation of the graft survival when the renal subcapsular space was the site for implantation of islets. Thus, the present study indicates that in different transplant sites different immunosuppressive regimes are needed for the control of rejection by FK506 in rat islet allografts.

Predominant role of the Fab fragment in delaying hyperacute rejection in guinea pig-to-rat xenotransplantation.

BACKGROUND: Human intravenous immunoglobulin G delayed xenogeneic hyperacute rejection (HAR) in the guinea pig-to-rat combination. We investigated the respective roles of the Fc and Fab fragments of the IgG molecule in this inhibitory effect. METHODS: By using a guinea pig-to-rat heart transplantation model, the efficiency of IgG, Fab, and Fc in prolonging the grafted heart's survival time (ST) was compared. RESULTS: A dose-dependent increase in the ST was observed with Fab (r=0.74, P < 0.0001), IgG (r=0.57, P < 0.001), and Fc (r=0.51, P < 0.01). The linear regression slopes with Fab and with IgG were, respectively, sevenfold and fourfold steeper than with Fc. The ST was significantly longer than controls (23+/-7 min) after infusion of 2 g/kg IgG (147+/-42 min) or 1 g/kg Fab (176+/-38 min), whereas the highest dose of Fc (1.5 g/kg) did not induce significant prolongation of ST. In terms of equivalent functional doses, 1 g/kg Fab was significantly more potent in prolonging the ST than 1.5 g/kg IgG (87+/-25 min) or 0.5 g/kg Fc (33+/-14 min). Analysis of the rejected hearts evidenced edema, necrosis, and rat C3 deposits characteristic of HAR. CONCLUSION: These results indicated that the delaying action of intravenous immunoglobulin G on HAR in the guinea pig-to-rat combination is mostly mediated through the Fab fragment.

Successful intracerebroventricular allotransplantation of parathyroid tissue in rats without immunosuppression.

The lateral ventricle of the brain may be an immunoprivileged site for viable allografts. Allotransplanted parathyroid tissue from histoincompatible ACI rats survived and remained functional for more than 3 months in the cerebroventricles of recipient F344 rats. Microscopic examination proved that the allotransplanted parathyroid tissues retained normal histological features. In sharp contrast, when the parathyroid was placed beneath the renal capsule the allografted parathyroid tissue uniformly lost its capacity to liberate parathyroid hormone within one month, and only residual scar tissue remained at the transplantation site. After allotransplantation of parathyroid tissue into the cerebroventricle, the serum concentrations of both Ca++ and parathyroid hormone were maintained at levels similar to those before parathyroidectomy, until the time of sacrifice. During the 3-month period of post-transplantation observation, no neurological symptoms were noted in any of the F344 rats.

Promotion of rat cardiac allograft survival by intrathymic inoculation of donor splenocytes.

Donor-specific unresponsiveness to LEW heterotopic cardiac allografts was induced in WF rats following intrathymic inoculation of LEW splenocytes in conjunction with a single intraperitoneal dose of antilymphocyte serum. In contrast, LEW cardiac allografts were promptly rejected in WF recipients pretreated with an intravenous inoculation of donor splenocytes. Without transient immunosuppression with antilymphocyte serum neither intrathymic nor intravenous inoculation of splenocytes led to allograft survival. Substitution of antilymphocyte serum by a short course of cyclosporine did not permit allograft survival, suggesting that a T-cell-depleting regimen is crucial to tolerance induction by this protocol. The unresponsive state could be transferred to secondary syngeneic hosts by spleen cells from long-term recipients of intrathymic splenocytes and cardiac allografts but not by spleen cells from recipients of intrathymic splenocytes alone. This suggests that persistence of donor alloantigen from the graft is necessary for maintenance of the tolerant state. The unresponsive state after intrathymic inoculation of allogeneic splenocytes may be mediated through interaction of maturing host thymocytes with donor alloantigen.

Prevention of recurrent autoimmune diabetes in the BB rat by islet transplantation under the renal capsule.

Pancreatic islet grafts transplanted into subjects with spontaneous autoimmune diabetes are threatened by two immune responses, allograft rejection and the recurrence of autoimmune insulitis. To examine the recurrent autoimmune response to transplanted islets it is necessary to exclude islet allograft rejection. The BB rat is a unique model of spontaneous diabetes with clinical and pathological characteristics identical or similar to those found in human insulin dependent diabetes mellitus (IDDM). In this study we demonstrate permanent acceptance of histocompatible islet grafts in chemically induced diabetes and a lack of intracolony tissue antigen rejection in our BB rat colony. Therefore the vigorous destruction of transplanted BB islets in the liver of spontaneously diabetic BB rats is due to recurrence of diabetes. This recurrence can be prevented by transplantation of islets under the renal capsule. This may be important for clinical application in IDDM, particularly with regard to host and donor tissue matching.

T-cell and major histocompatibility complex requirements for obliterative airway disease in heterotopically transplanted murine tracheas.

BACKGROUND: One third of human lung allografts develop chronic rejection manifested as obliterative bronchiolitis. Heterotopically transplanted allogeneic murine tracheas develop obliterative airway disease (OAD) leading to a lesion resembling human obliterative bronchiolitis. The purpose of this study was to determine the T-cell and major histocompatibility complex (MHC) molecule requirements of murine OAD. METHODS: BALB/c allografts and C57BL/6 (B6) isografts were transplanted into B6 severe combined immunodeficient (SCID) and B6 wild-type (WT) recipients. MHC class I-discrepant bm1 grafts, class II-discrepant bm12 grafts, and F1(bm1 x bm12) (F1) grafts also were transplanted into B6 WT recipients. Grafts were harvested between days 5 and 56 following transplantation and evaluated histologically. RESULTS: Complete MHC-disparate allografts placed in WT recipients had significantly more disease than similar allografts in SCID recipients, and the latter were indistinguishable from isografts in either WT or SCID recipients, indicating a lymphocyte dependence on the disease development. Pathology was significantly more severe in bm1 and F1 allografts than in isografts recovered from B6 recipients, but bm12 allografts appeared no different than isografts. T-cell infiltrates in these bm12 allografts contained only CD4+ cells, whereas infiltrates in the BALB/c, bm1, and F1 allografts manifesting OAD contained both CD4+ and CD8+ cells. No grafts had significant B-cell infiltration. CONCLUSIONS: These findings suggest that OAD relies on a host T-cell response that includes CD8+ cells, directed against allo-class I-bearing donor cells within the graft.

Immunosuppressive properties of the benzothiazepine calcium antagonists diltiazem and clentiazem, with and without cyclosporine, in heterotopic rat heart transplantation.

In vitro studies have shown that calcium channel blockers (CCB) exert inhibitory effects on immunocompetent cells but conflicting results have been reported as to the translation of these effects into significant in vivo immunosuppression. In this study we evaluated the effects of the benzothiazepine-like calcium blockers diltiazem and clentiazem, given alone or associated with cyclosporine on survival improvement of heterotopic rat heart transplants. Inbred male rats of the Lewis strain were used as recipients and Wistar-Furth as donors. Following abdominal implantation of the graft, recipients were randomly divided into 9 groups (n = 5). Group 1 were control isografts (Lew-Lew); group 2 were control allografts (WFu-Lew), and group 3 were allografts treated with low-dose oral cyclosporine 2 mg/kg/day. Groups 4 and 5 were allografts treated with oral diltiazem 0.25 and 2.50 mg/kg/day. Groups 6 and 7 were treated with oral clentiazem, 0.25 and 2.50 mg/kg/day. Groups 8 and 9 consisted of allografts receiving low-dose cyclosporine with either diltiazem or clentiazem 2.50 mg/kg/day, all drugs were administered daily by gavage. Graft function was monitored by transabdominal palpation, and rejection was considered to be complete when no contraction of the graft could be detected. Mean survival time of untreated allografts was 6.4 +/- 0.5 days. Cyclosporine alone increased the mean survival time to 10.6 +/- 2.7 days (P < 0.05 vs. group 2). At all doses studied, diltiazem and clentiazem significantly increased mean survival time of allografts, clentiazem being slightly more potent than diltiazem. In addition, the observed beneficial effects of the benzothiazepine-like calcium channel blockers were dose-dependent. When combined with cyclosporine, diltiazem and clentiazem interacted synergistically (mean survival time increased to 16.8 +/- 3.4 days for diltiazem and 15.8 +/- 1.4 days for clentiazem). These results demonstrate that the benzothiazepine-like calcium channel blockers, as opposed to phenylalkylamines or dihydropyridines, afford significant immunosuppression when used alone. These observations, and the fact that they beneficially interact with cyclosporine, suggest that the benzothiazepine-like calcium antagonists should be considered the drugs of choice when CCBs are to be selected in transplanted patients.

Factors determining prolongation of rat heart allograft survival by perioperative injection of donor spleen cells.

Previously, we have shown that a perioperative injection of donor mononuclear cells in combination with cyclosporine treatment on day 2 after transplantation prolongs heart allograft survival in rats. In this study we determined whether the efficacy of this treatment was influenced by the same factors that have been shown to affect the efficacy of preoperative administration of donor cells. The effect of the following factors were investigated: dosage and repetition of the donor cell injection, viability of the donor cells, immunosuppressive drugs other than cyclosporine, and the rat strain combination. We found that there was an optimal dosage of donor cells; dosages of 4 x 10(7) or 1 x 10(8) cells gave the best heart graft survival. Repetition of the donor cell injection was not useful. Reducing viability of the cells by irradiation did not abrogate the prolonged graft survival, whereas killing of the cells did. Methylprednisolone, azathioprine, or cyclophosphamide in combination with the perioperative donor cell injection did not prolong heart graft survival in comparison with treatment with the drug only. The efficacy of this treatment was also influenced by the rat strain combination. In some combinations, this treatment prolonged graft survival, whereas in others an effect was absent or undetectable. Importantly, this treatment never adversely affected graft survival. We conclude that the efficacy of this treatment is influenced by similar factors as found for preoperative treatment with donor cells. A major advantage of this treatment over preoperative blood transfusions is that it avoids sensitization.

Enhanced CD4 reconstitution by grafting neonatal porcine tissue in alternative locations is associated with donor-specific tolerance and suppression of preexisting xenoreactive T cells.

BACKGROUND: Donor-specific xenograft tolerance can be achieved by grafting fetal porcine thymus tissue to thymectomized (ATX) mice treated with natural killer (NK) and T-cell-depleting monoclonal antibodies plus 3 Gy of total body irradiation (TBI). Grafting of neonatal, instead of fetal, thymus, along with neonatal pig spleen, leads to a lower level of mouse CD4 cell reconstitution, with less reliable tolerance induction. For a number of reasons, it would be advantageous to use neonatal rather than fetal pigs as donors. We therefore investigated the possibility that grafting larger amounts of neonatal porcine thymus tissue to different sites could allow improved outcomes to be achieved. MATERIALS AND METHODS: Multiple or single fragments of neonatal porcine thymus tissue were grafted with a splenic fragment to different sites (mediastinum, mesentery, and kidney capsule) of ATX B6 mice treated with T- and NK-cell-depleting antibodies and 3Gy TBI. Mice also received an intraperitoneal injection containing 1 x 10(7) donor splenocytes. Donor-specific skin graft tolerance was evaluated, and CD4 reconstitution and mouse anti-donor xenoantibodies were followed by flow cytometry. RESULTS: Peripheral repopulation of CD4+ cells occurred by 7 weeks after transplantation in mice grafted with four fragments of neonatal porcine tissue in either the mediastinum or the mesentery, but not in mice grafted under both kidney capsules with the same amount of tissue. The level of CD4 reconstitution correlated with skin graft tolerance and an absence of induced anti-donor xenoantibodies. Seventy-five percent of mice with >20% of CD4+ cells among peripheral blood lymphocytes (PBL) by 13 weeks posttransplantation accepted donor porcine skin, while rejecting either non-donor neonatal porcine or mouse BALB/c skin allografts. In contrast, only 29% of grafted mice with <20% CD4+ cells in the peripheral blood at 13 weeks accepted donor porcine skin. Grafted mice with poor reconstitution showed either low or high levels of anti-pig xenoantibodies of the IgM, IgG1, and IgG2a isotypes. Grafted mice with >20% CD4+ cells all had low levels of anti-pig xenoantibodies of these isotypes and displayed mixed lymphocyte reaction (MLR) tolerance to donor pig major histocompatibility complex (MHC), with responsiveness to allogeneic mouse stimulators. CONCLUSION: Grafting neonatal porcine thymus into either the mediastinum or mesentery provides earlier and more efficient reconstitution of the CD4 compartment than does grafting under the kidney capsule. Good CD4 reconstitution was associated with optimal donor-specific skin graft tolerance and avoidance of the anti-donor xenoantibody responses observed in mice with poor CD4 reconstitution. These results also suggest that there is a suppressive component to the porcine xenograft tolerance induced with this approach.

Different kinetics of obliterative airway disease development in heterotopic murine tracheal allografts induced by CD4+ and CD8+ T cells.

BACKGROUND: Both T and B cells have been shown to be implicated in the pathogenesis of bronchiolitis obliterans syndrome, which is considered to represent chronic lung allograft rejection. However, the relative contributions of T cells and alloantibodies in the pathogenesis of the disease are still unknown. In this study, we used an heterotopic murine tracheal transplantation model to determine the contribution of these components of the immune system in the pathogenesis of posttransplant obliterative airway disease (OAD). METHODS: Tracheal allografts from BALB/c and HLA-A2-transgenic (HLA-A2+) mice were heterotopically transplanted into C57BL/6, CD4-knockout (KO), CD8-KO, Ig-KO, and Rag1-KO mice. In additional experiments, recipient mice were pretreated with depleting antibodies against CD4+, CD8+, and NK1.1+ cells. Development of OAD was determined by histopathology at days 10, 30, 60, 90, and 180 after transplantation. RESULTS: HLA-A2+ allografts transplanted into C57BL/6, CD8-KO, and Ig-KO mice demonstrated OAD lesions by day 30. In contrast, allografts transplanted into CD4-KO mice showed no OAD lesions at day 30, partial OAD development by days 60 and 90, and complete OAD development by day 180. No OAD development was observed in allografts transplanted into Rag1-KO mice. Treatment with anti-NK1.1 antibody did not show any effect on posttransplant OAD development. In contrast, anti-CD4+ or anti-CD8+ antibody treatments partially reduced the OAD histopathology and combined anti-CD4/CD8 antibody treatment further abrogated the histopathology of the disease. CONCLUSION: These results show that both CD4+ and CD8+ T cells have a role in the pathogenesis of OAD and that natural killer cells and alloantibodies are not necessary for the development of this disease.