Lewis-Brown Norway Research Articles

Short-term therapy with TCR and FK-506 leads to long-term laryngeal transplant function

Problem: A mouse anti-rat T-cell receptor monoclonal antibody (TCR) has great potential in the development of long-term immunologic tolerance. Combination therapy with FK-506 and TCR may enable the establishment of long-term immunotolerance while reducing dose-related toxicities. The use of combined TCR and FK-506 in a completely allogeneic model permits a rigorous evaluation of potential synergism. Methods: Nineteen Brown Norway larynges were transplanted to Lewis recipients. Treatment consisted of 7 days of FK-506 (1.2 mg/kg/day) alone and in combination with TCR (0.5 mL/day). Control groups consisted of untreated animals, as well as 11 Lewis-Brown Norway larynges transplanted to Lewis recipients and treated with FK-506 monotherapy. Each group consisted of 6–11 rats. Median duration of engraftment was 44 days (range, 14–106 days). Graft histopathology was graded according to an established 31-point scale in a blinded fashion. Long-term grafts (>75 days) were followed with serum PTH levels. Results: Untreated controls experienced almost complete rejection (mean score, 27, SD 0). Allogeneic transplants treated with FK-506 monotherapy experienced near-complete rejection (mean score, 25.2, SD 2.1). Allogeneic transplants treated with combination therapy and followed for a median duration of 100 days demonstrated significantly better rejection scores than controls (mean score, 11.1, SD 1.7; P = 0.003). Combination therapy was also significantly more effective in preventing acute rejection than monotherapy with FK-506 in a semi-allogeneic model (mean score, 22.1, SD 4.6; P < 0.04). Conclusion: Short-term combination therapy with FK-506 and TCR prevents rejection in an allogeneic model for up to 100 days. This regimen was associated with significantly better histopathologic rejection scores than monotherapy with either agent, or monotherapy with FK-506 in a semi-allogeneic model. Significance: The blockade of primary antigen recognition with TCR may prevent the need for daily immunosuppressive therapy, thereby limiting treatment-associated toxicities. Pulsing protocols may also provide a mechanism for intermittent immunomodulation with this regimen. Support: None reported.

The interaction between FK778 and tacrolimus in the prevention of rat cardiac allograft rejection is dose dependent.

The synthetic malononitrilamide FK778 inhibits T- and B-cell responsiveness, phagocyte effector function, exerts inhibitory activity against cytomegalovirus, and is thus one of the most promising new immunosuppressive drugs. The aim of this study was to evaluate the combination of FK778 and tacrolimus in a high-responder rat cardiac transplantation model. The Brown Norway-Lewis rat strain combination was used to investigate graft survival after 10 days of posttransplant oral therapy with FK778 (5 or 20 mg/kg), tacrolimus (2 or 8 mg/kg), or combination regimens at varying doses (5+2 mg/kg, 10+1 mg/kg, or 20+8 mg/kg). Grafts were harvested after cessation of cardiac contractions. Combination indices (CI) were calculated for drug combinations. In untreated recipients, allograft survival was 6.2+/-0.4 days. FK778 at 20 mg/kg and tacrolimus at 2 or 8 mg/kg significantly prolonged graft survival to a mean survival time (MST) of 17.0+/-2.8, 18.5+/-2.7, and 25.0+/-2.5 days, respectively. The two low-dose drug combinations achieved a graft survival of 23.2+/-2.9 and 25.2+/-3.1 days, which was significantly longer compared with FK778 at 5 mg/kg, FK778 at 20 mg/kg, and tacrolimus at 2 mg/kg (P </=0.003) and was similar to that of high-dose tacrolimus therapy (8 mg/kg). The drugs showed synergistic interactions (CI values of 0.62 and 0.52, respectively). With the high-dose drug combination, MST was 30.0+/-4.1 days, which was significantly longer compared with all other groups (P </=0.012). However, drugs showed only additive interaction (CI=0.95), and recipients suffered enhanced toxic side effects. This study provides evidence that FK778 and tacrolimus show supportive interaction in their immunosuppressive potency that is synergistic in low-dose combinations and additive in higher doses.

Absence of graft-versus-host disease in the isolated vascularized bone marrow transplant.

An isolated vascularized bone marrow transplant (iVBMT) model was developed to study the contribution of the bone marrow component in a composite tissue allograft. We hypothesized that the iVBMT would be functional and cause graft-versus-host disease (GVHD) in a fraction of the recipients. Lewis iVBMT grafts were transplanted to Lewis-Brown Norway recipients. Animals were sacrificed at various times from 1 to 14 weeks. Polymerase chain reaction for microchimerism was performed on the host's marrow. No animals exhibited signs of GVHD at death. Histologic examination of the grafts showed a normal mix of hematopoietic and fatty elements and appeared to be functional. Tissues usually affected-tongue, ear, liver, and gut-also showed no evidence of disease. Polymerase chain reaction demonstrated microchimerism in both groups. These findings suggest that the vascularized bone marrow within a composite tissue allograft is not the component that causes GVHD; rather, it may serve an immunomodulatory function for tolerance induction.

Immunosuppression with FK778 and mycophenolate mofetil in a rat cardiac transplantation model.

FK778 blocks the dihydro-orotate dehydrogenase, necessary for pyrimidine synthesis, and mycophenolate mofetil (MMF) inhibits the inosine monophosphate dehydrogenase, a crucial enzyme for purine biosynthesis. Beneficial immunosuppressive effects have been suggested for the combination of both drugs. The Brown Norway-Lewis rat heterotopic heart transplantation model was used. FK778 (5 and 20 mg/kg/day), MMF (10 and 40 mg/kg/day), or a combination of both drugs for 10 days was used for prevention of acute graft rejection. Grafts of untreated animals were rejected after 6.2 +/- 0.4 days. Low-dose FK778 and low-dose MMF administration did not result in a significantly prolonged graft survival (6.7 +/- 0.8 and 8.7 +/- 1.4 days; P=not significant). Grafts of rats treated with high-dose FK778 or high-dose MMF survived significantly longer (17.0 +/- 2.8 and 20.7 +/- 3.8 days; P<0.01). Concomitant use of low-dose FK778 with low-dose MMF produced synergistic interactions (mean survival time 12.3 +/- 2.9 days; P<0.01; combination index=0.85). High-dose drug combination (mean survival time 24.0 +/- 1.4 days) showed antagonistic drug interaction (combination index=1.55) with increased toxic side effects.

Met-RANTES Inhibition of Mucosal Perfusion Failure in Acute Intestinal Transplant Rejection – Role of Endothelial Cell-Leukocyte Interaction

Acute rejection-induced microvascular injury results in graft dysfunction, ultimately leading to graft loss. Infiltration of T cells and monocytes as a consequence of an enhanced endothelial cell-leukocyte interaction appears to play an important role in this deleterious process. Recruitment of these pro-inflammatory cells to the vessel wall is mediated by chemokines such as RANTES. Heterotopic small bowel transplantation was performed in rats with the fully allogeneic Brown Norway-Lewis strain combination and, as a control, the syngeneic Lewis-Lewis strain combination. Intravital microscopy was performed from postoperative day 1–7 in both groups. The percentages of perfused villi and villus stasis, mucosal and muscular functional capillary densities, red blood cell velocities, and finally, firm adherence of leukocytes in postcapillary submucosal venules were assessed. Syngeneic small bowel transplantation revealed homogeneous perfusion of villi and muscle layers over the whole study period. Allogeneic small bowel transplantation showed a decline in perfusion from postoperative day 1 until complete failure on postoperative day 7. This was accompanied by a continuous increase in endothelial cell-leukocyte interaction which reached a plateau on postoperative day 5. Met-RANTES treatment at 200 µg/day for 5 days markedly attenuated both the decrease in functional capillary density and the increased endothelial cell-leukocyte interaction in rats following allogeneic small bowel transplantation. We conclude that blocking chemokine receptors, thereby limiting endothelial cell-leukocyte interaction, may constitute a useful therapeutic approach to the prevention of microcirculatory perfusion failure in acute transplant rejection.

A model for allogenic extremity transplantation in the rat with possible microcirculatory monitoring

A rat model for monitoring allograft rejection of transplanted rat hindlimbs at the microcirculatory level is described. The well-established rat cremaster flap model is combined with a rat hindlimb transplantation procedure at a level proximal to the neurovascular pedicle of the cremaster muscle. The cremaster serves as a microcirculatory monitor for in vivo evaluation of graft rejection by measuring skeletal muscle perfusion. Donor animals are male Lewis Brown Norway rats and recipients are Lewis rats. The right cremaster muscle of the donor animal is dissected as a tubular island flap and preserved in a subcutaneous tunnel in the hindlimb. Afterwards, the right hindlimb including the cremaster is amputated at the mid level of the common iliac vessels and transplanted to the recipient at the level of the external iliac vessels. Over a time period of five days, the cremaster muscle of the composite transplants showed appropriate tissue quality for intravital microscopical observations. This transplantation model allows evaluation of allograft rejection in vivo at the microcirculatory level.

Microcirculatory window for early detection of allograft rejection.

The purpose of this study is to introduce a technical detail on a transplantation model for in vivo evaluation of microcirculatory changes during the acute phase of allograft rejection. The cremaster muscle is incorporated and transplanted along with the hind limb to detect and study ischemia/reperfusion injury and the acute phase of allograft rejection in rats. Thirty-six animals were studied in three experimental groups of 12 animals each. Each group was divided into subgroups and microcirculatory measurements were taken at two different time periods: 24 and 72 hours. In the ischemic control group (N = 12), cremaster muscles were denervated, prepared as a tube flap, and submitted to the same interval of ischemia as the other groups but without transplantation. In the isograft group (N = 12), rat hind limb-cremaster grafts were transplanted between genetically identical Lewis rats (RT11). In the allograft group (N = 12), 12 transplantations were performed across a major histocompatibility barrier between Lewis Brown-Norway (RT-11+/-n) and Lewis (RT 11) rats. The diameters of first-, second-, and third-order arterioles and venules; red blood cell velocities; and functional capillary density were recorded at 24 and 72 hours after transplantation. Daily follow-up observations were continued until 3 days after the first clinical signs of graft rejection. The mean number of perfused capillaries in the two transplantation groups was significantly lower than in the control group at both 24 hours and 72 hours (p < 0.05). Those results were as follows: 8.2 +/- 2.1 at 24 hours, 7.7 +/- 0.85 at 72 hours in the ischemic control group; 5.4 +/- 0.9 at 24 hours, 6 +/- 0.6 at 72 hours in the isograft group; and 5 +/- 0.9 at 24 hours, 5.5 +/- 0.3 at 72 hours in the allograft group. Red blood cell velocities and vessel diameters in the main arteries were also decreased in transplant groups at 24 hours (p < 0.05) but returned to normal 72 hours after the operation (p > 0.05). The composite rat hind limb-cremaster model presented in this study introduces a reproducible in vivo approach to monitor the differences in microcirculatory hemodynamics of ischemia/reperfusion injury and acute graft rejection. The model allows the study of the timing, sequence, and correlation between clinical and hemodynamic signs during the acute phase of allograft rejection.

In vivo microscopic assessment of cremasteric microcirculation during hindlimb allograft rejection in rats.

Experimental and clinical studies of vascular allogenic extremity transplantation have yielded disappointing results and have not been clinically useful. With recent advances in transplantation immunology, considerable interest has focused on the understanding of leukocyte-endothelial interaction at the microcirculatory level. The objective of this study was to characterize the alterations in leukocyte-endothelial interaction in the early stages of rat hindlimb allograft rejection. To study the changes at the microcirculatory level, a new microsurgical model was developed; the cremaster muscle was incorporated into the transplanted hindlimb. The purpose of this study was to report on the microcirculatory changes during rat hindlimb allograft rejection. A total of 24 transplantations were performed among the four experimental groups. In a control group, 12 rat hindlimb-cremaster grafts were transplanted between genetically identical animals, Lewis to Lewis. Microcirculatory measurements of graft survival were taken at 24 hours (group 1A, n = 6) and at 72 hours (group 1B, n = 6). In the rejection control group, 12 transplantations were performed across a major histocompatibility barrier between Lewis-Brown Norway and Lewis rats. Microcirculatory measurements were taken at 24 (group 2A, n = 6) and 72 hours (group 2A, n = 6) as above. The following parameters were evaluated to discover the leukocyte-endothelial interaction: endothelial edema index and the number of rolling, adherent, and transmigrating leukocytes and lymphocytes in the postcapillary venule. Physical signs of limb rejection, such as edema, erythema, scaling, plaque formation on the skin, hair loss, and skin surface temperature, were monitored. Microcirculatory signs of rejection included the following. There was a significant increase in the number of adherent leukocytes in allograft transplants at both 24 hours (205 percent; 2.05 +/- 0.38) and 72 hours (431 percent; 9.11 +/- 3.41) when compared with isograft controls (1.00 +/- 0.89 at 24 hours; 2.11 +/- 0.34 at 72 hours) (p < 0.05). The activation of leukocyte transmigration increased more than 7-fold in muscle allografts at 24 hours (0.55 +/- 0.25 versus 4.16 +/- 1.89) and more than 6-fold at 72 hours (0.72 +/- 0.38 versus 4.38 +/- 1.28) after transplantation (p < 0.05). Endothelial edema index, a measure of endothelial swelling and cellular deposit accumulation, increased more than 119 percent in the allograft group 72 hours after transplantation (1.23 +/- 0.07 versus 1.46 +/- 0.09) (p < 0.05). The first clinical signs of limb rejection were scaling of the skin or hair loss; they were observed between the seventh and ninth postoperative days. The composite rat hindlimb-cremaster model presented in this study introduces a new in vivo approach to monitor acute graft rejection using the intravital microscopy system. This is a valuable model for defining the timing, sequence, and correlation between immunologic events and clinical signs during the acute phase of allograft rejection.

Site-Specific Immunosuppression using a New Formulation of Topical Cyclosporine A with Polyethylene Glycol-8 Glyceryl Caprylate/Caprate

Purpose.Dermal application of immunosuppressants can be an effective means of achieving site-specific immunosuppression (SITE) on skin allografts in burn wound management and in the treatment of various immune skin disorders. We have previously reported success with topical cyclosporine A (tCsA) in the treatment of skin allograft rejection in rats. Using a new tCsA formulation with a penetration enhancer (PE), polyethylene glycol-8 (PEG-8) glyceryl caprylate/caprate (Labrasol, Gattefossé, St. Priest, France), in a trinary drug delivery system, we hypothesized that we would induce SITE and significantly delay rejection of dual skin allografts in rats.Methods.Dual rat skin allografts from Lewis × Brown–Norway (LBN) donors were grafted to Lewis (Lew) recipients. Experimental animals (EXP,n= 7) received a 10-day course of systemic cyclosporine (sCsA, 8 mg/kg/day) followed by topical application. One of the two allografts on each experimental animal received tCsA/PE application (5 mg/kg/day) until sacrifice (tCsA/PE-treated). The other allograft received vehicle only (vehicle-treated). Allogeneic controls (ALLO-CON,n= 9) received no sCsA or tCsA. First signs of rejection were determined based on the initial observation of erythema, hair loss, flakiness, and/or scabs.Results.The mean time to rejection for ALLO-CON allografts was 6.3 ± 0.7 days (ttest,P= 0.0013); for vehicle-treated allografts, 12.3 ± 3.8 days (pairedttest,P= 0.0146); and for tCsA/PE-treated allografts, 25.6 ± 5.4 days. The disparity of days to rejection between dual allografts in the ALLO-CON group was 0.0 ± 0.0 day and that between the tCsA/PE- and vehicle-treated dual allografts was 13.3 ± 3.9 days (ttest,P= 0.0016).Conclusions.A new formulation of tCsA in a trinary drug delivery system is successful at delaying the onset of rejection in dual skin allografts in rats by SITE, and PEG-8 glyceryl caprylate/caprate may represent a potentially effective transdermal penetration enhancer.

Microcirculatory hemodynamics during the acute phase of free vascularized muscle allograft rejection.

This study investigated the microcirculatory pattern during acute rejection of a vascularized free muscle graft. Using a new rat cremaster muscle free flap model, isograft transplantation was done between genetically identical Lewis rats (N = 23) and allograft transplantation across a major histocompatibility barrier (Lewis Brown Norway [RT-11 + n] to Lewis [RT-11] rats; N = 24). At days 1, 3, 5, and 7 posttransplantation, microcirculatory measurements were taken of vessel diameter, red blood cell velocity, endothelial edema index, leukocytic-endothelial interaction (rolling, adhering, and transmigrating leukocytes), and capillary perfusion. In the allografts at 5 days the lumen obstruction was 27% greater (p < 0.05) than onset. The number of perfused capillaries decreased by 36% compared with isografts at day 5 (p < 0.05). The number of adhering leukocytes increased significantly in allografts both at day 1 (71%; p < 0.05) and at day 3 (77%; p < 0.05). At day 3, transmigrating leukocytes rose significantly (p < 0.05). Allograft histology showed myocytic destruction and lymphocytic infiltration at day 5. Our results suggest that the microcirculatory signs of acute muscle allograft rejection occurred 24 hours after transplantation, and preceded histological signs of rejection. The critical time for microvascular survival of muscle allografts was 3 days. This procedure allows us to distinguish events related to transplantation trauma from rejection phenomena.

Anti-ICAM-1 antibodies protect allografts against microvascular and parenchymal cell damage

Anti-ICAM-1 (anti-intercellular adhesion molecule-1) monoclonal antibodies (CD54) were tested for treating composite tissue allografts in the rat hindlimb-cremaster transplantation model for intravital microcirculatory studies. Twenty-four transplantations were carried out across major histocompatibility barriers between Lewis-Brown Norway and Lewis rats. Isograft control transplants were compared to nontreated allograft control transplants and to allografts treated with 1 mg/kg of anti-ICAM-1 monoclonal antibodies. At 24 and 72 hours, microcirculatory vessel diameters, red blood cell velocities, functional capillary perfusion, endothelial edema index, and leukocyte-endothelial interactions were measured. At 24 and 72 hours, the number of sticking leukocytes, sticking lymphocytes, transmigrating leukocytes, and the endothelial edema index in the treated allografts were significantly decreased more than in the other 2 groups (p < .05 for all variables). Anti-ICAM monoclonal antibodies significantly reduced leukocyte-endothelial interactions, protecting the allografts from acute microvascular and parenchymal injury.

Effects of vesnarinone, a novel orally active inotropic agent with an immunosuppressive action, on experimental cardiac transplantation in rats.

Vesnarinone (VES) has been used for treatment of patients with congestive heart failure. In addition to inotropic effects, it seems to have immunosuppressive action. We tested the hypothesis that VES suppresses graft rejection, inotropic dysfunction caused by early rejection, and chronic coronary obstruction in a heterotopic rat cardiac transplantation model. (1) To study acute rejection, hearts from Lewis-Brown Norway (LBN) rats were transplanted into Lewis rats, which were treated with or without VES (50 or 100 mg/kg/day orally). (2) In a functional study, LBN hearts with or without VES (100 mg/kg/ day) were isolated and perfused on day 3 after transplantation to assess inotropic response to isoproterenol (3 x 10(-8) M). (3) To study chronic rejection, Lewis hearts were transplanted into Fisher 344 rats, which were treated with or without VES (50 mg/kg/day) for 90 days. Coronary obstructive disease was assessed by morphometric analysis. There were five to six animals in each group. (1) VES (100 mg/kg/day) prolonged LBN heart survival (11.7 +/- 0.7 vs. 9.6 +/- 0.7 days in control; P < 0.05). (2) Left ventricular developed pressure was depressed in transplanted hearts regardless of VES treatment (84 +/- 12, 90 +/- 8 vs. 144 +/- 16 mmHg in untransplanted hearts; P < 0.01). The developed pressure after administration of isoproterenol in VES-treated hearts (184 +/- 20 mmHg) was higher than transplanted hearts without VES (118 +/- 16 mmHg; P < 0.05), and similar to untransplanted hearts (203 +/- 27 mmHg; P = NS). (3) Transplanted hearts treated with or without VES showed similar grades of rejection (2.0 +/- 0.3 vs. 2.6 +/- 0.2; P = NS), intimal area (6,996 +/- 3,186 vs. 13,441 +/- 5,165 microns2; NS), and coronary luminal obstruction (45 +/- 16% vs. 67 +/- 14%; NS). VES produces mild prolongation in survival of rat heart grafts, but has no significant effect on chronic graft atherosclerosis. VES preserves the positive inotropic effects of isoproterenol that are otherwise deteriorated by early acute rejection.

The behaviour of ED1- and ED2-positive cells in the rat iris and choroid following penetrating keratoplasty and cyclosporin A therapy.

The presence, morphology and distribution of ED1 and ED2+ cells have been recently reported in detail in the uveal tissues of the rat. These cells, particularly those of dendritic morphology, are possibly capable of antigen presentation and, therefore, may play an important role in immune processes of uveal and other ocular tissues. Using the whole-mount technique, the distribution of ED1+ and ED2- cells in the rat iris and choroid was investigated following penetrating keratoplasty (PKP) and following treatment with cyclosporin A (CsA). Lewis (LW) rats received corneal buttons from Lewis-Brown Norway (LW-BN) donors and were randomly assigned to one of two groups: (I) operated, untreated (n = 24); (II) operated, CsA treated (10 mg/kg i.m.: n = 22). Four groups served as controls: normal LW rats (n = 13); (IV) unoperated, CsA treated (16 days' treatment; n = 8); (V) eight corneal sutures only, representing a simulated or "sham" operation (n = 4); (VI) syngeneic operated (LW to LW: n = 4). Animals of groups I and II were killed on the 5th, 9th and 13th postoperative days and on appearance of the corneal rejection (group I, day 13; Group II, day 16). Both eyes were enucleated, immediately fixed, and iris-choroid flat mounts were examined for ED1+ and ED2+ cells using APAAP immunohistochemistry. In the normal LW rat iris, ED1+ and ED2+ cells of both non-dendritic and dendritic morphology were observed. The placement of sutures in the cornea and PKP with or without treatment resulted in a reasonably regular response in both the iris and the choroid in the operated and partner eye. These included: (a) an increase in round iridal ED1+ and ED2+ cells in the operated eye and in round ED1+ cells in the partner eye; (b) a decrease in dendritiform ED2+ cells in the iris of the operated eyes as well as in the partner eye; and (c) a decrease in the dendritiform ED2+ cells in the choroid of the operated and partner eye. CsA treatment alone in unoperated animals resulted in significant decreases in the number of dendritiform ED1+ cells in the iris and in the dendritiform ED2+ cells in the choroid. Corneal transplantation in the Lewis rat results in responses in ED1+ and ED2+ cells in uveal tissues in both the operated eye as well as in the partner eye. The differences in cell behaviour supports the idea that distinct immune-competent cell populations are present within uveal tissues and that they may have differing roles in the pathological eye.

Tolerance to Cardiac Allografts Requires a Time Lag between Intrathymic Treatment and Transplantation

Permanent tolerance to an experimental heterotopic cardiac allograft can be achieved by pretreatment with antilymphocyte serum (ALS) and intrathymic inoculation of donor cells. Most successful experimental protocols have employed a time lag of 2 to 3 weeks between intrathymic pretreatment and transplantation, which makes this treatment strategy impractical for clinical heart transplantation. In these experiments we modified the standard protocol by giving ALS 24 hr prior to both intrathymic injection of donor cells and heterotopic transplantation. Seven Lewis rats had intraperitoneal injection of 1 ml of ALS and 24 hr later underwent intrathymic injection of 5 × 107donor Lewis-Brown Norway (LBN) splenocytes and heterotopic cardiac transplantation using an LBN donor. Mean graft survival was 24.4 days, significantly longer than the 7.8-day graft survival observed in untreated Lewis recipients (n= 5) (P< 0.02). However, graft survival was not different from that observed in Lewis rats pretreated with ALS alone (n= 5) (25.8 days,P= NS). Permanent graft survival was produced in two rats receiving only ALS and in one rat receiving both ALS and intrathymic inoculation. In these experiments it appears that prolongation of graft survival may have been due to the effect of ALS alone. These results suggest that there is a critical time period between intrathymic inoculation and transplantation that is needed for permanent tolerance to be induced consistently. This may be due to the kinetics of the effects of ALS on alloreactive T-lymphocytes or to a time-dependent requirement for antigen processing in the thymus.

Durability of donor-specific and organ-specific heart transplant tolerance induced by intrathymic pretreatment with allogeneic spleen cells

Permanent acceptance of an experimental cardiac allograft can be achieved in the rat by pretreating the recipient with antilymphocyte serum and intrathymic donor lymphocytes. We investigated the durability and specificity of the tolerance produced by this pretreatment in a rat model of heterotopic heart transplantation with Lewis-Brown Norway donors and Lewis recipients. Pretreated Lewis rats received 1 ml antilymphocyte serum intraperitoneally and 5 × 10 7 Lewis-Brown Norway splenocytes intrathymically, followed 21 days later by Lewis-Brown Norway cardiac transplantation. The first Lewis-Brown Norway cardiac allograft survived long term (mean 140 days) in pretreated recipients who were given no subsequent immunosuppression. After 60 days with a beating Lewis-Brown Norway allograft, tolerant Lewis recipients underwent a second cardiac allograft with either a Lewis-Brown Norway heart or a third-party Wistar-Furth heart. The second Lewis-Brown Norway cardiac allograft was not rejected (mean survival 76 days), but that from the third-party Wistar-Furth donor was rejected in a normal fashion (mean survival 10.4 days). The presence of second grafts did not affect survival of first grafts. Tolerant Lewis recipients of two Lewis-Brown Norway heart grafts underwent subsequent transplantation with Lewis-Brown Norway skin. Skin allograft survival in this group (mean 8.4 days) was not different from that in Lewis recipients without pretreatment. Rejection of skin grafts had no effect on the heart grafts. These data suggest that tolerance to cardiac allografts produced by intrathymic pretreatment is durable and extends to a second heart graft from a genetically identical donor. Tolerant rats reject third-party hearts and primary donor skin grafts normally, and tolerance to previously placed heart grafts is not abrogated by this rejection. Non–major histocompatibility complex skin antigens not present on cardiac cells may account for the tissue specificity of the tolerance produced by intrathymic treatment in this model. (J T HORAC C ARDIOVASC S URG 1996;111:429-31)

The influence of penetrating keratoplasty and cyclosporin A therapy on MHC class II (Ia)-positive cells in the rat iris and choroid.

The presence of Ia-positive cells (MHC class II equivalent) has been previously reported in the iris and choroid of various species. They have been reported to have both round and dendritic morphologies; the latter may represent classic dendritic cells, potent antigen-presenting cells (APCs). It is possible that the dendritic-like cells play a important role in (auto)immune processes of uveal and other ocular tissues. Using the flat or whole mount technique, the distribution of Ia-positive cells in the rat iris and choroid was investigated following penetrating keratoplasty (PKP) and following treatment with cyclosporin A (CsA). Lewis (LW) rats received corneal buttons from Lewis-Brown Norway (LW-BN) donors and were randomly assigned to the following groups: (i) operated, untreated (n = 24); (ii) operated, CsA-treated (10 mg/kg i.m.; n = 22). Controls were groups (iii) normal LW rats (n = 13); (iv) unoperated, CsA-treated (16 days' treatment; n = 8); (v) anterior perforation of the anterior chamber (n = 3); (vi) eight corneal sutures only (n = 4); (vii) syngeneic operated (LW to LW; n = 4). Animals of groups (i) and (ii) were killed on the 5th, 9th and 13th postoperative days and on appearance of the corneal rejection (group i, day 13; group ii, day 16). Both eyes were enucleated, immediately fixed, and iris-choroid flat mounts were examined for Ia-positive cells using APAAP immunohistochemistry. In the normal Lewis rat iris, scattered Ia-positive cells of both nondendritic and dendritic morphology were observed. CsA treatment in the unoperated rat did not result in a significant decrease in the percentage of dendritic cells in the iris or choroid. Anterior chamber perforation, the placement of sutures in the cornea and syngeneic PKP resulted in a moderate increase in iris Ia-positive cells. Allogeneic transplantation resulted in a large increase in both types of Ia-positive cells, particularly on day 13 with corneal rejection. In group ii, an initial decrease in Ia-positive cells until day 13 was observed; upon rejection (day 16), the histological picture was similar to that of untreated animals. Alterations in the operated choroid were also apparent following CsA treatment. Corneal transplantation in the Lewis rat results in an increase in Ia-positive cells in the iris; CsA therapy can delay but not prevent this reaction. Changes in choroidal Ia-positive cells following PKP were not apparent, their numbers being affected only by CsA treatment following grafting.

Suppressor cells and intrathymic inoculation of donor alloantigens in cardiac transplantation

Donor-specific tolerance to a rat heterotopic cardiac allograft has been achieved by the pretransplantation intrathymic injection of donor splenocytes and a single intraperitoneal injection of antilymphocyte serum (ALS). Permanent tolerance is achieved without subsequent immunosuppression therapy. This study investigated the mechanisms responsible for maintenance of the tolerant state. Tolerance was produced in Lewis rats by the administration of 1 mL of ALS intraperitoneally and 5 x 10(7) Lewis Brown Norway (LBN) splenocytes intrathymically 21 days before heterotopic transplantation using an LBN donor. In tolerant Lewis rats bearing LBN allografts for more than 100 days, rejection could not be produced by the intravenous injection of naive Lewis spleen cells (5 x 10(7) cells x 1 day, n = 5; 5 x 10(7) cells x 3 days, n = 5) or cells from Lewis rats sensitized to LBN tissues (5 x 10(7) cells x 3 days, n = 5). Naive Lewis recipients were pretreated with ALS and 6 days later with intravenous spleen cells (25 x 10(7), n = 5) or lymphoid cells (10 to 15 x 10(7), n = 5) from a tolerant animal bearing a viable LBN graft. When transplantation with an LBN donor was done the next day, significant prolongation of LBN allograft survival (mean survival time 32.8 days, p < 0.01; and 22.2 days, p < 0.01; respectively) was seen. Wistar-Furth allograft survival was not prolonged by treatment with ALS and intravenous spleen (n = 5) or lymph node (n = 5) cells from rats tolerant to LBN tissues (mean survival time 8.6 and 9.2 days, control 9 days; p = not significant). The administration of ALS alone (n = 5) did not prolong LBN graft survival (mean survival time 11.8 days). These data suggest that transferable suppressor cells specific for the donor strain are at least in part responsible for the maintenance of long-term allograft survival after intrathymic pretreatment with allogeneic cells.

Impaired survival and growth in immunosuppressed young rats with lethal short gut syndrome and a small bowel transplant: An effect of cyclosporine

Neonates and growing individuals have increased nutritional demands as compared with adults. To determine the functional ability of an intestinal graft to allow survival and growth, an otherwise lethal short gut model should be used (resection of both the entire small bowel and the cecum). In this study the authors investigated the survival and growth in young rats (80 to 125 g) with this lethal short gut syndrome (SGS) and either syngeneic or allogeneic segmental small bowel transplantation (SBTx). Additionally they sought to determine the effect of therapeutical doses of cyclosporine (CyA) in young, growing rats. To avoid total parenteral nutrition in rats undergoing SBTx, surgery was carried out in two steps: after segmental SBTx of a 25-cm jejunal graft, SGS was created 2 weeks later. Lewis rats underwent 1: Syngeneic segmental SBTx + SGS (n = 7); 2: Allogeneic segmental SBTx (donor: Lewis Brown Norway F 1) + SGS + CyA (15 mg/kg/d for 7 days then every other day for 21 days) (n = 9); 3: Syngeneic segmental SBTx + SGS + CyA as in group 2 (n = 5); 4: SGS alone (n = 5): 5: small bowel resection alone (n = 5); 6: sham laparotomy twice (n = 5); 7: sham laparotomy twice + CyA as in group 2 (n = 6). Weight, general condition, and nutritional serum variables were followed up regularly for 4 months. Rats with resection of small bowel survived but did not grow. Rats with small bowel resection + cecectomy died within 5 days. Shamoperated rats had a normal weight development compared with CyA-treated sham-operated rats who grew significantly slower ( P < .05). Rats with syngeneic SBTx and SGS survived and grew consistently. Rats with syngeneic or allogeneic SBTx, SGS + CyA treatment had poor weight development and died within 2 weeks after the short gut procedure. Compared with sham-operated rats, CyA-treated shamoperated rats had lower levels of albumin and triglycerides ( P < .05) and higher levels of urea and creatinine ( P < .05). These results suggest that the short gut model used is indeed lethal and that syngeneic segmental SBTx permits survival and growth in young rats with this lethal SGS. They also suggest that when given over a 4-week period, a standard dose of CyA impairs growth in young rats, and that this may be caused by the intestinal and renal side effects of CyA. Lastly, we attribute the failure of CyA treated rats with SBTx and SGS to survive and grow in great part to the side effects of CyA.

Tolerance to experimental cardiac allografts produced by neonatal intrathymic injection of donor cells

Intrathymic inoculation of allogeneic cells after systemic administration of antilymphocyte serum in adult experimental animals has produced donor-specific tolerance to cardiac allografts. We investigated whether thymic injection of allogeneic cells without antilymphocyte serum in neonatal Lewis rats (day 1 of life) with immature immune systems also produced tolerance to cardiac grafts. Intrathymic or intraperitoneal injection of 5 × 10 7 Lewis (control) or Lewis-Brown Norway (allogeneic) spleen cells in Lewis neonates was followed by heterotopic cardiac transplantation using Lewis, Lewis-Brown Norway, or Wistar Furth (third-party allograft) hearts at 6 to 8 weeks of age. Graft survival was prolonged with both intraperitoneal and intrathymic allogeneic cells. Recipt ents of cells by the intrathymic route had longer graft survival, and 2 of 5 animals achieved permanent graft acceptance (longer than 100 days). As expected, Lewis isografts survived indefinitely, whereas third-party Wistar Furth allografts were rejected in the usual time frame. Intrathymic introduction of allogeneic cells in a neonatal recipient with an immature immune system can produce donor-specific tolerance to a subsequent graft without the need for a systemic immunosuppression regimen, even transiently.

Development of tolerance to experimental cardiac allografts in utero

“Actively acquired tolerance” to foreign cells was described in 1953 by Medawar and colleagues and formed the basis for subsequent efforts in organ transplantation. We have applied these historic principles of intrauterine immune manipulation in a vascularized cardiac allograft model. Allogeneic Lewis-Brown Norway (LBN) splenocytes (0 to 5 × 10 7 cells) were injected intraperitoneally into each fetus of a pregnant Lewis rat at day 14 to 16 of gestation, when T cells are “educated” to distinguish self from foreign. This manipulation causes fetal attrition inversely proportional to the number of cells injected. Heterotopic transplantation using an LBN heart was carried out in each surviving fetus at 6 to 8 weeks of age. Untreated Lewis rats rejected LBN hearts within 7.6 days. Rats receiving LBN splenocytes in utero demonstrated prolongation of graft survival proportional to the number of cells given. Surviving animals exposed to 5 × 10 7 allogeneic cells in utero (n = 4) had graft survivals of 24 to more than 150 days (mean, 88.C days), significantly longer than control animals (6 to 10 days; mean, 7.6 days; p < 0.02). Significant prolongation of cardiac allograft survival and in some cases complete tolerance can be achieved by intrauterine exposure to allogeneic cells at a critical period of immunologic development. Because many serious cardiac defects amenable to treatment by cardiac transplantation can be detected by ultrasound early in gestation, treatment based on this strategy may become useful in pediatric heart transplantation.