The use of skin allograft with donor-specific tolerance in a rabbit model of full-thickness burn

Previous studies in our laboratory demonstrated that portal vein or intraosseous administration of donor bone marrow cells for modulation of the central immune system is likely to be beneficial for allograft tolerance induction in rabbits. We recently reported that the severity of host conditioning for donor-specific tolerance induction was reduced without loss of efficacy by using a single intraosseous injection of donor bone marrow cells without immunosuppressive agent administration or T cell depletion. We now further investigated the feasibility and effectiveness of skin allografting using donor-specific tolerance to treat full-thickness burns. MATERIALS AND METHODS: Dutch rabbits were used as recipients, and Japanese white rabbits were used as donors. Three experimental groups of burned rabbits were studied. Group I, allograft control; group II, allograft plus total-body irradiation; group III, allograft plus total-body irradiation and bone marrow infusion. Allograft survival times were determined by macroscopic and histopathologic examinations. RESULTS: Mean (S.D.) skin allograft survival was as follows: group I, 13.2 (4.1) days; group II, 15.2 (3.7) days; group III, 108.4 (14.3) days. CONCLUSION: We have shown the potential to perform long-term skin allografts by the induction of donor-specific tolerance in a rabbit model of burn.     

Trafficking of donor-derived bone marrow correlates with chimerism and extension of composite allograft survival across MHC barrier

We proposed to evaluate differences between recipient's immune response to vascularized skin and combined vascularized skin/bone allografts, under a 7-day alphabeta-TCR plus cyclosporine (CsA) treatment protocol. Thirty-six transplantations were performed in six groups: group I (isograft control-vascularized skin graft; n=6); group II (isograft control-combined vascularized skin/bone graft; n=6); group III (allograft rejection control group-vascularized skin graft; n=6); group IV (allograft rejection control-combined vascularized skin/bone graft; n=6); group V (allograft treatment-vascularized skin graft; n=6); and group VI (allograft treatment-combined vascularized skin/bone graft; n=6). Isograft transplantations were performed between Lewis rats and allografts were transplanted across the MHC barrier from Brown Norway to Lewis rats. In the allograft treatment group, a combined alphabeta-TCR+CsA protocol was applied for 7 days. All groups were compared clinically, immunologically and histologically. Statistical significance was determined with two-tailed Student's t test. Indefinite graft survival was achieved in the isograft control group (>300 days). Allograft rejection controls rejected within 5 to 9 days posttransplant; chimerism levels were undetectable (<.5%). Allografts under the alphabeta-TCR+CsA protocol had significantly extended survival when skin was combined with bone (61-125 days) compared to vascularized skin allografts (43-61 days). Lymphoid macrochimerism was significantly higher in group VI than group V. Histology confirmed skin and bone viability. Combined vascularized skin/bone allografts had higher and sustained levels of donor-specific chimerism and extended allograft survival.     

The relationship between interferon-gamma and keratinocyte alloantigen expression after burn injury.

BACKGROUND: Cultured keratinocyte (CK) and cadaveric skin allografts have prolonged survival in patients with massive thermal injury. It is unclear if this delayed rejection is due to impaired host responsiveness or decreased graft immunogenicity. Although burn injury has been shown to decrease parameters of allograft response, no studies have examined the effect of burn injury on alloantigen expression. This study investigated the effect of burn size on class II antigen expression in CK allografts as well as on tissue levels of interferon-gamma (IFN-gamma), the principle regulator of alloantigen expression. METHODS: Anesthetized CBA mice (n = 64) received a 0%, 20% partial-thickness (PT), 20% full-thickness (FT), or 40% FT contact burn. Forty-eight hours later, wounds were partially excised and covered with CK allografts from C57BL/6 donors. Five days after burn injury, grafts were analyzed for donor-specific class II antigen. Protein expression was determined by Western immunoblotting and quantified with video densitometry. Wound, serum, and unburned skin levels of IFN-gamma were determined by enzyme-linked immunosorbent assay. Groups were compared by Fisher's analysis of variance. RESULTS: As burn size increased, class II antigen expression decreased (p < 0.001). This corresponded with decreased wound and skin levels of IFN-gamma after 40% burn (p < 0.05); however, wound IFN-gamma was significantly elevated after 20% PT and FT burns (p < 0.01). Serum IFN-gamma increased as burn size increased (p < 0.01). CONCLUSIONS: Burn injury decreases the antigenicity of CK allografts, which partly explains delayed allograft rejection after burn injury. Although wound IFN-gamma increases after minor thermal injury, the profound decrease in wound and skin IFN-gamma after a major burn corresponds with diminished class II antigen expression. The decreased availability of IFN-gamma after major thermal injury provides a mechanism for limited allograft tolerance.     

Urinary amylase monitoring for early diagnosis of pancreas allograft rejection in dogs

The diagnosis of pancreas allograft rejection is usually made on the basis of blood glucose concentration, a late indicator of rejection. We performed segmental pancreas transplants in totally pancreatectomized dogs with the exocrine secretions drained into the bladder (ductocystostomy). We directly measured exocrine pancreatic secretions (urinary amylase), in an attempt to find a sensitive indicator for early rejection. Five groups were studied: (I) autografts; (II) autografts immunosuppressed with cyclosporine (CsA), azathioprine and prednisone; (III) allografts without immunosuppression; (IV) allografts immunosuppressed with CsA alone; (V) allografts immunosuppressed with CsA, azathioprine, and prednisone. The control groups (I, II) maintained high urine amylase concentrations indefinitely (mean +/- SE of 125,544 +/- 36,931 u/liter). Rejection, as diagnosed by rise of serum glucose to greater than 150 mg/dl, occurred at a mean (+/- SE) of 9.0 +/- 0.2 days in nonimmunosuppressed recipients of Group III, at 9.3 +/- 0.7 days in cyclosporine-treated dogs of Group IV, and at 28.0 +/- 8.3 days after transplantation in dogs immunosuppressed with triple therapy of Group V. In all allograft recipients, urine amylase declined precipitously (less than 1000 u/liter) before the onset of hyperglycemia, by 1.3 +/- 0.2 days in Group III, 3.3 +/- 1.0 days in Group IV, and 9.4 +/- 2.8 days in Group V. In a further experiment, nine dogs with pancreas allografts received cyclosporine for prophylactic immunosuppression; further antirejection therapy with azathioprine and antilymphocyte globulin was given for 5 days beginning the first day that rejection was diagnosed. In five dogs (Group A) rejection was diagnosed when serum glucose rose to greater than 150 mg/dl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclosporine and skin allografts for the treatment of thermal injury. II. Development of an experimental massive third-degree burn model demonstrating extensive graft survival

We have previously reported the successful treatment and apparent development of skin allograft tolerance in a patient sustaining massive burns, utilizing skin allografts and cyclosporine. We now report the experimental correlate via successful achievement of a 75% body surface area (BSA) scald burn cyclosporine-skin allograft model in Lewis (LEW) rats. Cyclosporine (8 mg/kg/day) was given to the experimental animals daily for the first 20 days and then three times a week thereafter. Two experimental groups were studied: one received standard posttrauma care and the second critical posttrauma care. Controls (n = 22) and experimental groups 1 (n = 28) and 2 (n = 4) had average survival times of 13.8 +/- 12.8 days, 44.2 +/- 132.5 days, and 172.0 +/- 19.4 days, respectively. The allografts on the surviving experimental animals appeared normal and healthy and had nearly perfect hair growth. These results indicate that the model follows the clinical burn wound course, and treatment of massive burns with primary excision, skin allografts, and low doses of cyclosporine could provide immediate and complete functional repair of the burn wound.     

Tolerance in a concordant nonhuman primate model

BACKGROUND: We have previously demonstrated that induction of mixed lymphohematopoietic chimerism resulted in donor specific renal allograft tolerance without the need for chronic immunosuppression in nonhuman primates. Here we have tested whether tolerance can be similarly induced for baboon to cynomolgus renal xenografts. METHODS: After preconditioning with anti-thymocyte globulin (ATG), nonlethal total body irradiation, and thymic irradiation, cynomolgus monkeys underwent splenectomy, native nephrectomies, and baboon marrow and renal transplants. Postoperative cyclosporine was given for 28 days. RESULTS: In Group 1 (n=2, survival= 13, 14 days), both animals developed anti-donor immunoglobulin G, had biopsy findings consistent with humoral rejection, and showed rapidly progressive xenograft failure. In Group 2 (n=5, survival=1, 16, 33, 112, 190 days), 15-deoxyspergualine was added to the regimen (Day 0-13). In one long-term survivor, donor specific hyporesponsiveness was first observed (mixed lymphocyte culture [(MLR]) on Day 48. MLR reactivity returned on Day 64 together with the development of anti-donor antibody and subsequent xenograft failure on Day 112. Donor specific T-cell hyporesponsiveness was detected in the other long-term survivor for the first 133 days, after which a donor-specific skin xenograft was placed, (survival 24 days). Following the skin graft rejection, a rise in the MLR, development of anti-donor antibody and progressive rejection of the renal xenograft were observed. CONCLUSIONS: Antibody-mediated rejection seems to constitute the major difference between concordant xenografts and allografts. Addition of 15-deoxyspergualine for 2 weeks posttransplant extended concordant primate xenograft survival to 6 months without chronic immunosuppression. In contrast to the allogeneic model, renal transplant acceptance in this xenogeneic system was interrupted by placement of a donor-specific skin graft.     

Indefinite survival of rat islet allografts following infusion of donor bone marrow without cytoablation

We have tested the effect of donor bone marrow cell (DBMC) infusion on the survival of pancreatic islet allografts in the rat, without the use of cytoablative recipient conditioning. Lewis and diabetic Brown Norway rats were used as donors and recipients, respectively. Donor islets were placed beneath the left renal capsule. Infusion of DBMC and temporary immunosuppression followed by delayed islet transplantation resulted in indefinite survival of all islet grafts (MST >180 days). Control animals demonstrated recurrent hyperglycemia (islet allografts rejection). Donor bone marrow derived cells were detected in the spleen and cervical lymph nodes of BN recipients of LEW bone marrow but not in the recipients of islet transplants alone. Second set full thickness skin grafts were performed in normal BN and in recipients of a previously successful ITX. Donor specific skin grafts were accepted in the animals that had received DBMC 40 days before the islet allograft, while animals receiving DBMC at the time of the islet allograft rejected the donor specific skin graft similarly to the controls. However, these animals did not reject a second set donor-specific islet transplant. The results indicate that radiation conditioning of the recipients was not necessary to induce microchimerism and graft acceptance in this rodent model of islet allotransplantation.     

The early influence of albumin administration on protein metabolism and wound healing in burned rats.

Hypoalbuminemia is often claimed to impair wound healing, and therefore albumin has traditionally been administered to derive beneficial effects on general physiologic conditions including the nutritional state. However, the influence of albumin administration on systemic protein metabolism and wound healing is still unclear. Therefore, the objective of this study was to investigate the influence of albumin administration on protein metabolism and wound healing in burned rats. After receiving basic total parenteral nutrition (TPN) for 4 days, Sprague-Dawley rats underwent a 6-cm skin incision in the back and a burn involving 20 percent of the whole body surface. The rats were divided into three groups. Group I continued to receive basic TPN. Group II was given basic TPN, but 20 percent of the total nitrogen was replaced by albumin. Group III was administered basic TPN plus albumin equivalent to 20 percent of the total nitrogen of basic TPN. Group IV had the skin incision but no burn, receiving only basic TPN. All the groups were euthanized 4 days after the burn or skin incision. The wound healing potential in terms of tensile strength was enhanced by replacement and addition of albumin (groups II and III, respectively) after a 20 percent burn. Hydroxyproline levels in the wound tended to increase in group II, and significantly increased in group III. Whereas albumin replacement (group II) did not remarkably change the protein metabolism, albumin addition (group III) significantly increased both protein synthesis (S) and breakdown (B) with the S/B ratio and nitrogen balance remaining the same as with albumin-free nutrition (group I). The urinary 3-methyl-histidine/creatinine ratio significantly increased after burn in group III. We conclude that intravenous albumin administration enhanced incisional wound healing in burned rats. Increased protein synthesis with concurrent myolysis and protein breakdown by albumin addition (group III) was observed during wound healing.     

Prolongation of impure murine islet allografts with antilymphocyte serum and donor-specific bone marrow.

The inclusion of contaminating nonislet tissue in allografts of purified islets of Langerhans has been shown to decrease allograft survival times in unimmunosuppressed mice. Our current work examines the effectiveness of antilymphocyte serum and donor bone marrow cell infusion on the survival of very impure islet allografts. Treatment of B6AF1 mice with peritransplant antilymphocyte serum plus posttransplant infusion of donor (C3H/HeJ) bone marrow was very effective at prolonging contaminated allograft survival. ALS plus BMC was also effective for even more immunogenic allograft situations; mice that received contralateral transplants of adult and neonatal tissue, and mice that received dual transplants of adult islets. Long-term graft-bearing mice that originally received ALS and BMC were challenged with C3H/HeJ skin grafts. These mice exhibited several different types of response. Four of thirteen appeared sensitized to donor antigen, four rejected the skin grafts in approximate first-set fashion, and five showed limited prolongation, with skin grafts surviving 20-48 days. Long-term graft-bearing mice were also tested in in vitro proliferative assays. As responders in mixed lymphocyte reactions, splenocytes (SPC) from only one of eight appeared to show specific unresponsiveness to donor cells. Four of eight had a normal proliferative response and stimulation index to stimulation with C3H/HeJ SPC. SPC from three did not produce a normal stimulation index, but had an abnormally high (3x normal) background proliferation. SPC from seven of eight of these mice were able to suppress the proliferative response of naive B6AF1 SPC to C3H/HeJ cells. Our conclusions are that while ALS plus BMC are very effective in prolonging islet allograft survival, this treatment does not appear to be uniformly inducing a state of immunologic unresponsiveness in these mice. It appears that multiple mechanisms are acting to maintain islet allografts in this system, either independently or sequentially.     

Renal subcapsular islet cell transplantation

This study was directed towards improving islet cell allotransplantation (ICTx) by testing the renal subcapsular region (RSC) as an alternative site for graft placement. In addition, a simplified, non-collagenase method was assessed for preparation of the allograft from the donor pancreas. Five groups of pancreatectomized mongrel dogs were followed. Group I (n = 10) were not transplanted and survived 5.0 +/- 2.92 days (M +/- SD). Five of six animals in Group II (n = 6) which received an ICTx prepared without collagenase in the RSC survived until allograft removal by nephrectomy at greater than 90 days (P less than .0005). No immunosuppression was given to this group. Recipients in Group III (n = 11) were transplanted as in Group II, but were given minimal immunosuppression with azathioprine. They also demonstrated excellent graft function until removal of the graft by nephrectomy at between 3 weeks and greater than 90 days. Animals in Group IV (n = 6) received an intrasplenic (IS) ICTx prepared without collagenase and survived only 4.16 +/- 1.16 days. In Group V (n = 6) poor survival was also noted (7.66 +/- 4.58 days) after IS-ICTx of a collagenase prepared allograft. All animals in Group IV and V received minimal immunosuppression as in Group III. These results indicate the potential for utilization of the RSC as an alternative site for ICTx. In addition, the collagenase-free method was satisfactory for ICTx preparation.     

Monitoring antidonor alloantibodies as a predictive assay for renal allograft tolerance/long-term observations in nonhuman primates

BACKGROUND: In an effort to define reliable assays that might predict postimmunosuppressant-withdrawal development of chronic rejection (CR), despite conditioning for tolerance induction, we evaluated various immunological responses in nonhuman primate renal allograft recipients. METHODS: Fourteen Cynomolgus monkeys received low dose total body irradiation, thymic irradiation, antithymocyte globulin, and peritransplant CD154 blockade, followed by a one-month course of cyclosporine. Recipients underwent major histocompatibility complex mismatched kidney transplantation with donor bone marrow infusion (Group A, n=8), without donor cell infusion (Group B, n=2), or with donor splenocyte infusion (Group C, n=4). RESULTS: All Group A recipients developed mixed chimerism and four of them survived long-term without rejection. The remaining four rejected their kidney allografts either chronically or acutely. All recipients in Groups B and C failed to develop chimerism and rejected their allografts. Among various in vitro assays, detection of anti-donor alloantibody (ADA) by flow cytometry (FCM) was the most relevant to long-term outcome. All five recipients that developed both anti-T cell and B cell IgG ADA in Groups A, B and C, developed histological evidence of CR within 200 days of the appearance of ADA. One of two recipients that developed only anti-B cell IgG ADA eventually developed CR over two years following discontinuation of immunosuppression and 1.5 years after ADA development. Another recipient with very low anti-B cell ADA has never developed CR. CONCLUSION: ADA monitoring with FCM assay appears to be useful in predicting the failure of tolerance prior to the development of functional or histologic abnormalities of the renal allograft.     

Mixed hematopoietic chimerism prevents allograft vasculopathy.

BACKGROUND: Mixed hematopoietic chimerism has been shown to induce long-term acceptance of transplant organs. We determined whether mixed chimerism prevented allograft vasculopathy, using the rat aortic allograft model. METHODS: Mixed chimeras were prepared by reconstituting lethally irradiated (1100 cGy) WF rats with a mixture of T-cell depleted (TCD) syngeneic (WF) plus TCD allogeneic (ACI) bone marrow. Donor-specific (ACI) or third-party (F344) aortic grafts were transplanted into mixed chimeric animals 1 to 2 months after bone marrow reconstitution. No immunosuppressive drugs were administered. At 30 days postoperatively, aortic allografts were harvested for histology and measurement of cytokine mRNA by semiquantitative RT-PCR. Some aortic grafts were harvested at 90 and 180 days after transplantation for histological analysis. The degree of intimal hyperplasia and cytokine gene expression were compared among 4 groups: I (syngeneic; ACI donors to ACI recipients), II (allografts; ACI to WF), III (donor specific; ACI donor to chimeras) and IV (third-party; F344 to chimeras). RESULTS: There was no difference in the degree of intimal hyperplasia (IH) between groups I and III. Groups II and IV had significantly more IH than group I. Compared to group I, levels of mRNA for IFN-y, IL-2, IL-10 and iNOS in groups II and IV were higher, while there was no difference in mRNA levels between group I and III. CONCLUSIONS: These data suggest that mixed chimerism prevents allograft vasculopathy. Mixed chimerism holds great promise in clinical transplantation as a means to prevent allograft vasculopathy.     

Allograft vein patency in a canine model. Additive effects of cryopreservation and cyclosporine

Autogenous saphenous vein is the preferred conduit for many cardiovascular operations. Attempts to use allograft veins for arterial reconstruction have had poor results. To define circumstances under which allograft veins might prove to be acceptable vascular conduits, dogs underwent femoral artery bypass using reversed saphenous veins. Veins were transplanted fresh or after cryopreservation. Group I dogs received a fresh autograft to replace one femoral artery (group I F) and a cryopreserved (CP) autograft (group I C) to replace the other. Group II dogs received fresh allograft veins, and group III received CP allograft veins, neither group receiving additional treatment. Group IV received fresh allograft veins and Group V received CP allograft veins; both groups received cyclosporine 15 mg/kg. Animals were maintained until grafts occluded or until six months elapsed. Patency was observed in all group I F grafts throughout the observation period. Six-month patency rates in the other groups were: group I C, 9/10 (P = NS vs. group I F); group II, 0/10 (P less than 0.01), group III, 0/10 (P less than 0.01), group IV, 1/10 (P less than 0.01), group V, 7/11 (P = NS). In a separate series of observations 10 cryopreserved allograft veins were implanted in 10 dogs that received CsA for 30 days. CsA was then discontinued. All of these grafts occluded within 30 days of discontinuing the CsA. Long-term patency of saphenous vein allografts was achieved only with the combination of cryopreservation and immunosuppression with continued CsA.     

Preoperative low-dose irradiation promotes long-term allograft acceptance and induces regulatory T cells in a porcine model of pulmonary transplantation

BACKGROUND: A simplified conditioning protocol including single-dose preoperative thymic and low-dose whole body irradiation with or without subsequent donor bone marrow transplantation (BMTx) can be applied in porcine lung transplantation. We hypothesized that this protocol would prolong allograft survival. METHODS: Left-sided single lung transplantation from major histocompatibility complex (MHC)-mismatched donors was performed in 27 minipigs. Recipients received whole body (1.5 Gy) and thymic irradiation (7 Gy) before transplantation (IRR group, n=6), intravenous immunosuppression with methylprednisolone, cyclosporine, and azathioprine for 27 postoperative days (IS group, n=5) or both (IRR+IS group, n=10). BMTx group recipients were treated with irradiation, immunosuppression and a donor bone marrow infusion on postoperative day 1. Peripheral blood leukocyte phenotype and donor cell chimerism were monitored by flow cytometry. Purified CD25+ T cells from long-term survivors or rejecting animals were used for in vitro MLR suppression assays. RESULTS: Median graft survival was: IRR 12 days, IS 55 days, IRR+IS 239 days, and BMTx 80 days (P<0.0001). Early peripheral blood macrochimerism was substantial in both the IRR+IS and the BMTX group but was lost in all groups after day 80. The frequency and suppressive function of CD4+CD25+ T cells were enhanced in IRR+IS group long-term survivors. CONCLUSION: Although donor bone marrow infusion was not beneficial in our model, a substantial proportion of the animals treated with irradiation and a 28-day course of immunosuppression accepted their lung allografts long term. The mechanism involved in maintaining allograft tolerance may be based on peripheral T-cell regulation.     

Applications of bilateral vascularized femoral bone marrow transplantation for chimerism induction across the major histocompatibility (MHC) barrier: part II

Bilateral vascularized bone marrow transplant (VBMT) model was designed to induce chimerism across the major histocompatibility (MHC) barrier under combined alphabeta T-cell receptor monoclonal antibody and cyclosporine A (alphabeta-TCRmAb/CsA) protocol. Seventeen transplants were performed between BN(RT1) donors and Lewis(RTI) recipients. Group I, isograft controls; Group II, allografts rejection controls; Group III, allografts under 7-day protocol of alphabeta-TCRmAb/CsA. Donor bilateral femoral bones were bilaterally anastomosed to the abdominal aorta and inferior vena cava of recipient. At day 7 posttransplantation, all bone flaps were viable. Groups I and III survived without signs of rejection. In Group III, peak level of chimerism in peripheral blood was evaluated at day 21 (24.2%), at day 63 declined to 1.5%, and was maintained at this level thereafter. Donor-derived cells were present in the bone marrow of recipients at 28.2% at day 21 posttransplant. Histology confirmed viability of bone marrow cells in isograft during the entire follow-up and up to 35 days in treatment Group III. Bilateral VBMT induced donor-specific chimerism across the MHC barrier under the immunomodulatory protocol of alphabeta-TCRmAb/CsA.     

Cyclosporine and skin allografts for the treatment of thermal injury. I. Extensive graft survival with low-level long-term administration and prolongation in a rat burn model

The hypothesis tested in the present and accompanying study is that an effective treatment for severe burns involves early excision of necrotic tissue followed by skin allografting and cyclosporine (CsA) immunosuppressive therapy. LEW (RT1) rats served as recipients of thermal injury and/or skin allografts. BN x LEW F1 (LBN, RT1(l+n)) rats served as skin donors. LEW burn recipients received a hot water (90 degrees C for 10 sec) 30% body surface area (BSA) full-thickness burn. As expected, LEW recipients treated with CsA (25 mg/kg/day for 20 days) demonstrated significant graft prolongation compared with controls (P less than 0.005). Skin graft survival was similarly prolonged in LEW recipients undergoing burn injury, primary wound excision, and CsA administration compared with burn-skin allograft controls (P less than 0.001). Mortality was not increased in the thermal injury-CsA-treated recipients compared with burn controls. A final experiment was initiated to investigate how low-level long-term (greater than 100 days) maintenance CsA treatment influenced skin allograft survival for possible future consideration in burn trauma. Recipients receiving skin allografts plus CsA (20 days, 8mg/kg/day, followed by every other day thereafter) did not reject their grafts. However, a possible early sign of rejection (a single small ulcerative lesion) was noted in five of these long-term CsA-treated animals at a mean of 34 +/- 11 (SD) days. The lesion in these animals did not progress any further during CsA administration. Histopathologic study of selected animals removed from the CsA maintenance regimen for greater than 50 days following long-term administration revealed a number of interesting chronic lesions similar to those previously reported in the skin component of composite tissue (limb) allografts following long-term low-level CsA intervention. In conclusion, CsA was very successful in preventing rejection of skin allografts in a rat burn model without apparent adverse effects.     

Infusion of Lin- bone marrow cells results in multilineage macrochimerism and skin allograft tolerance in minimally conditioned recipient mice

Donor-specific immunological tolerance using high doses of donor bone marrow cells (BMC) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease (GVHD) remains a risk. In the present study, we demonstrate that the infusion of low numbers of donor Lin(-) bone marrow cells (Lin(-) BMC) 7 days post allograft transplantation facilitates high level macrochimerism induction and graft tolerance. Full-thickness BALB/c skin allografts were transplanted onto C57BL/6 mice. Mice were treated with anti-CD4 and anti-CD8 mAbs on day 0, +2, +5, +7 and +14 along with low dose busulfan on day +5. A low dose of highly purified Lin(-) BMC from BALB/c donor mice was infused on day +7. Chimerism and clonal cell deletion were evaluated using flow cytometry. Donor-specific tolerance was tested by donor and third-party skin grafting and mixed leukocyte reaction (MLR). Lin(-) BMC infusion with minimal immunosuppression led to stable, mixed, multilineage macrochimerism and long-term allograft survival (>300 days). Mixed donor-recipient macrochimerism was observed. Donor-reactive T cells were clonally deleted and a 130% increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) was observed in the spleen. Tolerant mice subsequently accepted second donor, but not third-party (C3H), skin grafts and recipient splenocytes failed to react with allogeneic donor cells indicating donor-specific immunological tolerance was achieved. We conclude that the infusion of donor Lin(-) BMC without cytoreductive recipient conditioning can induce indefinite survival of skin allografts via mechanisms involving the establishment of a multilineage macrochimeric state principally through clonal deletion of alloreactive T cells and peripherally induced CD4(+)Foxp3(+) Tregs.     

Induction of Unresponsiveness to Major Transplantable Organs in Adult Mammals: A Recapitulation of Ontogeny by Irradiation and Bone Marrow Replacement

Transplantation of renal allografts obtained from prospectively selected genotypically DLA-identical donors into supralethally irradiated dogs reconstituted with their own stored bone marrow has produced a state of unresponsiveness to these kidneys in the recipients. Eleven of 18 kidneys transplanted at 12 hours after marrow replacement currently survive with normal function and maintain life in the recipients for 757, 800, 825, 978, 1062, 1092, 1136, 1282, 1373, 1380, and 1381 days, respectively. Similar results occurred in eight of 13 allografts transplanted at 28 hours after marrow replacement, which currently survive for 349, 363, 377, 407,436,470, 485, and 513 days, respectively, and in eight of 13 kidneys grafted at 36 hours after marrow replacement, which are surviving for 197, 247, 298, 324, 337, 396, 443, and 472 days, respectively. Achievement of optimal results is dependent on the specific timing and sequence of each procedure. Only four of 16 recipients of kidneys transplanted at the time of marrow replacement were unresponsive to their allografts. Similarly, only five of 19 recipients of kidneys placed in irradiated dogs at 40 hours before marrow replacement accepted such allografts. When kidney transplants were placed into the recipients 20 hours before removal of marrow, irradiation, and reconstitution with stored marrow, only three of 21 dogs became unresponsive to such ailografts. In five of 12 instances, the recipients were also unresponsive to skin allografts obtained from their respective kidney donors. Such skin grafts currently survive for 606, 673, 687, 701, and 708 days, respectively. The remaining seven skin grafts were rejected at 28, 39,42, 84, 90, 92, and 115 days, respectively. Second- and third-set skin grafts from the same kidney donor were rejected by six of these dogs at 19, 20, 21, 29, 29, and 30 days, and at 21, 22, 23, 24, 27, and 27 days, respectively. Rejection of these skin grafts had no detectable effect on the function and survival of kidney allografts from the same source. Seven of eight skin grafts obtained from other DLA-identical donors were rejected at 13,14,16,25,28,38, and 84 days, respectively; one allograft continues to survive for 708 days. Eleven DLA-incompatible skin allografts placed on the recipients at the same time were rejected within 11-20 days. Supralethal total body irradiation and bone marrow replacement can establish in the adult canine host a privileged phase of immunological reactivity during which exposure to alloantigens produces specific long-term unresponsiveness rather than sensitization. The use of stored autologous rather than allogeneic bone marrow for reconstitution of the irradiated recipient eliminates the hazards of GVH complication usually associated with this procedure. This consideration and the apparent capacity of the tolerant host to maintain a long-term state of unresponsiveness without any further immunosuppressive therapy point to the potential relevance of the results to human transplantation.     

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 x 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 (t test, P = 0.0013); for vehicle-treated allografts, 12.3 +/- 3.8 days (paired t test, 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 (t test, 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.