Functional recovery of rat hind-limb allografts

Few papers have assessed the long-term functional recovery of animal limb allografts. In this study, the functional recovery of rat limb allografts was serially and quantitatively investigated for a period of 1 year. The donor's hind limb was orthotopically transplanted into the recipient. Fifteen recipients with allografts were treated with FK506. Functional recovery of the grafted limb was assessed serially by cutaneous reaction test, walking track analysis, and electrophysiologic evaluation. Sensibility improved to a similar extent in both isografts and allografts, and the recovery rate at 1 year was 68 percent, compared to the normal side. Sciatic function index significantly improved to - 70 points after 1 year. The amplitude recorded from the gastrocnemius muscle significantly improved, and the ratio compared to the normal side was 43 percent. Limb isografts and allografts treated with FK506 showed no significant differences in functional recovery. The data can be used as a reference standard for future investigations.     

Efforts to enhance survival of limb allografts by prior administration of whole blood in rats using a new survival end- point

Injecting whole blood into the recipient before surgery can significantly prolong renal transplant survival in rats. Therefore, experiments were performed in rats to study the effects of prior administration of whole blood on the survival of limb allografts. Tests to quantitate survival of the allografts included monitoring the internal temperature of the leg, assaying serum creatine kinase levels, and testing for alloantibodies. Lewis recipients of (BN × LEW)F₁ limb transplants that received 1 ml of BN or (BN × LEW)F₁ whole blood before surgery had mean survival times that were longer compared with controls as measured by a 10 F change in temperature. In a test- retest experiment, decline of temperature proved to be a reliable quantitative determination of limb allograft survival since a difference of only 5.6% was observed in the mean number of days of graft survival between two separate groups of control Lewis recipients. Moreover, combined data demonstrated that control Lewis recipients of (BN × LEW)F₁ limb allografts averaged 24.0 days of graft survival based on a 10 F decline in temperature with a 95% confidence interval of ± 6.3 days. It is concluded that prior administration of whole blood can produce significant prolongation of survival in organ transplantation, but it is not as effective in enhancing survival of limb allografts. It is also concluded that internal temperature measurement of limb allografts is an easy, effective, and quantitative method of monitoring rejection.     

Combined donor leucocyte administration and immunosuppressive drug treatment for survival of rat heart allografts

BACKGROUND: Donor leucocytes (DL) play an important role in rat liver transplant tolerance and their postoperative administration can convert rejection to tolerance. They appear to induce early activation, altered patterns of infiltration and death of recipient alloreactive T cells. The ability of immunosuppressive drugs to combine with DL administration was examined in a rat heart transplant model. METHODS: Immediately after PVG to DA heterotopic heart transplantation, 6 x 10(7) spleen DL were injected. Cyclosporine A (CsA), 1.5 mg/kg/day, or methotrexate (MTX), 0.1 or 0.2 mg/kg/day, were given from day (d) 0 to d4 (early) or from d3 to d7 (delayed). Castanospermine (CAST) was administered from d0 to d7 at 100 or 300 mg/kg/day. In a separate experiment, transplanted hearts and recipient spleens were collected from treatment groups for analysis of infiltrate and cytokine mRNA expression. RESULTS: Delayed treatment with CsA or early treatment with MTX but not CAST combined with DL to result in prolonged graft survival. Recipients treated with DL and delayed CsA had a reduced level of intra-graft interleukin (IL)-2, interferon (IFN)-gamma, IL-4, and IL-4R mRNA expression and reduced infiltrate compared to DL alone. Early MTX plus DL led to almost complete inhibition of all markers of inflammation during treatment followed by a rapid increase after cessation. In combination with DL, CsA was more effective than MTX for induction of donor-specific tolerance at the dose and administration regimens tested. CONCLUSIONS: Delayed CsA or early MTX combine with DL to prolong heart allograft survival. Early and extensive inhibition of rejection by MTX was less effective than delayed and partial inhibition of the response by CsA for induction of transplant tolerance.     

Primary observation of prolonged survival of cultured epidermal allografts]

It is still controversial that cultured epidermal allografts can survive long. In this study, seventy-nine pieces of cultured epidermal sheets were grafted on the wounds after taking for autografts. The wounds grafted with allogeneic cultured epidermis healed with mean time of 7.2 +/- 1.4 days, while the wounds uncovered with cultured epidermis healed with the mean time of 12.8 +/- 2.5 days (P less than 0.005). No evident signs of allogeneic rejection were found either by clinical or histological observation from 20 days to one year's follow-up. In order to prove the existence of cultured epidermal allografts on grafted area, two methods were established: 1) indirect enzyme conjugated SPA assay to detect A or B blood group antigens with McAb; 2) polymerase chain reaction (PCR) to amplify Y chromosome specific DNA sequence after the female were grafted with cultured male epidermis. In four patients grafted with ABO blood group mismatched cultured epidermis, the donor antigens were found in the grafted area as long as 35 post graft day (PGD). The recipient antigens appeared on 19 PGD. Y chromosome specific DNA was detected in five samples taken from two female patients grafted with cultured male epidermis. Of these five samples, the biopsy time was on 11, 19, 30, 35, 92 PGD respectively. Combining the clinical and histological observation with the results of two methods, it can be concluded that the survival time of cultured epidermal allografts were definitely prolonged.     

Induction of donor-specific tolerance in rat hind-limb allografts under antilymphocyte serum and cyclosporine A protocol

Composite tissue allograft (CTA) transplantation became a clinical reality despite major side effects associated with the administration of chronic immunosuppression. Development of new treatment modalities eliminating life-long immunosuppression is essential for the future of CTA transplantation. In this study, combined use of cyclosporine A (CsA) and antilymphocyte serum (ALS) was tested for the potential to induce tolerance in the rat hind-limb allograft recipients across a major histocompatibility (MHC) barrier (Lewis-Brown-Norway [LBN, RT1(l+n)] to Lewis [LEW, RT1(l)] rats). Thirty transplantations were performed in 5 experimental groups. Animals received CsA and ALS 12 hours before surgery for 21 days thereafter. Although the allograft controls rejected their limbs at day 7 combined treatment of CsA and ALS resulted in indefinite survival (over 420 d) in all allograft recipients. Long-term survivors showed 35% to 42% of donor-specific chimerism in the peripheral blood. Clinical tolerance was confirmed by acceptance of the donor-specific skin grafts and immunocompetence was confirmed by rejection of the third-party grafts. Mixed lymphocyte reaction revealed suppressed response against donor-type antigens and increased response to third-party antigens. Donor-specific tolerance across MHC barrier was induced in CTA allografts under 21 days protocol of ALS/CsA.     

Cotransplanted hepatic stellate cells enhance vascularization of islet allografts

Islet transplantation is an alternative to whole pancreas transplantation in curative therapy of diabetics. The outcome of engraftment of islet, however, remains disappointing. Rapid and adequate islet revascularization is crucial for the survival and function of transplanted islets. In this study, hepatic stellate cells (HSC) were cotransplanted with islet allografts, achieving marked prolongation of islet allografts. This was associated with enhanced revascularization within islet grafts as determined by anti-CD31 antibody staining.     

Suppressor cells and their role in the survival of immunologically enhanced rat kidney allografts

A search has been made for suppressor cells in the spleens of AS rats bearing long-surviving, enhanced AUG strain kidney allografts. The assay consisted of an adoptive transfer of splenocytes from AS rats with enhanced AUG kidneys to normal AS rats that also received test grafts of AUG kidneys. The critical feature of the AUG kidney test grafts was that the native population of passenger cells had been replaced by AS passenger cells--thus reducing, but not eliminating, immunogenicity of the graft. With this assay, it was shown that 2.7-3.5 X 10(8) spleen cells transferred substantial and statistically significant suppression of graft rejection. Suppression was also transferred by spleen cells that did not adhere to nylon wool. It is concluded that suppressor cells are one of the mechanisms ensuring continued survival of enhanced kidney allografts.     

Long-term survival and immunological tolerance of human epidermal allografts produced in culture

Human epidermal cells from a small skin specimen can be grown in culture into multilayered sheets suitable for the permanent coverage of large burn wounds when used as epidermal autografts. We report here on the long-term survival of such cultured epidermal sheets used as epidermal allografts (EAG) across a major histocompatibility barrier in three nonimmunosuppressed adult patients, suffering from large chronic grafted leg ulcers, where the EAG have been placed to cover the conventional split-thickness skin autograft donor site. The absence of rejection was based upon clinical, histological, and immunopathological observation of the allografted sites at various intervals after grafting of the EAG. The identity of the epidermal cells on the grafted area with cultured cells from allogeneic donor was then established after blood substance typing by indirect immunofluorescence. Furthermore, epidermal cells from cultured sheets, but not control human cells from freshly excised normal epidermis, failed to stimulate the recipient peripheral blood cells in the mixed epidermal cell lymphocyte culture reaction, a finding that is related to the complete absence of class-II-antigen-bearing cells in cultured epidermis. This absence of T cell stimulation was noted not only on the day of grafting but throughout the follow-up. Altogether, these findings show that Langerhans cell and other class-II-antigen-bearing cell-depleted cultured epidermal allografts, are tolerated in unrelated recipients. EAG may serve as a skin substitute in patients with large wounds or burns. Since EAG may be grown continuously, the coverage of burns may not then be limited by the availability of the donor site, or by the time necessary to produce epidermal tissue in cultures.     

Indefinite survival of rat parathyroid allografts without postoperative immunosuppression

Methods that avoid long-term immunosuppression must be developed for human parathyroid allotransplantation to be feasible. Pretransplant treatment of the graft to eliminate passenger cells is one such method. An alternative approach is short-term treatment of the recipients with cyclosporine (CsA). In this study, parathyroid glands from Lewis X Brown Norway rats were cultured for 1 week and treated with antiserum directed against class II major histocompatibility complex antigens. Treated glands were transplanted into hypocalcemic Wistar-Furth recipients that previously received 30 mg/kg of CsA once a day for 3 days before transplantation. At 280 days after transplantation, 67% of the recipients had functional parathyroid allografts. Control rats (no CsA; fresh, untreated glands) rejected these grafts within 28 days. Control rats given 3 days of CsA and transplanted with fresh, untreated glands had functional grafts for greater than 56 days (median survival, 80.5 days). Prolongation of allograft survival with short-term, preoperative CsA demonstrates the efficacy of immunosuppression given at the time of antigen presentation. This course of CsA is even more effective when the recipient receives a graft whose passenger cells are eliminated.     

Failure of pentoxifylline to enhance skin flap survival in the rat

The effect of pentoxifylline, a hemorheological agent that increases erythrocyte flexibility and augments capillary blood flow, on the survival of the dorsal skin flap of the rat was studied. An experimental group received intraperitoneal injections of pentoxifylline twice daily, 50 mg/kg/day, and a control group was given identical volumes of saline. Standard McFarlane flaps sized 4 x 9 cm (n = 22) and narrower 2 x 6-cm flaps (n = 18) were constructed 14 days later. After a postoperative observation period of 7 days, no significant difference in tissue survival between the pentoxifylline-treated animals and the controls was found in either flap model. Fourteen days preoperative treatment with pentoxifylline appeared to have no effect on flap survival.     

Extension of survival of rat parathyroid allografts by depletion of Ia donor cells plus preoperative cyclosporine

Methods that avoid chronic immunosuppression of transplant recipients must be developed to eliminate the various risk factors associated with such treatment (e.g., increased infections and malignancies). Pretransplant treatment of the graft with anti-Ia serum plus complement to eliminate "passenger cells" is one such method. An alternative approach is short-term treatment of the recipients with cyclosporine (CsA). In this study, parathyroid glands from Lewis X Brown Norway rats were cultured for one week at 37 degrees C and treated with anti-Ia and complement. Treated glands were transplanted into parathyroidectomized, hypocalcemic Wistar-Furth recipients that had received 30 mg/kg of CsA once a day for the three days prior to transplant. At 1 year posttransplant, 67% of the recipients had functional parathyroid allografts. Control rats (no CsA; fresh, untreated glands) rejected their grafts within 28 days. Controls given three days of CsA and transplanted with fresh, untreated glands all had functional grafts for greater than 56 days (median survival: 80.5 days). Prolongation of allograft survival with short-term, preoperative CsA demonstrates the efficacy of immunosuppression given only at the time of antigen presentation. This course of CsA allowed for indefinite graft survival when the recipient received a graft previously cultured and treated with Ia antiserum. These results are encouraging and should be evaluated further to determine whether similar approaches will be useful in human transplants.     

Peripheral donor leukocytes prolong survival of rat renal allografts.

BACKGROUND: The development of strategies to enhance the survival of transplanted organs and to potentially lower or even discontinue immunosuppressive therapy would represent a significant advancement in post-transplant patient care. METHODS: We studied the effect of pretransplant infusion of donor leukocytes alone or in combination with a short course of cyclosporine on the long-term outcome of a rat model of kidney allograft. RESULTS: A single intravenous infusion of donor peripheral blood leukocytes (100x10(6) cells) from Brown-Norway (BN) rats into major histocompatibility complex (MHC) incompatible Lewis recipients largely failed to prolong kidney allograft viability from the same donor transplanted 60, 40, or 30 days after cell infusion. A short course of cyclosporine (per se, unable to prolong graft survival) was started at the same day of donor leukocyte infusion, but instead was able to prolong the survival of the BN kidney transplant-performed 40 days later-but not of a Wistar Furth (WF) third party, with some animals even developing tolerance. A mixed lymphocyte reaction of host cells from long-term surviving rats to BN stimulator cells was significantly reduced as compared with controls. Donor BN DNA was detected in the peripheral blood of Lewis rats until day 40 after BN leukocyte infusion. Microchimerism persisted (60 to 70 days post-transplant) in most long-term graft recipients. Reducing the time interval between donor leukocyte infusion and subsequent kidney transplant to 10 days still prolonged graft survival. Donor peripheral blood mononuclear cells, but not polymorphonuclear cells, in the leukocyte preparation contributed to prolong kidney allograft survival. CONCLUSIONS: Pretransplant donor leukocyte infusion under the appropriate conditions can tip the immune balance toward improved graft acceptance. This result could be relevant to the achievement of donor-specific tolerance of the graft with the maintenance of an intact response to third-party antigens.     

Graft-versus-host reactivity and renal allograft survival in rats given allogeneic spleen cells or spleen allografts.

Selective recruitment of antigen-sensitive cells (ASC) into the spleen as a method of inducing specific suppression was attempted by intravenous injection of either DA or Lewis spleen cells 24 hr before a (DA X Lewis)F1 renal allograft into a Lewis or DA recipient, either with or without a splenectomy. This led to suppression of rejection in the DA recipient and delayed rejection in the Lewis recipient. Splenectomy produced a minimal augmentation effect. Assay of graft-versus-host (GVH) reactions in (DA X Lewis)F1 rats by a popliteal node assay showed that injection of allogeneic DA or Lewis spleen cells 48 hr before the assay significantly reduced the reaction produced by node lymphocytes but not spleen lymphocytes, suggesting a loss of ASC from the lymph nodes. Lewis spleen allografts did not produce such a significant reduction in the GVH reactivity of DA node lymphocytes as intravenous Lewis cells, whereas DA spleen allografts led to an increased GVH reactivity of Lewis node lymphocytes. From these studies, it is not possible to attribute the suppression produced by the intravenous injection of allogeneic cells to selective recruitment of antigen-sensitive cells to the spleen.     

Infiltration patterns of macrophages and lymphocytes in chronically rejecting rat kidney allografts

The migration of circulating leukocytes to sites of inflammation or antigen is based, at least in part, on the activities of adhesion molecules. In the context of organ transplantation, some of these have been shown to be upregulated during acute allograft rejection. As their role during chronic rejection has not been examined, we have used an established rat model to compare sequentially the presence of host cells within the grafts, as defined immunohistologically, with patterns of in vitro leukocyte binding and their dependence upon particular adhesion molecules. Various donor populations of peripheral blood lymphocytes (PBL), lymph node lymphocytes (LNL), and splenic monocytes were interacted with snap-frozen sections of allografted, isografted, and native kidneys at serial intervals up to 24 weeks after transplantation. Monocyte binding in the allografts rose at 8 weeks and peaked at 12 weeks, a period preceding the maximum numbers of macrophages noted immunohistologically in the chronically rejecting grafts at 16 weeks. Lymphocyte binding and infiltration patterns were similar, remaining stable throughout the follow-up period and consistently greater than those noted in isografts. In vitro binding of the monocytes was inhibited by mAbs against ICAM-1, LFA-1, CD18, and MAC-1; MAC-1 did not influence lymphocyte binding, although the other mAbs were effective. We conclude that adhesion molecules are responsible, at least in part, for patterns of cell populations infiltrating chronically rejecting renal allografts.