Kirby S. Black

COMPOSITE TISSUE (LIMB) ALLOGRAFTS IN RATS: II. INDEFINITE SURVIVAL USING LOW-DOSE CYCLOSPORINE

Cyclosporine has reawakened interest in transplantation of peripheral composite tissue allografts (CTA) of skin, muscle, bone, vessel, and nerves. The purpose of this study was to examine whether cyclosporine could produce indefinite survival of CTA. Two groups of LEW recipients of LBN limb transplants were given different long-term treatments of cyclosporine. Tolerance was achieved in many of the animals. Several possibilities for the mechanism of this tolerance are discussed.

Transcutaneous PO2 in flaps: a new method of survival prediction.

SUMMARY We have shown a new clinical method for flap monitoring. This new method is noninvasive, continuous, quantitative, easy, and commercially available. If any oxygen can be detected or if there is a response to increased FIO2, flap survival is assured.

Development of stable mixed T cell chimerism and transplantation tolerance without immune modulation in recipients of vascularized bone marrow allografts

A consistent majority (62.5%) of immunologically unmodified rat recipients transplanted with vascularized hind-limb bone marrow allografts across a semiallogeneic transplant barrier developed tolerance with absence of graft-versus-host disease. A minority of recipients (37.5%) demonstrated lethal GVHD. Transplantation tolerance in the majority was associated with the induction of stable low-level mixed T cell chimerism, including donor CD5+, CD4+, and CD8+ lymphocytes. Chimeras were specifically immune nonresponsive to host alloantigenic determinants. These results emphasized a potentially important mechanism for low-level stable mixed lymphoid chimerism (SMLC) in tolerance induction, independent of immune suppressive effects due to irradiation or immunopharmacologic intervention. These vascularized bone marrow transplantation (VBMT) results may establish the experimental foundation for a novel approach to stem cell transfer and bone marrow transplantation.

Composite tissue (limb) allografts in rats. III: Development of donor-host lymphoid chimeras in long-term survivors

Eight LEW rat recipients possessing long-term-surviving (206-701 days) LBN vascularized hind limb allografts (CTAs) were tested for donor-host lymphoid chimerism. The recipients received various cyclosporine (CsA) treatment protocols in order to induce indefinite CTA acceptance. Histological examination of long-term-surviving CTAs demonstrated normal-appearing bone marrow in the donor limb. Lymphocytes isolated from host hemopoietic tissues (peripheral blood and/or spleen) by ficoll-hypaque density gradient centrifugation were tested against LEW-anti-BN antisera. Comparisons were made to standard curves employing various known concentrations of LBN and LEW cell combinations. The level of lymphocyte agglutination (dependent variable) showed a significant (P less than 0.025-0.005) linear relationship to the concentration of LBN donor cells (independent variable) present. Lymphocyte suspensions isolated from long-term CTA host peripheral blood and/or spleen showed a mean of 19.7% (+/- 9.7-95% confidence interval) donor LBN mononuclear cells present. Thus, it appeared that lymphoid cells originated from, and/or were released from LBN donor bone marrow into the circulation, resulting in chimeric repopulation of hemopoietic tissues. The presence of donor immunocytes in these limb allograft recipients may have been beneficial, and thus could have helped contribute to the long-term CTA survival observed.

The Effects of Testicular Trauma on Fertility in the Lewis Rat and Comparisons to Isoimmunized Recipients of Syngeneic Sperm

Adult male Lewis (LEW) rats were used to investigate the effects of unilateral testicular trauma on fertility. Comparisons were made between normal and experimental rats immunized with syngeneic sperm in Complete Freunds Adjuvant (CFA). Matings within the three groups yielded offspring to all normal males, no offspring to the immunized rats, and 27% (3/11) fertility in the trauma group (p less than 0.001). The contralateral testis demonstrated decreased volumes, various degrees of aspermatogenesis and smaller seminiferous tubular diameters, in both the trauma and immunized groups compared to the controls. Similar histopathologic findings of chronic granulomatous inflammation within contralateral testes in both the trauma and immunized groups suggested a common immune etiology for infertility via possible disruption of the blood-testis barrier.

Composite tissue allografts in rats: IV. Graft-versus-host disease in recipients of vascularized bone marrow transplants

This laboratory has used a composite tissue allograft model as a vehicle for studies on a new type of bone marrow transplant, the vascularized bone marrow transplant. The model consists of a rat hind limb transplant that incorporates integumentary musculoskeletal, and lymphopoietic tissues. These transplants, in comparison with conventional marrow transplants, have the advantage of providing a syngeneic microenvironment and immediate engraftment of both mature and progenitor hemopoietic cells at the time of transplantation. The characteristics of graft-versus-host disease were studied in this model. Lewis X Brown Norway F1 (LBN RT-1(1+n)) rats received hind limbs from Lewis (LEW RT-1(1)) donors (n = 19). Animals were observed daily for signs of graft-versus-host disease. Necropsies were performed. A minority of animals developed lethal disease (7 of 19 recipients) and demonstrated cachexia with concomitant histopathologic changes of the disease. Acute and chronic groups emerged with distinct clinical courses, which are similar to other models of this disease. Recipients of vascularized bone marrow transplants (limb transplants) showed clinical and histopathologic changes of the disease. The transplants may be used as a model of graft-versus-host disease in humans. Most interestingly, the transplant has a lower incidence of disease compared with other methods of bone marrow transplantation and represents an alternative to conventional bone marrow transplantation, which deserves further exploration. It may be possible to develop a new technique for bone marrow transplantation based on this surgical approach. It is proposed that the transfer of vascularized blocks of bone/marrow into prospective recipients as opposed to cellular bone marrow transplants may be preferable.

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 and effective treatment for severe burns involves early excision of necrotic tissute followed by skin allografting ad cylosporine (CsA) immunosuppressive therapy. LEW (RR11) rats served as recipients of thermal injury and/or skin allografts. BNxLEW F1M (LBN, RT11+n) rats served as skin donors. LEW burn recipients received a hot water (90oC 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 singnificant graft polongation compared with controls (P<0.005). Skin graft survival was similarly prolonged in LEW recipeints undergoing burn unjury, primary wound excision, and CsA administration comkpared with not increased in the thermal injury—CsA-treated recipients comkpared with burn controls. A final experiment was initiated to investigate how low-level long-term (>100 days) maintenace CsA treatment influenced skin allograft survivalfor possible future consideration in burn trauma. Recipioents receiving skin allografts plus CsA (20 days, 8mg/kg/day, followed by every other day therafter) did not reject their grafts. However, a possible early sign of rejection ( a gingle small ulceratiove lesion) was noted in five of these long-term CsA-treated animals at a meanof 3411 (SD) days. The lesion in these animals did not progress any further during CsA administration. His-topathologic study of selected animals removed from the CsA maintenance regimen for greter than 50 days following long-term administration revealed a number of interesting chronic lesions similar to thosse previously reported in the skin comonent 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.

A pair of five-day flaps: early division of distant pedicles after serial cross-clamping and observation with oximetry and fluorometry.

In each of 2 recent patients with distant pedicles (one a groin flap and the other a cross-leg flap), we were able to perform the final division and detachment of the flap on the fifth postoperative day. Cross-clamping was used to create intermittent periods of ischemia. The periods of ischemia were progressively increased until the time of division. Fluorometry with intravenous fluorescein played a role in deciding when to divide the flap. The patients were discharged from the hospital on the sixth and seventh days, respectively. Trimming was done on an outpatient basis.

Reconstructive Allotransplantation: Considerations Regarding Integumentary/Musculoskeletal Grafts, Cyclosporine, Wound Coverage in Thermal Injury, and the Immune Response

With the advent of cyclosporine, a powerful and selective immunosuppressant, comes resurgence of a long-sought goal: to transplant modules of allointegumentary/musculoskeletal tissues or components thereof for the repair of peripheral tissue defects. Because these modules of integumentary and/or musculoskeletal tissue are actually composites of various tissues, they are also known as composite tissue allografts. The immediate goal of the studies reviewed herein is to lay the foundation in transplant immunobiology for the clinical exploitation of composite tissue allografts. The objective of these continuing studies is to induce permanent acceptance of composite tissue allografts. The value of such grafts lies in their potential for complete functional and cosmetic restoration in the surgical reconstruction of tissue after full-thickness burn injury. The initial results of basic experiments with cyclosporine are extremely encouraging in regard to the clinical potential for integumentary/musculoskeletal grafts in reconstructive allotransplantation.

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 tlerance in a patient sustaining massive burns, utilizing skin allowgrafts and cyclosporine. We now report the experimental correlate cia successful achievement of a 75% body surface area (BSA) scald burn cyclosporaine—skin allograft model in Lewis (LEW) rats. Cyclosporaine (8 mg/ kg/day) was given to the experimental animals daily for the first 20 days and then three times a week therafter. Two experimental groups were studied: one reveived stadard posttrauma care and the second critical posttrauma care. Controls (n=22) and experimental groups 1 (n=28) and 2 (n=4) had average survival time sof 13.812.8 days, 44.2132.5 days, and 172.019.4 days, respectively. The allografts on the surviving experimental animals appeared normal and healthy and had nealy 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.