The extended V-Y flap.

The extended V-Y flap is a modification of the V-Y advancement flap, which is very useful in closing defects following excision of facial lesions. The modification involves the addition of an extension limb onto the advancing edge of the standard flap. This limb is located adjacent to the area requiring reconstruction and is hinged down as a transposition flap on the end of the V-Y advancement flap to close the most distal portion of the defect. The extended V-Y flap has been found to be very effective in closing large defects in areas that typically have inadequate subcutaneous tissue to allow extensive mobilization of the standard V-Y advancement flap. It has been used effectively with excellent cosmetic results in the temporal, scalp, forehead, and nasal areas, providing a well-contoured and aesthetically pleasing reconstruction.

Mechanism of tolerance following class I--disparate renal allografts in miniature swine. Cellular responses of tolerant animals.

Previous studies utilizing a recombinant MHC haplo-type in our partially inbred miniature swine herd have demonstrated that some recipients matched only for SLA class II show long-term acceptance of renal allografts without exogenous immunosuppression. Such animals have been shown to develop systemic tolerance as evidenced by prolonged rejection times of subsequent donor-specific, but not third-party, skin grafts. In the present studies in vitro cellular responses of long-term tolerant animals and of 7 animals studied sequentially are presented. Long-term tolerant animals demonstrated responses consistent with the absence of the class I reactive helper populations normally present in naive controls. Animals studied sequentially segregated into two groups based on cellular reactivity and survival. All animals showed complete loss of antidonor class I cell-mediated lymphocytolytic (CML) reactivity by postoperative day 10. However, animals surviving less than 20 days maintained CML reactivity to donor class I plus third-party class II in the posttransplant period, while animals surviving greater than 40 days lost such reactivity. Addition of exogenous interleukin 2 could not reverse this loss. These studies suggest that tolerance induction to a renal allograft across a class I only difference involves effects on both helper and killer class I reactive cell populations.

Effects of T cell depletion in radiation bone marrow chimeras. III. Characterization of allogeneic bone marrow cell populations that increase allogeneic chimerism independently of graft-vs-host disease in mixed marrow recipients.

The opposing problems of graft-vs-host disease vs failure of alloengraftment severely limit the success of allogeneic bone marrow transplantation as a therapeutic modality. We have recently used a murine bone marrow transplantation model involving reconstitution of lethally irradiated mice with mixtures of allogeneic and syngeneic marrow to demonstrate that an allogeneic bone marrow subpopulation, removed by T cell depletion with rabbit anti-mouse brain serum and complement (RAMB/C), is capable of increasing levels of allogeneic chimerism. This effect was observed in an F1 into parent genetic combination lacking the potential for graft-vs-host disease, and radiation protection studies suggested that it was not due to depletion of stem cells by RAMB/C. We have now attempted to characterize the cell population responsible for increasing allogeneic chimerism in this model. The results indicate that neither mature T cells nor NK cells are responsible for this activity. However, an assay involving mixed marrow reconstitution in an Ly-5 congenic strain combination was found to be more sensitive to small degrees of stem cell depletion than radiation protection assays using three-fold titrations of bone marrow cells. Using this assay, we were able to detect some degree of stem cell depletion by treatment with RAMB/C, but not with anti-T cell mAb. Nevertheless, if the effects of alloresistance observed in this model are considered, the degree of stem cell depletion detected by such mixing studies in insufficient to account for the effects of RAMB/C depletion on levels of allogeneic chimerism, suggesting that another cell population with this property remains to be identified.

Antiviral T cell competence and restriction specificity of mixed allogeneic (P1 + P2----P1) irradiation chimeras.

Mixed irradiation bone marrow chimeras were prepared by reconstituting lethally irradiated C57BL/10 (B10) or B10.D2 mice with T cell-depleted bone marrow cells of B10 plus B10.D2 origin. These chimeras were healthy and survived well under conventional housing conditions and after experimental laboratory infections. Of a total of 17 chimeras tested, 2 died spontaneously or from the injected virus. Twelve of fifteen chimeras mounted a measurable cytotoxic T cell response to virus. Despite approximately equal percentages of B10 and B10.D2 lymphocytes in chimeras, cytotoxic T cell responses to vaccinia virus and lymphocytic choriomeningitis virus were mediated variably by either syngeneic or allogeneic donor lymphocytes; thus the H-2 type of effector T cells frequently did not correspond to the 50:50 distribution of spleen or peripheral blood lymphocytes. Cytotoxic responses were restricted exclusively to recipient H-2 type. All mixed chimeras examined were able to mount a good IgG response to vesicular stomatitis virus. These results confirm previous data suggesting that such mixed chimeras are healthy and immunocompetent and demonstrate strict recipient-determined restriction specificity of effector T cells; they also suggest that if T help is necessary for induction of virus-specific cytotoxic T cells, it does not require host-restricted interactions between helper T cells and precursor cytotoxic T cells.

Multiple mixed chimeras: reconstitution of lethally irradiated mice with syngeneic plus allogeneic bone marrow from multiple strains.

Reconstitution of lethally irradiated B10 mice with a mixture of 5 x 10(6) B10 plus 15 x 10(6) B10.D2 T-cell-depleted (TCD) bone marrow (BM) cells has previously been shown to produce stable, mixed chimeras which are specifically tolerant to donor skin grafts; the inclusion of TCD syngeneic marrow in the inoculum leads to improved immunocompetence in the resulting chimeras. In order to determine whether this method of transplant tolerance induction could be extended to multiple simultaneous allogeneic donors, we have investigated the engraftment capacity of combinations containing syngeneic and more than one allogeneic source of bone marrow. B10 mice were lethally irradiated and reconstituted with a mixture of (B10 + B10.D2 + B10.BR) or (B10 + B10.RIII + B10.BR) TCD BM. Analysis of each group of animals by flow microfluorometry provided evidence for stable multiple mixed chimerism in the majority of animals. All animals which exhibited such multiple chimerism were also tolerant of skin grafts from both allogeneic donors and promptly rejected fourth party skin grafts. An attempt to produce chimerism with TCD marrow from 5 allogeneic plus syngeneic BM cells was less successful. When animals were given non-TCD allogeneic BM from 2 allogeneic donors along with TCD syngeneic BM, they reconstituted as fully allogeneic chimeras in which one or the other allogeneic donor prevailed. These results indicate that (1) multiple allogeneic donor BM cells can engraft simultaneously in the mixed marrow model, but there may be a limit to the number of marrow strains which can repopulate a single animal; (2) multiple allogeneic engraftment confers transplantation tolerance to multiple donors; and (3) TCD is essential to permit multiple mixed chimerism to develop.

The recovery of resistance to alloengraftment following lethal irradiation and administration of T cell-depleted syngeneic bone marrow.

Recent studies from this laboratory have shown that unmanipulated, MHC-mismatched allogeneic bone marrow (BM) engrafts and produces complete allogeneic chimerism when administered to recipient mice 8 days following lethal irradiation and reconstitution with T cell-depleted (TCD) syngeneic bone marrow. Host lymphopoietic recovery thus appears to be insufficient by 8 days after irradiation and TCD syngeneic bone marrow transplantation (BMT) to resist alloengraftment. In the present studies we have examined the development of such resistance to alloengraftment by determining the limits of the time period permitting engraftment, and have assessed the role of allogeneic T cells in achieving chimerism after delayed allogeneic bone marrow transplantation. Our results indicate that increasing the delay for more than 8 days following irradiation and TCD syngeneic BMT leads to a rapid loss of the ability to achieve alloengraftment by non-TCD allogeneic bone marrow. Removal of T cells from allogeneic BM inocula administered 8 days after irradiation and TCD syngeneic BMT resulted in loss of the ability to achieve alloengraftment. Repopulation patterns in host spleens following delayed reconstitution suggest that active elimination of engrafted syngeneic lymphohemopoietic elements is necessary to permit engraftment of allogeneic marrow administered after such a delay.