Skin Graft Donor Research Articles

Bone marrow transplantation across major histocompatibility barriers in mice. Effect of elimination of T cells from donor grafts by treatment with monoclonal Thy-1.2 plus complement or antibody alone.

The current studies were designed to evaluated optimal conditions for reduction of graft-versus-host disease (GVHD) by removal of donor T cells from bone marrow inoculum. A model was used in which the addition of spleen cells to donor marrow heavily favored the development of lethal GVHD. Treatment of donor bone marrow plus spleen cells with monoclonal anti-Thy-1.2 antibody plus complement protected lethally irradiated recipients from GVHD across major histocompatibility barriers better than donor cells treated with the same dilution of antibody alone. Engraftment was demonstrated by the presence of high percentages of donor cells in the peripheral blood of these animals and the long-term survival of donor skin grafts. These results may be important in light of the development of new antihuman T cell monoclonal antibodies which may be used in the treatment of donor marrow in clinical transplantation.

Interaction of H-2Db with mutant histocompatibility gene H (KH-11) in the mouse.

The detectable presence of H (KH-11)b, a mutant non-H-2 histocompatibility gene, was previously shown to depend upon the simultaneous presence, in the skin-graft donor, of both the mutant gene and the H-2b haplotype. The experiments reported here demonstrate that H-2Db is the essential element of H-2b for this interaction. Of two H-2Db histocompatibility mutations, H-2bm13 can replace H-2Db in this interaction, but H-2bm14 cannot.

Tolerance to allogeneic and to xenogeneic heart grafts provided by thymectomy of adult mice combined with donor cell and cyclophosphamide inoculation.

A new method of induction of tolerance to allogeneic and to xenogeneic cells is presented. It includes thymectomy of adult mice followed 1 month later by the injection of 1 X 10(8) spleen cells i.v. and i.p. administration of 200 mg of cyclophosphamide per kg 1 day after cells. This method induced prolonged survival of heterotopically transplanted neonatal C57BL/6 murine heart grafts (more than 8 months) and of August rat heart grafts (more than 2 months) in CBA mice. Tolerance to allo- or xenoantigens was formed at the cell level. Experimental animals did not produce allo- or xenohemagglutinins after graft implantation. Spleen cells of mice with surviving C57BL/6 heart grafts did not respond to C57BL/6 cells in mixed lymphocyte culture (MLC) reaction. Lymphoid cell chimerism was not observed in animals tolerant to alloantigens.

TRANSPLANTATION IN MINIATURE SWINE

Renal allografts were performed between and among animals from three herds of miniature swine that were selectively inbred to homozygosity at the major histocompatibility complex, MSLA. The results suggest several genetic factors which influence the survival of renal allografts in these animals. As expected, the major histocompatibility complex (MHC) was of dominant importance, and all MSLA-mismatched grafts were rejected promptly (12 +/- 3.7 days). Some MSLA-matched grafts were also rejected (30 +/- 15.0 days), indicating that non-MSLA loci also determine antigens which can lead to kidney rejection. Other MSLA-matched grafts were accepted indefinitely. At least one immune response gene that determined ability to reject kidneys across non-MSLA differences seemed to be segregating in our swine population. Animals that had accepted MSLA-matched renal grafts for extended periods demonstrated markedly prolonged survival of subsequent donor skin grafts compared to skin graft survival across the same non-MSLA difference in normal animals. This finding suggests that failure to reject kidneys across non-MSLA differences indicates systemic tolerance, and that there may be a relationship between the induction of such tolerance and the proposed immune response gene controlling rejection.

The monkey skin graft model as an assay of human antilymphocyte globulin.

SUMMARY Thirteen lots of horse antihuman lymphocyte globulin (ALG), produced using several different antigen sources and methods of preparation, were tested in the primate skin allograft assay system. Each ALG tested showed significant prolongation of skin allograft survival (range, 15-44+ days; average, 22.79 ± 8.93 days) compared to the control (9.95 ± 1.46 days). No antigen source, method of preparation, or route of administration produced consistently longer survival and none prevented the formation of specific antibody against the skin graft donors. I.v. administered ALG did not lead to the fixation of antiglomerular basement membrane (GBM) antibody as assessed by conventional immunofluorescent techniques and resulted in a lower incidence of antihorse IgG antibody production than that following administration by the s.c. route. One lot of ALG, however, caused fatal disseminated intravascular coagulation in four monkeys, only when administered i.v. A positive correlation between primate skin allograft survival and rosette inhibition (RI) titers was not demonstrated.

Modification of graft-versus-host disease by neuraminidase treatment of donor cells. Decreased tolerogenicity of neuraminidase-treated cells.

A lethal graft-versus-host disease (GVHD) was induced in cyclophosphamide (Cy)-treated C3H mice by i.v. injections of C57 bone marrow (20, 30, or 50 X 106) or spleen cells (5 X 106) which had been pretreated in vitro with Vibrio cholerae neuraminidase (VCN) or heat-inactivated VCN. The surviving C3H recipients were grafted with C57 skin to evaluate the presence of tolerance or immunity to the donor cells. The acute lethality of high doses (400 mg/kg) of cyclophosphamide was prevented equally well by VCN-treated or unmodified allogeneic bone marrow cells. The acute lethal GVHD, attributable to high doeses (400 mg/kg) of cyclophosphamide and allogeneic spleen cells, could be induced equally well by VCN-treated and unmodified spleen cells. The results suggest that VCN-treated cells maintain their viability, can reconstitute cyclophos-phamide-treated mice, and can induce GVHD in severely immunosuppressed mice. At more moderate doses of cyclophosphamide (300–350 mg/kg), VCN treatment of donor bone marrow and spleen cells delayed the onset and reduced the lethality of the GVHD when compared with cells treated with heat-inactivated VCN. Surviving animals who had received donor cells treated with heat-inactivated VCN were frequently tolerant of donor (C57) skin grafts. Surviving mice who had received VCN-treated cells were immune to the donor skin grafts. VCN treatment appears to render cells less tolerogenic than normal so that mice do not become tolerant and the GVHD cannot develop. The results are compatible with our previous findings that VCN increases the immunogenicity of normal lymphoid cells, both in unmodified hosts and in vitro.

The influence of histocompatibility upon the corneal immune reaction after interlamellar allotransplantation in rabbits.

The influence of the RL‐A (R abbit L eukocyte — locus A) histocompatibility system upon the corneal immune response to interlamellar allotransplantation was studied in 75 rabbits. The evaluation of histocompatibility was based upon reactions with 18 different lymphocytotoxic rabbit isoantisera obtained by pairwise immunization of outbred rabbits. These sera were tested against pure lymphocyte suspensions from the potential corneal donors and recipients.The evaluation of the immune reaction was performed by clinical observation, by photographs, and histologically in sections of the eyes and in conjunctival scrapings.The immune reaction to allotransplantation was weak and consisted of lymphocyte infiltration around the graft. Immune reaction was seen only in histoincompatible transplantations, compatible ones showed no reaction. The lymphocyte infiltrations were more pronounced when the noncompatible animals were presensitized by donor skin grafts. In contrast to this no reaction could be produced in compatible animals presensitized by donor skin grafts. Histocompatibility evaluation may be of clinical importance in corneal transplantations if the recipients have been presensitized to histocompatibility antigens by previous transplantations, transfusions, or pregnancies, especially if the recipient cornea is vascularized.

Prolonged survival of H-2 incompatible skin allografts on F1 animals treated with parental lymphoid cells

The survival times of H-2 incompatible skin allografts were studied in F1 hybrids treated with parental strain lymphoid cells. The injection of 50×106 A/Sn lymphoid cells into A/Sn×CBA F1 hybrids greatly prolonged the survival of 5M skin allografts applied 7 days later. Suppression of allograft rejection was observed also in F1 recipients that were actively or adoptively immunized against the skin graft donor. The immunocompetence of the sensitized F1 hosts was supressed to a greater degree than that of normal non-sensitized recipients. It is unlikely that immuno-incompetence is caused by basic effects of the graft-versus-host reaction or by antigenic competition since overt signs of a graft-versus-host reaction were not a necessary prerequisite for immunosuppression. It is suggested that a general inhibition of expression of immunity develops as a consequence of the immune reaction, possibly mediated by humoral factors.

Demonstration of Cytotoxic Humoral Substances during the Course of First Set Skin Allograft Rejection

SummaryCytotoxic humoral substances, which are most likely produced by the stimulation of first set skin allograft, were demonstrated by utilizing Millipore diffusion-chamber technique. The cytotoxic effect of the substance was much stronger in the animals which had rejected their grafts, whereas it was least in the animals bearing viable grafts. The presence of such cytotoxic humoral substances was observed as early as 8 days after grafting, and lasted for at least 6 weeks. The results obtained at the twelfth week of grafting suggested the virtual disappearance of the cycotoxic substances. Lymphocytes originating from a rat other than the skin graft donor were also killed rapidly when implanted into recipient rats which had rejected grafts, indicating cross-reactivity within the animals used in this study.

Time of development and duration of the effect of a guinea pig transfer factor

The time of development and the duration of the effect of a guinea pig antihomograft transfer factor has been studied. The factor is demonstrable in the lymph nodes by the seventh or eighth day after grafting. The capacity for causing a cutaneous inflammatory reaction in the donor and inducing the accelerated rejection of donor skin grafts arises and declines at the same rate. By injecting the factor prior to grafting it is shown that it has a short-lived effect. The data are consistent with the characteristics of the development, duration, and passive transfer of antibodies.

The effect of skin homograft rejection on recipient and donor mixed leukocyte cultures.

The lymphocyte proliferation in repeatedly studied mixed leukocyte cultures of peripheral white blood cells from a skin graft donor and 2 recipients was significantly increased at the time of graft rejection. This was determined from the increased proportions of mononuclear cells labeling with tritiated thymidine, increased mitotic indices, and the appearance of increased numbers of transformed lymphocytes after rejection of 1st and 2nd skin grafts. The temporarily enhanced response occurred sooner and was of shorter duration after the second than after the first graft, but was quantitatively similar each time. The cell proliferation in the mixed leukocyte cultures of the two recipients was similarly affected by the homograft rejections. The cultures containing three cell populations usually manifested a greater lymphocyte response than corresponding cultures of leukocytes from only two unrelated subjects. An increase in the ratio of female recipient to male graft donor metaphases in the cultures at the time of enhanced lymphocyte transformation indicated that proliferation of the graft recipient lymphocytes was responsible for the above findings. Unmixed, unstimulated control cultures grown in autologous, the other subjects plasma, or heterologous calf serum failed to support significant lymphocyte transformation. The role of humoral factors and relationship of the in vitro cellular responses to the in vivo homograft reaction are discussed.

SPECIFIC SUPPRESSION OF WOUND HEALING IN MICE BY GRAFT-VS-HOST REACTION.

SUMMARY Young adult (C57L x A) F1 mice bearing full-thickness skin grafts from A/HeJ mice received a single dose of 500 rad X-radiation, followed by i.p. injection of 6 x 106 lymph node cells from normal A/HeJ donors; controls were injected with inactivated A/HeJ lymph node cells (i.e., 2000 rads in vitro). Circular punch skin wounds through the donor skin graft and adjacent host skin were made 8 days later. The mice receiving the intact parental strain lymph node cells showed symptoms of transplantation disease, while the controls were free of such signs. Skin wound healing was assessed histologically in mice killed 3 and 6 days after wounding, permitting a comparison of healing in the donor skin versus host skin on the same mouse. The findings indicate a mild but definite specific inhibition of wound healing in LAF1 skin as compared with A skin in the mice suffering from transplantation disease. In each of seven mice examined 6 days after injection of A-strain lymphoid cells, healing in the grafted A strain skin was definitely greater than in the host's own (LAF1) skin. Thus, the LAF1 skin at this time showed only minimal to early healing, while the wounds in the grafted A-skin were completely closed in 4 of 7 cases, of which 2 were healed. The possible theoretical basis for the specific inhibition of wound healing in graft-versus-host disease is briefly discussed.

Further studies on the formation of phosphatidyl ethanolamine in liver preparations

We have previously reported immunity to donor antigens following in utero transplantation (IUT) of cytokine-stimulated allogeneic hematopoietic stem cells (sca+/lin−) (day 9 of gestation). Transplanted mice showed accelerated rejection of donor skin grafts and high anti-donor cytotoxic response, a finding not seen in the control mice. This was accompanied by an enhancement of Th1 over Th2 cytokine production and persistent donor microchimerism 1, 2. In order to assess the role of the thymus in allograft rejection, prenatal transplants were performed under similar experimental conditions at a later gestational age, when the thymus is more developed (day 13).Cytokine-stimulated stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF)–purified sca+/lin− cells of C57BL/6 (H-2b, 1E+) background were injected into MHC-mismatched BALB/c (H-2d, 1E−) fetal mouse recipients at day 13 of gestation. Chimerism was determined by highly sensitive (0.001%) semiquantitative polymerase chain reaction (PCR) 3, 4. Mixed lymphocyte reaction (MLR) and cytotoxic T-cell assay (CTL) were used to evaluate tolerance vs immunity. Cytokine levels were quantified in MLR supernatants using ELISA assay. The percent of T cells was determined by flow cytometry (FACS) and CD4/CD8 ratio calculated. Postnatal boosts (transplants without conditioning) were performed at 6 months of age to enhance donor chimerism and test the degree of tolerance.When assayed at 4 months of age, donor-type cells were not detected in the spleen or in the peripheral blood of BALB/c mice inoculated with C57BL/6 sca+/lin− cells. Transplanted but not control animals demonstrated high anti-donor MLR but not CTL responses. The increase of MLR reactivity was correlated with high levels of IL-2. Furthermore, transplanted mice showed higher resistance to postnatal boosts with allogeneic bone marrow (BM) cells, when compared to the control mice. The later resistance was accompanied by the expansion of host-type CD4 cells.These data demonstrate that transplantation of cytokine-stimulated sca+/lin− allogeneic cells at 13 days of fetal development leads to the allosensitization, characterized by an enhancement of MLR alloreactivity and by the rejection of postnatal boosts (transplants without conditioning). Host-type CD4 cells might play a central role in this rejection. These findings indicate that the late injection of allogeneic cells may result in the development of allosensitization with subsequent donor graft rejection. Precise conditions for the development of tolerance must be established before prenatal transplants in humans with conditions other than SCID can be done.