Production of H-Y Antibody by Female Mice that Fail to Reject Male Skin

WHEN inbred mice are grafted with skin from inbred donors that differ from the recipients only by a single minor histocompatibility antigen, it is commonly observed that some recipients will retain their skin grafts while others will reject them. This is true of incompatibility for H-Y antigen, which is responsible for the rejection of male grafts by otherwise histocompatible inbred females of the same inbred strain¹. Thus in the DBA/2 (DBA) strain, male-to-female skin grafts are rejected by only some recipients; in the C57BL (B6) strain, females always reject male skin; and C3H/An (C3H) females usually accept male skin grafts indefinitely.     

Full-thickness grafting of acute eyelid burns should not be considered taboo.

Split-thickness skin grafts are commonly used for the treatment of acute eyelid burns; in fact, this is dogma for the upper lid. Ectropion, corneal exposure, and repeated grafting are common sequelae, almost the rule. It was hypothesized that for acute eyelid burns, the use of full-thickness skin grafts, which contract less than split-thickness skin grafts, would result in a lower incidence of ectropion with less corneal exposure and fewer recurrences. The records of all patients (n = 18) who underwent primary skin grafting of acutely burned eyelids (n = 50) between 1985 and 1995 were analyzed retrospectively. There were 10 patients who received full-thickness skin grafts (12 upper lids, 8 lower lids) and 8 patients who received split-thickness skin grafts (15 upper lids, 15 lower lids). Three of 10 patients (30 percent) who received full-thickness skin grafts and 7 of 8 patients (88 percent) who received split-thickness skin grafts developed ectropion and required reconstruction of the lids (p = 0.02). No articles were found substantiating the concept that only split-thickness grafts be used for acute eyelid burns. The treatment of acute eyelid burns with full-thickness rather than split-thickness skin grafts results in less ectropion and fewer reconstructive procedures. It should no longer be considered taboo and should be carried out whenever possible and appropriate.     

Diminished cellular cytotoxicity in mice hyperimmunized prior to skin grafting

Although cellular cytotoxicity generated in response to primary skin grafts has been studied in the past by several investigators, responses to skin grafts after repeated alloantigeneic stimulation have not been thoroughly studied. Using in vivo and in vitro assays of cellular cytotoxicity mice hyperimmunized prior to skin grafting showed lesser responses in local lymph nodes and spleen than did mice receiving primary grafts. Control experiments showed that suppressed responses were largely specific, were not due to circulating alloantibody, and were reversed if intraperitoneal tumor rather than a skin graft was used to challenge hyperimmunized mice. Early destruction of skin grafts in the immune host before vascularization was complete and thereby causing early elimination of cellular antigen was suggested as the mechanism for diminished cellular cytotoxicity in response to skin grafts in hyperimmunized hosts.     

The use of skin grafts in postburn contracture release: a 10-year review

Postburn scarring and contracture affecting function remain the most frustrating late complications of burn injury. Various techniques are used to release contractures; the choice depends on their location and/or the availability of unaffected skin adjacent to the contracture or elsewhere. A retrospective review was carried out of the case notes of patients who had skin grafting for the release of postburn contracture at the Burns Unit, City Hospital, Nottingham between May of 1984 and August of 1994 to evaluate the experience over this period. Information was obtained about the burn injury, contracture site, interval between burn and release of contracture, indication, age at first release, intervals between releases, operative details (donor and graft sites), complications and nonoperative treatment, and follow-up to the end of the study period. A total of 129 patients underwent skin grafting for release of contractures as opposed to any other method of correction. Full-thickness skin grafts were used in 81 patients (63 percent) and split-thickness skin grafts in 26 (20 percent). Twenty-two patients (17 percent) had both types used on different occasions. Flame burns (41 percent) were the most common causes, followed by scalds (38 percent). Two hundred thirty-nine sites of contracture were released, with the axilla (59) and the hand/wrist (59) being the most common sites involved, followed by the head/neck region (42). It was found that for the same site, release with split-thickness skin grafts was associated with more rereleases of the contracture than with full-thickness skin grafts. Also, the interval between the initial release and first rerelease was shorter than with full-thickness skin grafts (p < 0.048). It was also noted that children required more procedures during growth spurts, reflecting the differential effect of the growth of normal skin and contracture tissue. Patients reported more satisfaction with texture and color match with the full-thickness skin grafts. There was comparable donor-site and graft morbidity with both graft types. The use of skin grafts is simple, reliable, and safe. Whenever possible, the authors recommend the use of full-thickness skin grafts in preference to split-thickness skin grafts in postburn contracture release.     

Transplantation and cellular reactions to tissue antigens in Streptococcus-immunized rabbits]

The immunization of rabbits with the cells and the disintegration products of fractions of the cytoplasmic membranes of group A streptococcus (type 1) in incomplete Freund adjuvant, introduced in a single injection into the pads of the paws, caused lesions in autoplastic skin grafts and accelerated the rejection of alloplastic skin grafts. The rabbits showed positive delayed-type skin reactions to streptococcus and homologous skin antigens, and lymphocytes specifically reacting with FITC-labeled homologous skin antigen were found in their blood. Prolonged intravenous immunization with streptococcus, which induced the formation of complement fixing antibodies to homologous skin antigens, did not influence the taking of autoplastic and alloplastic skin grafts. The injection of hyperimmune streptococcus rabbit antiserum containing antibodies to skin antigens to intact rabbits produced no lesions in the autoplastic skin grafts and prolonged the lift of the alloplastic skin grafts.     

The role of serum imbibition for skin grafts

Numerous studies of grafted skin suggest that full-thickness skin grafts are nourished by exudate from the recipient bed called a serum imbibition. However, whether serum imbibition by itself is sufficient for nourishment of skin grafts has not been shown definitely and directly. To clarify the role of serum imbibition, we performed a comparative study between 20 skin grafts and 20 musculocutaneous flaps. The nourishment of the cell in the skin graft is by serum imbibition. That in musculocutaneous flaps is mainly derived from blood supply. We evaluated the nourishment by means of the unique characteristics of the cell cycle. Once cells are put into a synthetic phase, they cannot reverse or stop the progress of the cell cycle. To take advantage of this characteristic of the cell cycle, prewounding methods (40 flaps were lifted once and put back to the original sites prior to the evaluation) were intended for the cells in pre-elevated skin to turn into a proliferating phase. Cells were examined by antibody against proliferating cell nuclear antigen immunohistologically, to determine whether they had turned into the proliferating phase or not. After 3 days, all flaps were reelevated; half (20 flaps) had their muscle layer and the neurovascular bundle removed to make a full-thickness skin graft. The rest (20 flaps) were only lifted. They were sutured back to the original sites. Ten skin grafts and musculocutaneous flaps each were harvested at 3 hours (1st day) and at 11 days (11th day) after the second operation. Bromodeoxyuridine, which is a thymidine analog and is taken into the cells in the synthetic phase, was introduced intraperitoneally 2 hours before the harvest. All flaps and grafts were evaluated histologically and immunohistologically. Proliferating cell nuclear antigen analysis showed that the prewounding method induced the cells of skin grafts and musculocutaneous flaps to proliferate before the implantation. Regarding the bromodeoxyuridine uptake, no significant differences could be seen between skin grafts and musculocutaneous flaps irrespective of their different nourishment. No structural changes, such as degenerative or necrotic, could be seen at the hair follicle and other glands even at the 11th day. Almost all of the layers of skin grafts survived as long as they were checked by light microscopy (hematoxylin and eosin stain). No differences could be seen between musculocutaneous flaps and skin grafts or between the 1st and 11th days in this study. We concluded that serum imbibition is sufficient for nourishment of skin grafts, just as blood supply is sufficient for nourishment of musculocutaneous flaps.     

Tissue engineering of cultured skin substitutes

Skin replacement has been a challenging task for surgeons ever since the introduction of skin grafts by Reverdin in 1871. Recently, skin grafting has evolved from the initial autograft and allograft preparations to biosynthetic and tissue-engineered living skin replacements. This has been fostered by the dramatically improved survival rates of major burns where the availability of autologous normal skin for grafting has become one of the limiting factors. The ideal properties of a temporary and a permanent skin substitute have been well defined. Tissue-engineered skin replacements: cultured autologous keratinocyte grafts, cultured allogeneic keratinocyte grafts, autologous/allogeneic composites, acellular biological matrices, and cellular matrices including such biological substances as fibrin sealant and various types of collagen, hyaluronic acid etc. have opened new horizons to deal with such massive skin loss. In extensive burns it has been shown that skin substitution with cultured grafts can be a life-saving measure where few alternatives exist. Future research will aim to create skin substitutes with cultured epidermis that under appropriate circumstances may provide a wound cover that could be just as durable and esthetically acceptable as conventional split-thickness skin grafts. Genetic manipulation may in addition enhance the performance of such cultured skin substitutes. If cell science, molecular biology, genetic engineering, material science and clinical expertise join their efforts to develop optimized cell culture techniques and synthetic or biological matrices then further technical advances might well lead to the production of almost skin like new tissue-engineered human skin products resembling natural human skin.     

Non-H-2 histocompatibility antigens encoded by Moloney-murine leukemia virus in Mov mouse strains are detectable by skin grafting and cytolytic T lymphocytes

The integration and expression of Moloney-murine leukemia virus (M-MuLV) into the germ line of Mov mouse strains on the C57BL/6 background results in the expression of a cell-surface Ag with characteristics expected from non-H-2 histocompatibility Ag: the ability to stimulate graft rejection and generation of CTL. However, both the previously studied Mov-3 and Mov-14 strains differ from the coisogenic C57BL/6 strain by different length segments of chromosome derived from the ICR strain in addition to the integrated M-MuLV genome. To conclusively demonstrate that an Ag encoded by M-MuLV is solely responsible for rejection of Mov skin grafts by coisogenic recipients, we have studied additional Mov strains that differ from coisogenic 129 or BALB/c backgrounds only by integration of an M-MuLV genome. A total of 129 strain recipients reject skin grafts from two viremic Mov strains, Mov-17 and Mov-18. A total of 129 strain hosts primed with either 1) multiple sets of Mov-17 and Mov-18 skin grafts or 2) single injections of Mov-17 and Mov-18 spleen cells produce M-MuLV-specific CTL that could be boosted in primary mixed lymphocyte culture. Generated CTL were reactive with Con A-stimulated lymphoblasts from all tested viremic Mov strains on the B6 and 129 backgrounds as well as B6 lymphomas. Further, we have observed that 129 strain mice reject Mov-9 skin grafts if these skin grafts are transplanted to virgin 129 recipients which have not received prior skin grafts from non-viremic Mov donors. In addition, skin grafts were transplanted from two viremic Mov strains, Mov-15 and Mov-16, to coisogenic BALB/c recipients; rejection of both sets of grafts was observed. However, BALB/c responders did not generate specific CTL after priming in vivo, with either multiple sets of allogeneic grafts or spleen cell injections, and boosting in vitro. These observations confirm the ability of integrated and expressed M-MuLV genomes to encode what is operationally defined as a non-H-2 histocompatibility Ag.     

The metabolism of skin grafts stored with excess carbon dioxide.

In Japan, the long-term storage of cereals and foods in the presence of excess carbon dioxide is already a practical reality. The present study was conducted to assess the metabolic changes in skin grafts during storage in the presence and absence of excess carbon dioxide, with the aim of seeking a simple and effective method to prolong skin-graft viability during storage. In experiment 1, 120 male Wistar rats weighing 250 to 300 gm were used. A split-thickness skin graft 450 microns in thickness was harvested from the back of each rat with a dermatome and was divided into two pieces for separate storage. One piece was stored in normal air at 4 degrees C (control grafts), and the other was stored in a gas mixture composed of 20% O2, 20% CO2, and 60% N2 at 4 degrees C (CO2 grafts). Metabolic changes in the skin grafts during storage were investigated by ATP and glucose assays. In experiment 2, 60 male Wistar rats were used. Collection and storage of the split-thickness skin grafts were performed as in experiment 1. In both groups, skin grafts were stored for 1, 2, or 3 weeks, and the oxygen consumption rate of each graft was determined. Experiment 3 used 80 male Wistar rats and the same procedure as in experiment 1; split-thickness skin grafts were harvested, divided into two pieces, and stored for 1, 2, or 3 weeks. In both groups, the stored skin grafts were regrafted onto the backs of the same donor rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstruction of the burned palm: full-thickness versus split-thickness skin grafts--long-term follow-up

The long-term results of full-thickness (N = 11) and split-thickness (N = 14) skin grafts for reconstitution of the palmar surface following release of palmar burn scar contractures in pediatric patients are compared. Patients treated with full-thickness skin grafts required 1.2 +/- 0.4 operations (mean +/- SD). Patients treated with split-thickness skin grafts required 1.3 +/- 0.6 operations (mean +/- SD). No significant difference in the number of operative procedures was noted. No functional difference existed between the two groups. The use of split-thickness skin grafts provided comparable function without increased operative procedures and was less deforming. Increased use of split-thickness skin grafts following release of palmar burn scar contractures in pediatric patients should be considered.     

Case report: Use of skin grafting to keep a scleroderma patient ambulant.

A case of scleroderma is presented in which there was extensive skin involvement with contractures, but without any systemic manifestation. This patient has been kept ambulatory by repeated skin grafting. The grafts were taken from the few areas which were not involved with the disease. The grafting was successful, and so far the skin grafts have remained free of disease.     

Inhibition of myofibroblasts by skin grafts.

The myofibroblast population was studied by electron microscopy in rat wounds healing by (1) contraction of granulation tissue, (2) by coverage with split-skin grafts, and (3) by coverage with full-thickness skin grafts. In all 3 types of wounds, myofibroblasts appeared early and reached a peak number at two weeks after wounding. At this time, 40 to 50 percent of the wound fibroblasts had myofibroblast characteristics. The granulating wounds contracted rapidly and completely, and had long persistence of myofibroblasts. Split-skin grafted wounds contracted less and had a more rapid decrease in myofibroblasts. The wounds covered with full thickness skin grafts had a minimum of contraction with a very rapid decrease in the number of myofibroblasts until by 4 weeks no myofibroblasts were present. Full-thickness skin grafts thus appeared to influence contracting wounds not by preventing the formation of myofibroblasts, but by speeding up completion of their life cycle.     

The effect of epinephrine in local anesthesia on the survival of full- and split-thickness skin grafts: an experimental study

The effect of local anesthesia containing epinephrine on the survival of split- and full-thickness skin grafts remains unclear. In this blinded study, Xylocaine with or without epinephrine was injected subdermally prior to harvesting of split-thickness and full-thickness skin grafts on the dorsum of rabbits. After procurement, the grafts were placed back into their original donor sites. Statistical analysis of graft survival 7 days postoperatively revealed a significant decrease in survival for the full-thickness skin grafts treated with Xylocaine with epinephrine as compared with similar grafts without epinephrine (p less than 0.0005). No significant difference was noted for split-thickness skin-graft survival in grafts treated with Xylocaine with and without epinephrine (p greater than 0.1).     

Antigraft responses to the H-28c antigen by B6 and B6D2F1 mice

The survival of minor H antigen-bearing skin grafts from donors congenic with C57BL/6 (B6) was compared in B6, B6D2, and AB6 hybrid recipients. In a case singled out for further study, B6 mice were found to reject HW 110 skin (H-28^c antigen) rapidly, whereas B6D2 mice rejected HW110 skin much more slowly and variably. Both major histocompatibility complex (MHC)-linked and non-MHC genes appeared to affect the survival of HW110 strain skin grafts on B6 and B6D2 recipients. Results of several experiments appear to rule out the sharing of H-28° epitopes between donors and recipients as an explanation for the relatively poor response of B6D2 mice to HW 110 skin grafts. Experiments involving bone marrow chimeras produced by the reciprocal exchange of bone marrow between irradiated B6 and B6D2 mice suggest that bone marrow-derived donor cells and non-bone-marrow-derived host cells each contribute to the immune response phenotype with respect to the H-28° antigen. An attempt was made to determine whether B6D2 mice that failed to reject HW110 strain skin grafts possessed suppressor cells specific for the H-28^c antigen. Spleen cells from poorly responsive B6D2 mice failed to suppress the rejection of HW 110 skin grafts when assayed in immunodeficient mice that were provided with cells from immune 136132 donors that were highly responsive to HW110 skin grafts.     

Bone marrow-derived T lymphocytes responsible for allograft rejection

Lethally irradiated mice reconstituted with syngeneic bone marrow cells were grafted with allogeneic skin grafts 6-7 weeks after irradiation and reconstitution. Mice with intact thymuses rejected the grafts whereas the mice thymectomized before irradiation and reconstitution did not. Thymectomized irradiated mice (TIR mice) reconstituted with bone marrow cells from donors immune to the allografts rejected the grafts. Bone marrow cells from immunized donors, pretreated with Thy 1.2 antibody and C', did not confer immunity to TIR recipients. To determine the number of T lymphocytes necessary for the transfer of immunity by bone marrow cells from immunized donors, thymectomized irradiated mice were reconstituted with nonimmune bone marrow cells treated with Thy 1.2 antibody and C' and with various numbers of splenic T lymphocytes from nonimmune and immune donors. Allogeneic skin graft rejection was obtained with 10(6) nonimmune or 10(4) immune T cells. The effect of immune T cells was specific: i.e., immune T cells accelerated only rejection of the relevant skin grafts whereas against a third-party skin grafts acted as normal T lymphocytes.     

Distinct in vivo CD8 and CD4 T cell responses against normal and malignant tissues

Normal tissue and tumour grafts expressing the same alloantigens often elicit distinct immune responses whereby only normal tissue is rejected. To investigate the mechanisms that underlie these distinct outcomes, we compared the responses of adoptively transferred HY-specific conventional (CD8 and CD4) or regulatory T (Treg) cells in mice bearing HY-expressing tumour, syngeneic male skin graft or both. For local T cell priming, T cell re-circulation, graft localization and retention, skin grafts were more efficient than tumours. Skin grafts were also capable of differentiating CD4 T cells into functional Th1 cells. Donor T cell responses were inversely correlated with tumour progression. When skin graft and tumour transplants were performed sequentially, contemporary graft and tumour burden enhanced CD8 but reduced CD4 T cell responses causing accelerated skin-graft rejection without influencing tumour growth. Although both skin grafts and tumours were able to expand HY-specific Treg cells in draining lymph node (dLN), the proportion of tumour-infiltrating Treg cells was significantly higher than that within skin grafts, correlating with accelerated tumour growth. Moreover, there was a higher level of HY antigen presentation by host APC in tumour-dLN than in graft-dLN. Finally, tumour tissues expressed a significant higher level of IDO, TGFβ, IL10 and Arginase I than skin grafts, indicating that malignant but not normal tissue represents a stronger immunosuppressive environment. These comparisons provide important insight into the in vivo mechanisms that conspire to compromise tumour-specific adaptive immunity and identify new targets for cancer immunotherapy.     

Sensitivity of long-standing xenografts of rat hearts to humoral antibodies.

Long-standing rat skin xenografts on immunosuppressed mice are known to become insensitive to destruction by mouse anti-rat serum. Our present experiments demonstrate that long-standing primarily vascularized rat-to-mouse cardiac xenografts, unlike skin, remain fully sensitive to antiserum-mediated destruction, even in mice also bearing a long-standing skin graft that is resistant to antiserum. It appears that skin grafts become resistant to antiserum-mediated destruction because of extensive replacement of the endothelium by cells of host origin. The hearts remain sensitive to antiserum since such an extensive endothelial replacement would not occur as readily in these whole organ grafts.     

Cultured epithelial autografts for giant congenital nevi

Eight pediatric patients with giant congenital nevi confluent over 21 to 51 percent body surface area were treated by excision and grafting. The nevus was excised to the muscle fascia, and the open wound was grafted with cultured epithelial autografts and split-thickness skin grafts. The patients have been followed from 17 to 56 months. Seventeen operations were performed in the eight patients, excising a mean of 6.9 percent body surface area at each procedure. The mean duration of anesthesia was 3.7 hours, and the mean operative blood loss was 12.3 percent estimated blood volume. The mean "take" for the cultured epithelial autografts was 68 percent, and for the split-thickness skin grafts, 84 percent. Epithelialization of open wound areas adjacent to the grafts was somewhat slower for the cultured epithelial autografts than for the split-thickness skin grafts, but it led to a healed wound in all patients except one. Ten of the 17 areas grafted with cultured epithelial autografts resulted in small open wounds that required regrafting. Wound contraction under the cultured epithelial autografts and under split-thickness skin grafts was similar and depended more on the anatomic site grafted than on the type of graft employed. in 16 of 17 operations, the cultured epithelium remained as a permanent, durable skin coverage. The use of cultured epithelial autografts allowed a larger area of excision than would have been possible with split-thickness skin grafts alone and, therefore, a more rapid removal of nevus. Cultured epithelial autograft are an important new technique in the care of patients with giant congenital nevi.     

Application of a high-level peracetic acid disinfection protocol to re-process antibiotic disinfected skin allografts

Skin allografts, derived from cadaveric donors, are widely used for the treatment of burns and ulcers. Prior to use in clinical situations, these allografts are disinfected using a cocktail of antibiotics and then cryopreserved. Unfortunately, this antibiotic disinfection procedure fails to decontaminate a significant proportion and these contaminated grafts can not be used clinically. We have investigated whether it is possible to apply a second, more potent disinfection procedure to these contaminated grafts and effectively to re-process them for clinical use. Cadaveric skin grafts, treated with antibiotics and cryopreserved, were thawed and a peracetic acid (PAA) disinfection protocol applied. The grafts were then preserved in a high concentration of glycerol or propylene glycol, and properties thought to be essential for successful clinical performance assessed. The cytotoxicity of the grafts was assessed using both extract and contact assays; damage to the skin collagen was assessed using a collagenase susceptibility assay and the capacity of the grafts to elicit an inflammatory response in vitro was assessed by quantifying the production of the pro-inflammatory cytokine TNF-alpha by human peripheral blood mononuclear phagocytes. PAA disinfection, in conjunction with either glycerol or propylene glycol preservation, did not render the grafts cytotoxic, pro-inflammatory, or increase their susceptibility to collagenase digestion. The rates of penetration of glycerol and propylene glycol into the re-processed skin were comparable to those of fresh skin. This study has demonstrated that PAA disinfection combined with immersion in high concentrations of either glycerol or propylene glycol was an effective method for re-processing contaminated skin allografts, and may justify their clinical use.     

Marrow allograft success inW/W v anemic mice: Relation to skin graft survival times

Genetically anemicW/W ^v mice were cured by marrow allografts from donors of 13 out of 18 tested strains that differed at non-H-2 histocompatibility alleles defined by skin or tumor grafting. They were also cured by donors from all four tested congenic lines whose antigenic differences had been defined by induction of serum antibodies. They were not cured acrossH-2 differences. Tail skin graft survival times on uncuredW/W ^v recipients were determined for all congenic lines used as marrow donors. The longest and shortest skin graft survival times predicted correctly marrow graft success or failure. NoW/W ^v mice were cured by marrow grafts from donors of the three congenic lines whose skin grafts were rejected in fewer than three weeks. Almost everyW/W ^v mouse grafted was cured by marrow grafts from donors of the 13 congenic lines whose skin grafts survived longest, from 11 to more than 25 weeks. Intermediate skin graft survival times failed to predict whether marrow grafts would succeed.W/W ^v mice were cured by marrow from four congenic lines with mean skin graft survival times of 4.2, 4.4, 8, and 9 weeks, while marrow grafts failed from other congenic lines with mean skin graft survival times of 3.3, 3.4, 4.8, and 8.7 weeks. The simplest explanation for these results is that the antigens specified by theH-2, H-3, H-4, H-25, andH-28 loci are strongly immunogenic on both marrow precursor cells and skin,H-17 andH-24 are strongly immunogenic on skin but not on marrow, andH-12 is strongly immunogenic on marrow precursor cells but less strongly on skin.