Cultured Keratinocyte Allografts Research Articles

Delayed reepithelialization and scarring deregulation following drug-induced toxic epidermal necrolysis

A 51-year-old Caucasian woman developed severe drug-induced toxic epidermal necrolysis (TEN) due to allopurinol. The withdrawal of the culprit drug was unfortunately delayed, and dramatic retardation of reepithelialization was observed. At that stage of disease evolution, an inflammatory cell infiltrate was present in the dermis. Coverage of eroded lesions by frozen cultured keratinocyte allografts failed to hasten reepithelialization compared to ungrafted sites. This unusual protracted TEN evolution was followed by the development of extensive hypertrophic and keloid scars. Several biopsies were taken over 6 months. The histologic presentation of the grafted and ungrafted eroded scar tissues looked similar. Both the number and size of the Factor XIIIa-positive dermal dendrocytes, as well as the number of α-actin-positive myofibroblasts showed a marked increase between weeks 2 and 12 after grafting. They were reduced after 6 months when the scarring process was stabilized. α1 [IV] collagen was never expressed over the eroded scars. Similar to burn patients, delayed reepithelialization might be a risk factor for abnormal scarring in TEN. Cultured keratinocyte allograft apparently offered no improvement in reepithelialization and did not prevent abnormal scarring in this TEN patient.

CD80, but not CD86, express on cultured murine keratinocyte stem cells

Although cultured keratinocyte allografts are initially accepted, grafted donor cells are gradually replaced by recipient elements. The precise mechanisms underlying this process are not clear. Keratinocyte stem cells (KSCs) were isolated and purified from neonatal C57BL/6 (H-2 b) and BALB/c (H-2 d) mice. Cultured KSCs were negative for IA/IE, indicating the absence of any contamination with Langerhans cells. Immunostaining showed that over a 7-day culture period, KSCs expressed increasing amounts of CD80, while CD86 could not be detected. RT-PCR results were consistent with these data. Mixed lymphocyte reaction assays showed that KSCs cultured in 10% serum for 7 days stimulated allogeneic rather than syngeneic T-cell proliferation. This study demonstrates that the costimulatory molecule CD80, but not CD86, may be expressed on cultured KSCs. The data indicate KSCs could act as antigen-presenting cells and thus provide an alternative explanation for the ultimate rejection of allogeneic keratinocytes.

Intrabody-mediated phenotypic knockout of major histocompatibility complex class I expression in human and monkey cell lines and in primary human keratinocytes.

Cultured keratinocyte allografts from unrelated donors can be readily grown as sheets in large-scale cell culture and have been used as an immediate skin cover for severely burned patients. Despite the absence of passenger leukocytes and the unlimited amount of material that can be obtained for permanent skin coverage, the allografts are susceptible to rejection. Since MHC class I (MHCI) antigens serve as targets for allograft rejection, we investigated whether 'phenotypic knockout' of human MHCI could be achieved through expression of an ER-directed anti-human MHCI single-chain intrabody (sFvhMHCI) that is directed against a monomorphic, conformational epitope, expressed across species lines, on the MHCI heavy chain. Co-immunoprecipitation of both MHCI heavy chain and beta2-microglobulin occurred in transfected monkey COS-1 cells, while Jurkat T cells stably expressing the ER-directed sFvhMHCI intrabody showed that complete phenotypic knockout of MHCI cell surface expression could be achieved. Infection of several human cell lines of divergent tissue sources and different HLA haplotypes resulted in marked down-regulation of MHCI expression, even under conditions where inflammatory cytokines (eg gamma-IFN) which up-regulate MHCI expression were used. Finally, when adenovirus encoding the anti-human MHCI intrabody was used to transduce primary human keratinocytes, a marked reduction of surface MHCI expression was observed. These in vitro studies set the groundwork for in vivo studies to determine if intrabody-mediated knockout of MHCI can impair alloantigen expression and prolong the survival of keratinocyte allografts.

Skin grafting for venous leg ulcers.

Venous leg ulceration is a common, recurring, disabling condition affecting up to 1% of adults. Treatment is aimed at healing with simple dressings and compression bandages / stockings. Unfortunately in some cases this treatment is unsuccessful with ulcers remaining open for months or years. In order to stimulate healing, some clinicians use skin grafts. These skin grafts may be taken from the patients own uninjured skin (e.g. thigh), may be grown from the patient's skin cells into a dressing, (both known as autografts) or applied as a sheet of bioengineered skin grown from a donor cells (known as an allograft). Preserved skin from other animals, e.g. pigs, have also been used and these are known as xerografts. To assess the effectiveness of skin grafts in the treatment of venous leg ulcers. We searched the Cochrane Wounds Groups specialised register (date of search October 1999), and reference lists of relevant articles. We hand searched relevant journals and conference proceedings, and contacted experts in the field. Randomised controlled trials evaluating skin grafts in the treatment of venous leg ulcers. There was no restriction on articles based on language or publication status. Data extraction and assessment of study quality was undertaken by two reviewers independently. Trials with similar patients, comparisons, and outcomes were pooled. Seven RCTs of skin grafts for venous leg ulcers were identified. In 6 trials patients also received compression bandaging. Two trials (98 patients) evaluated split thickness autografts, three trials (92 patients) evaluated cultured keratinocyte allografts, one compared tissue engineered skin (artificial skin) with a dressing (309 patients) and one compared it with a split thickness skin graft (7 patients, 13 ulcers). The trials comparing artificial skin with a dressing reported a significantly higher proportion of ulcers healing with artificial skin. There was insufficient evidence from the remaining trials to determine whether other types of skin grafting increased the healing of venous ulcers. There is limited evidence that artificial skin used in conjunction with compression bandaging, increases the chance of healing a venous ulcer compared to compression alone. Further research is needed to assess whether other forms of skin grafts increase ulcer healing.

Immunogenicity of cultured keratinocyte allografts deficient in major histocompatibility complex antigens.

Full-thickness (FT) and cultured keratinocyte (CK) allografts have been used as temporary skin replacements in patients with massive burns, but these grafts are ultimately rejected after restoration of host immunocompetence. Genetic engineering has permitted the creation of knockout (KO) mice deficient in class I or class II major histocompatibility antigens. This study examines the immunogenicity of such grafts to determine if these genetically modified keratinocytes could be used for permanent wound coverage. Host sensitization to alloantigen was assessed by second-set rejection. CBA mice (n = 111) were primed with flank grafts consisting of FT and CK allografts from normal C57BL/6 donors, FT and CK class I KO allografts, FT and CK class II KO allografts, and CK autografts. Three weeks later, hosts were challenged with normal tail allografts and observed for second-set rejection. Median graft survival was analyzed by chi2 and Wilcoxon rank tests. In the second experiment, cytotoxic T lymphocytes (CTLs) were harvested from CBA mice (n = 28) 3 weeks after flank grafting. CTL effectors were tested on radiolabeled targets at various ratios in a 51Cr release assay. Dilution curves of CTL activity were compared by analysis of variance. Hosts primed with CK or FT allografts demonstrated accelerated rejection of second-set tail grafts compared with hosts covered with CBA autografts. CK knockout grafts were less immunogenic than FT knockout skin; class II KO allografts were considerably less immunogenic than class I KO allografts. CTL activity against the knockout CK allografts was negligible compared with that of hosts primed with normal allografts or FT knockout allografts. Although full-thickness knockout skin retains substantial immunogenicity, cultured keratinocytes deficient in class II antigens fail to prime for accelerated second-set rejection and do not elicit a CTL response in the graft recipient. This lack of immunogenicity may permit the indefinite survival of allogeneic knockout keratinocytes in patients requiring massive wound excision and coverage.

Burn injury impairs second-set rejection and CTL reactivity in mice primed by cultured keratinocyte allografts.

Cultured keratinocyte (CK) allografts have limited antigenicity and have been used as a skin replacement in patients with massive thermal injury. Recent data indicate that CK grafts are more immunogenic than previously believed and could compromise wound healing in the immunocompetent host. The purpose of this study was to determine if the immunosuppression of burn injury might affect the alloantigen response and minimize sensitization to CK allografts. CBA mice received a 0%, 20%, or 40% burn that was partially excised three days later and grafted with a full-thickness (FT) skin allograft, CK allograft, or CK autograft. Two weeks postburn, mice received FT tail skin allografts, which were observed for rejection. We observed that FT and CK allografts primed the unburned host with equal efficacy. However, burn injury selectively minimized priming by CK allografts, resulting in delayed rejection of second-set allografts. With evidence that burn injury inhibits host sensitization to CK allografts, we then examined the effect of burn size on CTL alloreactivity. Additional CBA mice underwent burn injury, excision, and grafting as described above. Host splenocytes were harvested two weeks later and tested on radiolabeled targets for allospecific cytotoxicity. CTLs from unburned mice primed with FT allografts demonstrated the greatest CTL lysis, followed next by CTLs from unburned mice covered with CK allografts. Burn injury inhibited CTL activity as a function of wound size. Activity of CTLs from burned mice primed with CK allografts improved after in vitro allostimulation but remained below that of CTLs from unburned, unprimed mice. We conclude that burn injury selectively inhibits the allospecific response to CK allografts. The decreased immunogenicity of CK allografts, when used for burn wound coverage, may improve the long-term survival of allogeneic keratinocytes, enhancing their potential as a biologic skin replacement.

The Relationship Between Interferon- γ and Keratinocyte Alloantigen Expression After Burn Injury

Cultured keratinocyte (CK) and cadaveric skin allografts have prolonged survival in patients with massive thermal injury. It is unclear if this delayed rejection is due to impaired host responsiveness or decreased graft immunogenicity. Although burn injury has been shown to decrease parameters of allograft response, no studies have examined the effect of burn injury on alloantigen expression. This study investigated the effect of burn size on class II antigen expression in CK allografts as well as on tissue levels of interferon-gamma (IFN-gamma), the principle regulator of alloantigen expression. Anesthetized CBA mice (n = 64) received a 0%, 20% partial-thickness (PT), 20% full-thickness (FT), or 40% FT contact burn. Forty-eight hours later, wounds were partially excised and covered with CK allografts from C57BL/6 donors. Five days after burn injury, grafts were analyzed for donor-specific class II antigen. Protein expression was determined by Western immunoblotting and quantified with video densitometry. Wound, serum, and unburned skin levels of IFN-gamma were determined by enzyme-linked immunosorbent assay. Groups were compared by Fisher's analysis of variance. As burn size increased, class II antigen expression decreased (p < 0.001). This corresponded with decreased wound and skin levels of IFN-gamma after 40% burn (p < 0.05); however, wound IFN-gamma was significantly elevated after 20% PT and FT burns (p < 0.01). Serum IFN-gamma increased as burn size increased (p < 0.01). Burn injury decreases the antigenicity of CK allografts, which partly explains delayed allograft rejection after burn injury. Although wound IFN-gamma increases after minor thermal injury, the profound decrease in wound and skin IFN-gamma after a major burn corresponds with diminished class II antigen expression. The decreased availability of IFN-gamma after major thermal injury provides a mechanism for limited allograft tolerance.

CULTURED MOUSE KERATINOCYTE ALLOGRAFTS PRIME FOR ACCELERATED SECOND SET REJECTION AND ENHANCED CYTOTOXIC LYMPHOCYTE RESPONSE1,2

It has been reported that cultured keratinocyte (CK) allografts are not rejected in mice, unlike in other species. Several reports have suggested that mouse CK allografts are incapable of stimulating a primary alloresponse, including sensitization of recipients to alloantigens. In this study, we investigated the immunogenicity of mouse CK allografts in vivo by determining whether CK allografts primed for a second set rejection response. First, we grafted mice with either CK allografts, CK autografts, full-thickness (FT) allografts, or no graft at all. We then regrafted mice 4 weeks later with a tail skin allograft. Mice grafted with CK allografts rejected second allografts as rapidly and as vigorously as mice grafted with FT flank allografts. Next, we tested whether CK allograft primed recipients for enhanced CTL responses. We found that mice grafted with CK allografts generated a significantly enhanced CTL alloreactive response after in vitro stimulation. The response was similar to that of mice grafted with FT skin allografts. With evidence that CK allografts primed, we biopsied wounds immediately after CK allografting and, using Western immunoblotting, found that CK allografts had substantial expression of MHC class II antigens in vivo. We conclude from the results of our studies that mouse CK allografts unequivocally prime recipients to alloantigens in vivo and suggest that a possible mechanism for alloantigen priming may be CK allograft expression of MHC class II antigens.

Cultured keratinocyte allografts induce more rapid healing of skin graft donor sites

Cultured keratinocyte allografts induce more rapid healing of skin graft donor sites

Cultured keratinocyte allografts and wound healing in severe recessive dystrophic epidermolysis bullosa

Patients with recessive dystrophic epidermolysis bullosa (RDEB) frequently have painful erosions that are slow to heal. There is no definitive treatment; therefore any therapy that improves wound healing would be beneficial to these patients. Our purpose was to assess the effects of cultured allogeneic keratinocytes on wound healing in RDEB. Ten patients with RDEB and dermatome-induced superficial dermal wounds were studied. Cultured keratinocyte grafts were applied to part of the wound, with another part left ungrafted. Both sites were assessed clinically and microscopically, particularly with regard to basement membrane zone reconstitution. Apart from minor differences in keratinocyte differentiation and a moderate analgesic effect induced by the graft, there were no other distinguishing findings in wound healing in the grafted and nongrafted sites. There was little clinical benefit from cultured keratinocyte allografts in wound healing in RDEB. However, this study showed that RDEB keratinocytes have an inherent capacity to express some type VII collagen epitopes transiently during wound healing, although this was not associated with the detection of anchoring fibrils.

REJECTION OF CULTURED KERATINOCYTE ALLOGRAFTS IN PRESENSITIZED MICE

The fate of cultured keratinocyte (KC) allografts remains controversial. Although prolonged survival of cultured KC allografts in naive mice has been reported, the detailed mechanisms remain undetermined. Furthermore, it was also reported that in the human cultured KC allografts do not survive permanently, and they are rapidly replaced by recipient cells. In the present study, we have addressed this issue and obtained findings that cultured KC allografts survive for a prolonged period in naive mice under the conditions in which reepithelization by recipient cells is prevented. However, the same cultured KC allografts were rejected when they were grafted onto recipients primed with allogeneic spleen cells or full-thickness skin grafts. To clarify the mechanisms behind these findings, the allostimulatory ability of cultured KC and their susceptibility to alloreactive cytotoxic T cells were examined in vitro. In a mixed epidermal cell-lymphocyte reaction, cultured KC were unable to induce allospecific proliferative responses of naive T cells. On the other hand, primed T cells from presensitized mice showed weak but significant proliferative responses against allogeneic KC. It also was confirmed that cultured KC are susceptible to lysis by alloreactive cytotoxic T cells. These data indicate that prolonged survival of cultured KC allografts in naive mice is attributable to a defect in the afferent, but not the efferent, phase of the rejection process that is caused by the weak allostimulatory ability of cultured KC. This assumption was also supported by the finding that spleen cells from the recipient mice bearing long-surviving KC allografts retain in vitro responsiveness against stimulator cells syngeneic to the grafted KC. Taken together, these findings indicate that long-term survival of cultured KC allografts in naive mice may be due solely to the weak allostimulatory ability of cultured KC, but not to loss of susceptibility to alloreactive cytotoxic T cells after culture of KC or induction of tolerance in the recipient mice bearing KC allografts.

Variable rejection patterns of cultured keratinocyte allografts in the rabbit

There are many conflicting reports on the survival of cultured keratinocyte allografts (CKAs). Studies were performed in the rabbit model to elucidate further the fate of CKAs. Of 24 CKAs, 7 were identified as technical failures, 13 displayed classical macroscopic evidence of rejection with a prolonged mean survival time of 2.8 days, compared to non-cultured allograft controls (p < 0.01), and 4 failed to display typical macroscopic evidence of rejection. Furthermore, the time to eventual wound healing of the classically rejected CKAs were delayed by 6.0 days (p < 0.0001), compared to identical non-grafted control wounds.

A randomized controlled trial of cultured keratinocyte allografts for chronic venous ulcers

The efficacy of cultured keratinocyte allografts on the healing rate and pain relief of 30 small and medium-sized chronic venous leg ulcers was assessed in a prospective, randomized, placebo-controlled study. Measurements of surface area, ulcer bed appearance and pain severity were made at fortnightly intervals for 6 weeks. There was significant healing of ulcers in both the allografted and control groups (P = 0.001), but there was no significant difference between the two groups (P>0.05). There was also no significant difference between the two treatments in the amount of pain relief obtained. Thus, a single application of cultured keratinocyte allografts to chronic venous ulcers offered no advantage for wound healing or pain relief as compared with placebo. The low efficacy and the expense of cultivating and storing allografts does not allow a single application of cultured allografts to be recommended at present as a routine treatment for venous leg ulcers.

Epidermal allografts

The epidermis is the sheet of stratified epithelial cells covering the dermal connective tissues. In addition to the epithelial cells (known as keratinocytes) there exist within the epidermis two populations of migratory leukocytes: the class II-positive, dendritic Langerhans' cell, and gamma-delta receptor-positive T lymphocytes. Allografts consisting essentially of epidermis have been in use since the introduction of "pinch" grafts by L. Reverdin in 1872. However, the capacity to culture keratinocytes in vitro to produce sheets of stratified epithelial cells has opened new possibilities. Cultured keratinocyte layers are devoid of passenger leukocytes and were initially widely believed not to be susceptible to rejection. This, however, is clearly not the case, at least in rats and humans. Nevertheless, in spite of severe acute rejection, it is interesting that cultured keratinocyte allografts are very poor at evoking antibody responses in graft recipients. Where permanent survival of the keratinocyte is required, autografts must be used, since the administration of immunosuppression to protect allografts would not be justified. Clinical application of cultured keratinocyte allografts, without immunosuppression, is limited to their use as temporary dressings (e.g., in burns) or to stimulate the recipient's own skin cells to growth (e.g., on leg ulcers). Autografts have been used for patients with extensive burns and in the treatment of chronic mastoiditis.

Fresh and cryopreserved cultured keratinocyte allografts for wound healing

The use of cultured human epithelium for skin grafting has recently been developed and used successfully to treat burns and various smaller skin defects. Cultured epidermal allografts are replaced by the recipient's own skin. The use of cultured allografts has a healing effect in chronic ulcerations and in burn wounds, probably by releasing growth factors. We have recently demonstrated that cryopreserved allografts can also induce wound healing. Both an edge effect and complete healing were found in ulcers treated with fresh or cryopreserved cultured epidermal allografts. These findings could be important in patient care, since in wound-healing problems, cryopreserved allografts could be immediately available. The cryopreservation of human keratinocyte sheets may also open new perspectives in pharmacotoxicological research on topically applied substances.

Rejection of cultured keratinocyte allografts in the rat

Rejection of cultured keratinocyte allografts in the rat

REJECTION OF CULTURED KERATINOCYTE ALLOGRAFTS IN THE RAT

We have made a detailed analysis of the fate of Langerhans cell-free cultured keratinocyte allografts in two rat strain combinations, DA-to-PVG and DA-to-LEW, and compared the results with the rejection of conventional skin allografts in these strain combinations. The cultured keratinocyte layers were grafted both to the body surface using a technique to prevent wound contraction, and to the renal subcapsular site. Histological examination of grafts was made on days 2, 7, 10, 14, and 28 after transplantation. Donor-specific anti-class I MHC monoclonal antibodies were used to verify the donor origin of the keratinocytes. We report that the keratinocyte allografts are acutely rejected but, in contrast to the conventional allografts, do not evoke alloantibody responses. Rejection of the keratinocytes at the renal subcapsular site was as rapid as that of conventional skin grafts. However, rejection of keratinocyte grafts on the body surface was delayed by a few days when compared with conventional skin grafts. Immunosuppression with cyclosporine prevented the rejection of DA keratinocyte layers placed at the renal subcapsular site of PVG rats, but rejection followed soon after cessation of cyclosporine therapy. These data suggest that rejection is a major constraint for the clinical application of cultured keratinocytes, and that autografts must be used if permanent cover is required. Moreover, the findings have interesting theoretical implications relating to the much greater vulnerability to rejection of skin grafts compared with organ grafts. Our current and previous data also suggest that class II positive dendritic cells are the major stimulus to alloantibody production after tissue transplantation.

Cultured keratinocyte grafts are recognized, but not rejected by CD8+ T cells in vivo

It has been shown in the mouse that cultured keratinocyte allografts [fully major histocompatibility complex (MHC) mismatched] survive for at least 100 days without evidence of rejection. In an attempt to analyze the immune mechanisms underlying this phenomenon we have investigated the induction of tolerance to such grafts. Primary cultures of BALB/c keratinocytes were prepared using irradiated 3T3 feeder cells, and the cultured cell sheets were grafted, using a silicone transplantation chamber, onto CBA recipients. After the cultured grafts had been in place for 4-6 weeks full-thickness tail skin from BALB/c donors was grafted onto the dorsal flank opposite the cultured graft. The median graft survival time of these full-thickness allografts was 15.5 days compared to 13 days in the control group. These data show that in the absence of Langerhans cells and MHC class II expression, keratinocytes expressing class I and minor histocompatibility antigens induce a prolonged survival of full-thickness skin allografts. The results from experiments in which T cell subsets were depleted in vivo suggest that CD8+ cells (a) recognize the class I alloantigens of the cultured graft, (b) do not reject the cultured graft and (c) do not progress further in their maturation pathway. We propose that these CD8+ T cells might have been partially primed by receiving only the first signal of activation and that they may be equivalent to "poised" cytotoxic T cells. These CD8+ cells can reject non cultured full-thickness skin grafts, and do so more effectively after removing CD4+ cells.