Aicha Demidem

Long-term survival and immunological tolerance of human epidermal allografts produced in culture

Human epidermal cells from a small skin specimen can be grown in culture into multilayered sheets suitable for the permanent coverage of large burn wounds when used as epidermal autografts. We report here on the long-term survival of such cultured epidermal sheets used as epidermal allografts (EAG) across a major histocompatibility barrier in three nonimmunosuppressed adult patients, suffering from large chronic grafted leg ulcers, where the EAG have been placed to cover the conventional split-thickness skin autograft donor site. The absence of rejection was based upon clinical, histological, and immunopathological observation of the allografted sites at various intervals after grafting of the EAG. The identity of the epidermal cells on the grafted area with cultured cells from allogeneic donor was then established after blood substance typing by indirect immunofluorescence. Furthermore, epidermal cells from cultured sheets, but not control human cells from freshly excised normal epidermis, failed to stimulate the recipient peripheral blood cells in the mixed epidermal cell lymphocyte culture reaction, a finding that is related to the complete absence of class-II-antigen-bearing cells in cultured epidermis. This absence of T cell stimulation was noted not only on the day of grafting but throughout the follow-up. Altogether, these findings show that Langerhans cell and other class-II-antigen-bearing cell-depleted cultured epidermal allografts, are tolerated in unrelated recipients. EAG may serve as a skin substitute in patients with large wounds or burns. Since EAG may be grown continuously, the coverage of burns may not then be limited by the availability of the donor site, or by the time necessary to produce epidermal tissue in cultures.

Growth and differentiation of human epidermal cultures used as auto‐ and allografts in humans

Human keratinocytes from small skin specimens were grown on mouse 3T3 cell feeder layers into epidermal sheets free from Langerhans cells and MHC class II antigen. These were found to be suitable for the permanent coverage of wounds when used as autografts or allografts. We report here the ultrastructural differentiation of this cultured epidermis after grafting onto autologous or allogeneic recipients. The cultured epidermis was a thin but multilayered Malpighian epithelium composed of keratinocytes at different stages of differentiation. The dermo‐epidermal basement membrane was newly synthesized during the first few days following transplantation onto de‐epidermized wounds. The analysis of keratins and examination of various keratinocyte membrane antigens by immunofluorescence indicated that full terminal epithelial differentiation was only achieved after in vivo transplantation of the cultured epidermis. Langerhans cells, absent in cultures, progressively colonized the grafts, while melanocytes, not detectable in sections of the cultures, were identified among the keratinocytes 2 weeks after grafting.

Quantitative HRMAS proton total correlation spectroscopy applied to cultured melanoma cells treated by chloroethyl nitrosourea: Demonstration of phospholipid metabolism alterations

Recent NMR spectroscopy developments, such as high‐resolution magic angle spinning (HRMAS) probes and correlation‐enhanced 2D sequences, now allow improved investigations of phospholipid (Plp) metabolism. Using these modalities we previously demonstrated that a mouse‐bearing melanoma tumor responded to chloroethyl nitrosourea (CENU) treatment in vivo by altering its Plp metabolism. The aims of the present study were to investigate whether HRMAS proton total correlation spectroscopy (TOCSY) could be used as a quantitative technique to probe Plp metabolism, and to determine the Plp metabolism response of cultured B16 melanoma cells to CENU treatment in vitro. The exploited TOCSY signals of Plp derivatives arose from scalar coupling among the protons of neighbor methylene groups within base headgroups (choline and ethanolamine). For strongly expressed Plp derivatives, TOCSY signals were compared to saturation recovery signals and demonstrated a linear relationship. HRMAS proton TOCSY was thus used to provide concentrations of Plp derivatives during long‐term follow‐up of CENU‐treated cell cultures. Strong Plp metabolism alteration was observed in treated cultured cells in vitro involving a down‐regulation of phosphocholine, and a dramatic and irreversible increase of phosphoethanolamine. These findings are discussed in relation to previous in vivo data, and to Plp metabolism enzymatic involvement. Magn Reson Med 49:241–248, 2003.

Cultured human epidermal allografts are not rejected for a long period

Cultured epithelia prepared from autologous epidermal cells (EC) have been used with success as autografts in patients with burns and other large wounds [3, 4, 9]. Until now, nothing has been known about the tolerance to such epithelia in allogeneic human recipients. The present study deals with the in vivo behavior of human cultured epithelia after grafting onto a genetically dissimilar recipient and, in parallel, the response of allogeneic immunocompetent cells to the stimulatory effects of cultured EC. A skin sample was obtained from a male burnt patient (AM) and epidermal epithelia were prepared according to the method described by H. Green [4]. Epidermal sheets ready for grafting were placed basal side up on Vaseline gauze rectangles. A 76-year-old female patient (CI), suffering from a large chronic leg ulcer on the left ankle, received a 120 cm 2 area splitthickness graft taken from her left forearm and placed as an autograft on the leg ulcer. After 2 days, cultured Epidermal Allografts (EAG) were placed on the 120 cm 2 donor-site wound. The gauzes were removed 5 days after grafting. The dressing was then changed daily for 10 days. Thereafter, the grafts were left exposed. When formed in culture under these conditions, the epithelial sheets ranged in thickness from six to seven cell layers. When the Vaseline gauze was removed 5 days after grafting, the EAG were visible as a very thin translucent sheet. Over the next 2 weeks, grafts began to thicken, resemble a conventional thin split-thickness graft, spread outwards and merge with each other. These clinical observations were similar to

Bystander effects are induced by CENU treatment and associated with altered protein secretory activity of treated tumor cells A relay for chemotherapy

In a previous study, it was reported that secondary untreated melanoma tumors implanted several weeks after and at distance from primary chloroethylnitrosourea (CENU)‐treated tumors underwent differentiation and growth inhibition. To see whether the primary treated tumor released soluble factors that mediated the secondary tumor response, serum transfer experiments were performed in vivo. Administration of serum from CENU‐treated tumor‐bearing donors arrested tumor proliferation, decreased vessel formation and induced tumor metabolite alterations encompassing glutathione decrease and polyunsaturated fatty acid and phosphoethanolamine increase. These changes mimicked secondary tumor phenotype. To reproduce the model in vitro, cell culture supernatant transfer experiments were performed. CENU‐treated cell cultures showed polyploidy and reactive oxygen species (ROS) production. Cell cultures challenged by a conditioned medium of CENU‐treated cells underwent growth inhibition, cytoskeleton disorders, cytokinesis retardation, metabolite alterations, glutathione decrease and phosphoethanolamine increase, without ROS elicitation. Proteomics of CENU‐treated cell conditioned media revealed altered protein secretion activity by CENU‐treated cells. Among de novo secreted proteins, the most expressed were phosphatidylethanolamine‐binding protein (PEBP), cardiovascular heat shock protein (cHsp), Rho‐associated coiled‐coil forming kinase 2 (ROCK) and actin fragments. These proteins testified of cytoskeleton disorders, growth inhibition and metabolite alterations. This article demonstrates the release by CENU‐treated tumors of growth inhibitory differentiation‐inducing soluble factors. These factors mediate remote bystander effects and attest persistent biological activity of residual tumors after chemotherapy.

The effect of anti-CD4 monoclonal antibody treatment on immunopathological changes in psoriatic skin

Recent clinical studies which showed the therapeutic effect of cyclosporin A and of anti-CD4 MoAb emphasized the role of activated CD4+ T cells infiltrating the lesional skin in the pathogenesis of psoriasis. The aim of the present study was to analyze the mode of action of anti-CD4 MoAb in 3 psoriatic patients who experienced an anti-CD4 MoAb-induced clinical improvement maximal 3-4 weeks after the onset of an 8-day therapy. We evaluated the effect of anti-CD4 MoAb treatment on the phenotype of resident and passenger inflammatory skin cells in lesional skin samples. We observed a gradual improvement of 3 out of 4 histopathologic features including parakeratosis, papillomatosis and acanthosis. In the dermis there was no modification in the density of the dermal mononuclear cell infiltrate, which consisted mainly of CD3+, CD45RO+, TCR alpha beta+, CD11a+, HLA-DR+T cells with a CD4/CD8 cell ratio of 1.5/1. Therefore as previously observed for peripheral blood mononuclear cells, the number of CD4+ T cells infiltrating the dermis remained unaffected by the treatment. In contrast, CD4 MoAb treatment was associated with drastic changes in the epidermis. These included a decrease in both CD4+ and CD8+ epidermal T cell infiltrate, diminished numbers of ICAM-1+ and HLA-DR+ keratinocytes and restored numbers of CD1a+ epidermal Langerhans cells. We conclude from this study that clinical improvement of psoriasis by anti-CD4 MoAb therapy paralleled: (1) a decrease in epidermal T cells, and (2) a down-regulation of keratinocyte activation markers (ICAM-1 and HLA-DR). These results suggest that the observed changes are secondary to down-regulation of inflammatory cytokine production by T cells in situ.

Optimal methionine-free diet duration for nitrourea treatment: a Phase I clinical trial.

In animal models, methionine (MET) restriction in association with chloroethylnitrosoureas led to a substantial improvement. On this basis, we initiated a Phase I clinical trial of dietary MET restriction in association with chloroethylnitrosourea (cystemustine) treatment for patients with recurrent glioma or metastatic melanoma. Our purpose was 1) to determine the optimal MET-free diet duration for a maximum depletion of plasma MET and 2) to evaluate the feasibility of this association. A total of 10 patients received 4 cycles of 2 wk of an association of a MET-free diet of 1, 2, 3, or 4 consecutive days and cystemustine (60 mg/m2). For each cycle, plasma MET concentrations, nutritional status (weight, albumin, prealbumin) and toxicity were measured. Conversely, fed-state concentrations of plasma MET (12 AM) were reduced by dietary MET restriction, with an optimal depletion of 41% at the 1st day of MET-free diet without effect of the extending MET-free diet period. Indeed, we demonstrated the feasibility, that is, good diet acceptability and good tolerance (nutritional status and toxicity), of the association of a MET-free diet and cystemustine treatment. Based on these results, a Phase II clinical trial has been initiated to test the activity of the association of a 1-day MET-free diet with cystemustine treatment.

Loss of allogeneic T-cell activating ability and Langerhans cell markers in human epidermal cell cultures

Human Langerhans cells are the only epidermal cells that express the T6 and HLA-DR antigens and are responsible for the in vitro allogeneic T-cell proliferative responses in the mixed skin cell lymphocyte reaction (MSLR). To investigate the presence of Langerhans cells in normal human epidermal cell cultures, epidermal cell suspensions obtained from normal human skin specimens and from the subsequent epidermal cell cultures were analyzed by indirect immunofluorescence for the presence of T6 and HLA-DR determinants. In parallel, MSLRs were conducted with suspensions of cultured epidermal cells as stimulatory cells. These studies present evidence that when human epidermal cells are grown in culture, they loose both the ability to stimulate the proliferation of allogeneic T lymphocytes in vitro and their expression of HLA-DR and T6 antigens. The T6 antigens were lost during the first 2 weeks of culture, while HLA-DR determinants were still expressed by a small number of cells and were progressively lost through duration of cultures. The loss of HLA-DR antigens closely paralleled the progressive inability of human epidermal cells in culture to stimulate allogenic T cells in MSLR.

Methionine-dependence phenotype of tumors : Metabolite profiling in a melanoma model using L-[methyl-13C]methionine and high-resolution magic angle spinning 1H-13C nuclear magnetic resonance spectroscopy

Tumors frequently have abnormal L‐methionine (Met) metabolism, the so‐called Met‐dependence phenotype that refers to the inability to proliferate in the absence of Met. However, the origin of this phenotype is still unknown and may arise from one of several pathways of Met metabolism. To help characterize the metabolic features of Met‐dependent/independent phenotypes, the fate of the methyl carbon of L‐[methyl‐13C]Met was chased in a murine model of malignant melanoma (B16‐F1) in vitro and in vivo. Growth curves under Met restriction showed that melanoma cells in vitro were Met‐independent, whereas implanted melanoma tumors in vivo were Met‐dependent. Label‐assisted high‐resolution magic angle spinning 1H‐13C NMR spectroscopy metabolite profiling showed that, in vitro, creatine and phosphatidylcholine 13C‐enrichments were poor, but S‐adenosyl‐Met and posttranslationally N‐methylated protein signals were strong. In contrast, in vivo, creatine and phosphatidylcholine enrichments were strong but S‐adenosyl‐Met and N‐methylated protein signals were poor. In addition, in vivo, transsulfuration was very efficient, consumed one‐carbon units originating from the methyl carbon of Met, and yielded taurine labeling. From these data, the Met‐dependent/independent phenotypes appear closely related to the source of one‐carbon units. Thus, L‐[methyl‐13C]Met‐assisted NMR spectroscopy metabolite profiling allowed the discrimination between Met‐dependence and Met‐independence and provided novel mechanistic information on their origin. Magn Reson Med, 2006.

Dissociation Of Antigenicity And Immunogenicity Of Neonatal Epidermal Allografts In The Mouse

Cultured neonatal epidermal cells or sheets have been grafted successfully onto allogeneic recipients across an MHC barrier, while genetically identical skin allografts were rejected. Induction of specific allosensitization by prior priming with allogeneic spleen cells or allogeneic skin grafts did not prejudice the survival of subsequent cultured epidermal allografts. When cultured epidermis and adult skin of the same genotype were grafted simultaneously, as double grafts, cultured epidermis survived despite the presence of an ongoing allograft rejection reaction in the host. Furthermore, pretreatment of the recipients with cultured epidermis failed to protect against rejection of subsequent allogeneic adult skin grafts of the same genetic origin. These data indicate that cultured epidermis is neither immunogenic nor antigenic in allogeneic host. In companion experiments it was determined that neonatal (noncultured) epidermal allografts also survived indefinitely, implying that neonatal epidermis lacks antigenicity. However, in contrast to cultured epidermis, neonatal epidermal allografts evoked specific systemic immunity in their recipients, since they rejected a subsequent allogeneic adult skin grafts in accelerated fashion. These data demonstrate that in neonatal epidermis antigenicity can be dissociated from immunogenicity.