Wound healing of human skin transplanted onto the nude mouse. II. An immunohistological and ultrastructural study of the epidermal basement membrane zone reconstruction and connective tissue reorganization

The reconstruction of human epidermis during healing of human skin wounded after grafting onto the nude mouse was described in a previous paper (M. Démarchez, P. Sengel, and M. Pruniéras, 1986, Dev. Biol. 113, 90-96). The regeneration of the epidermal basement membrane zone (BMZ) and the reorganization of the connective tissue are the subjects of the present study. They were investigated by two complementary methods: electron microscopy to analyze the BMZ reorganization, and indirect immunofluorescence with species-specific and cross-reacting antibodies directed against laminin, bullous pemphigoid antigen, mouse or human collagens of types I or IV, human elastic fibers, fibronectin, fibrin, actin, and human vimentin, to examine the species origin and distribution of BMZ and connective tissue components during the regeneration process. It is reported that grafted human skin preserves its own immunological markers not only in the epidermis but also in the BMZ and dermis as well, and that, after injury, its regeneration proceeds according to the following sequence of overlapping events: production of a mouse granulation tissue; reepidermization by human cells; reconstruction of a BMZ with human characteristics; formation of a human neodermis. It is concluded that human skin grafted onto the nude mouse is able to regenerate its three structural compartments, namely, the epidermis, BMZ, and dermis. Interestingly, it appeared, also, that the connective tissue regeneration would be a two-step mechanism including the sequential formation of two tissues of distinct sources, namely, a granulation tissue and a neodermis.     

The effect of graded doses of fission neutrons or X rays on the stromal compartment of the thymus in mice

The effect of irradiation on the supportive role of the thymic stroma in T cell differentiation was investigated in a transplantation model using athymic nude mice and transplanted irradiated thymuses. In this model, neonatal CBA/H mice were exposed to graded doses of whole-body irradiation with fast fission neutrons of 1 MeV mean energy or 300 kVp X rays. The doses used varied from 2.75 up to 6.88 Gy fission neutrons and from 6.00 up to 15.00 Gy X rays at center-line dose rates of 0.10 and 0.30 Gy/min, respectively. Subsequently, the thymus was excised and a thymus lobe was transplanted under the kidney capsule of H-2 compatible nude mice. One and two months after transplantation, the T cell composition of the thymic transplant was investigated using immunohistology with monoclonal antibodies directed to the cell surface differentiation antigens Thy-1, Lyt-1, Lyt-2, MT-4, and T-200. Furthermore, the stromal cell composition of the thymic transplant was investigated with monoclonal antibodies directed to MHC antigens and with monoclonal antibodies defining different subsets of thymic stromal cells. To investigate the reconstitution capacity of the thymic transplant, the peripheral T cell number was measured using flow cytofluorometric analysis of nude spleen cells with the monoclonal antibodies anti-Thy-1, anti-Lyt-2, and anti-MT-4. The results of this investigation show that a neonatal thymus grafted in a nude mouse has a similar stromal and T cell composition as that of a normal thymus in situ. In addition, grafting of such a thymus results in a significant increase of the peripheral T cell number. Irradiation of the graft prior to transplantation has no effects on the stromal and T cell composition but the graft size decreases. This reduction of size shows a linear dose-response curve after neutron irradiation. The X-ray curve is linear for doses in excess of 6.00 Gy. The RBE for fission neutrons for the reduction of the relative thymic graft size to 10% was equal to 2.1. Furthermore, the peripheral T cell number decreases with increasing doses of irradiation given to the graft prior to transplantation. The present data indicate that the regenerative potential of thymic stromal cells is radiosensitive and is characterized by D0 values equal to 2.45 and 3.68 Gy for neutrons and X rays, respectively. In contrast, the ability of the thymic stromal cells to support T cell maturation is highly radioresistant.     

Immunoreactivity of six monoclonal antibodies directed against 1,25-dihydroxyvitamin-D3 receptors in human skin

Using confocal laser scanning microscopy, we tested the suitability of five monoclonal mouse antibodies (IVA7E7, IVB12G12, IVG9C11, VD2F12, and VIIID8C12) that had been raised against different domains of the porcine intestinal 1,25-dihydroxyvitamin-D₃ receptor (VDR), for the immunohistological detection of VDR in human skin. The VDR immunoreactivity of these antibodies was compared with the well-characterized VDR-staining pattern of the mouse monoclonal antibody 9A7 raised against chick intestinal VDR. All six antibodies revealed strong nuclear and qualitatively similar immunoreactivity in all cell layers of the viable epidermis. Our data demonstrate that the five mouse monoclonal antibodies are suitable for immunohistochemical detection of VDR in frozen sections. These antibodies show comparable staining patterns in human skin even though they had been raised against different functional domains of the 1,25-dihydroxyvitamin-D₃ receptor.     

Selective toxicity of neocarzinostatin-monoclonal antibody conjugates to the antigen-bearing human melanoma cell line in vitro

Summary Monoclonal antibodies (IgG₁) against high molecular weight antigen A-1-43 on human melanoma cell line A-375 were successfully linked to the anti-tumour protein neocarzinostatin (NCS) using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). The conjugate retained both the reactivity of the antibody and the toxicity of the drug. The antigen-bearing cell line A-375, antigen-lacking cell line MeWo and normal skin fibroblasts were exposed to NCS-monoclonal antibody conjugates. As negative control, cells were also treated with free NCS and NCS coupled to normal mouse IgG₁ antibodies. Inhibition of ³H-thymidine uptake after treatment was used to measure the biological activity of the cytotoxic drug complex or substance, respectively.Comparing the inhibition dose for 50% uptake (ID₅₀) it was found that the monoclonal antibody-drug complex is about 100 times more toxic for the antigen-bearing cell line than free NCS or normal mouse IgG₁-NCS. This high toxicity is due to a local increase of drug concentration on these cells. With the two cell lines lacking the appropriate antigen no significant differences in the ID₅₀ values were observed. A selectivity factor of 40–50 was obtained by comparing the cytotoxic effect of the monoclonal antibody-NCS conjugate upon the antigen-bearing as opposed to the antigen-lacking cell type. These data demonstrate, that the toxicity of NCS can be directed by monoclonal antibodies to human tumour cells carrying the corresponding surface antigen.     

Blood group substances, T6 antigen and heterochromatin pattern as species markers in the nude mouse/human skin model

Antibodies to blood group antigens and to human T6 antigen and the DNA-specific fluorochrome D287/170 were applied to human skin transplanted to nude mice and to the surrounding murine skin. It was found that the two epithelia maintained their characteristic, different blood group B antigen distributions and heterochromatin patterns. Furthermore, it was shown that human Langerhans' cells persisted in the grafted epidermis. During an observation period of until 48 weeks after transplantation only one of 32 grafts showed signs of invasion by murine epidermis.     

Immunohistochemical distribution of neuropeptide Y,peptide YY,pancreatic polypeptide-like immunoreactivity and their receptors in the epidermal skin of healthy women

Few studies have suggested that neuropeptide Y (NPY) could play an important role in skin functions. However, the expression of NPY, the related peptides, peptide YY (PYY) and pancreatic polypeptide (PP) and their receptors have not been investigated in human skin. Using specific antisera directed against NPY, PYY, PP and the Y₁, Y₂, Y₄ and Y₅ receptor subtypes, we investigated here the expression of these markers. NPY-like immunoreactivity (ir) in the epidermal skin could not be detected. For the first time we report the presence of positive PP-like ir immunofluorescent signals in epidermal cells, i.e. keratinocytes of skin from three areas (abdomen, breast and face) obtained as surgical left-overs. The immunofluorescent signal of PP-like ir varies from very low to high level in all three areas. In contrast, PYY-like ir is only expressed in some cells and with varied level of intensity. Furthermore and for the first time we observed specific Y₁ and Y₄ receptor-like ir in all epidermal layers, while the Y₂ and Y₅ subtypes were absent. Interestingly, as seen in human epidermis, in Episkin, a reconstituted human epidermal layer, we detected the presence of PP-like as well as Y₁-like and Y₄-like ir. These data have shown the presence and distribution of PYY, PP and Y₁ and Y₄ receptors in the human skin and Episkin, suggesting possible novel roles of NPY related peptides and their receptors in skin homeostasis.     

Distribution of intact and Fab1 2 fragments of anti-human chorionic gonadotrophin antibodies in nude mice bearing human choriocarcinoma xenografts

Summary A direct comparative study of the distribution of intact and Fab¹ ₂ fragments of mouse monoclonal anti-human chorionic gonadotrophin antibody, W14A, in nude mice bearing a human choriocarcinoma xenograft, indicates that the tumour:blood ratio is generally improved with fragments. Some parameters which may determine the distribution are outlined.     

Comparison of features of carcinogen-transformed human cells in vitro with sarcoma-derived cells

To define characteristics of chemically transformed phenotypes during and after progression to neoplasia and to assess their relationship to those phenotypes expressed by surgically removed sarcoma lesions, we compared the characteristics in the following manner. We investigated: (1) alterations in growth patterns; (2) anchorage-independent growth; (3) reactivity with monoclonal antibodies directed against surface antigen; (4) invasiveness in embryonic chick skin; (5) tumorigenicity in nude mice; and (6) karyology. Fifty different sarcoma cell lines were examined which exhibited different rates and absolute numbers of population doublings. With one exception, all sarcoma cell lines exhibited a finite life span ranging from 60 to 100 population doublings. Populations of these cells that exhibited anchorage-independent growth in soft agar also reacted positively with a monoclonal antibody (MoAb) 345.134S directed against a 115K-GP cell surface glycoprotein. Similarly, chemically transformed cells that grew in soft agar also reacted with the MoAb 345.134S, whereas cells with an inability to grow in soft agar did not. Cell lines established from human sarcoma and from chemically transformed human fibroblasts that reacted positively with the MoAb 345.134S were invasive for embryonic chick skin and formed tumors in nude mice. The selection medium used during culture of the carcinogen-treated cells resulted in the appearance of an altered phenotype that after at least 16 population doublings exhibited characteristics common to those cells derived from human sarcomas.     

Modified distribution of epidermal glycoproteins in the nude mouse

The nude mouse is an athymic mutant whose immunological deficiency has been exploited for transplantation of normal and diseased xenogeneic tissue. Histologically, its skin has no unusual features apart from the absence of hair. We report here a biochemical study of its epidermis, with comparison to the hairless mouse (which is devoid of hair but otherwise functionally normal). The epidermal glycoproteins were probed with the lectin, concanavalin A (Con A). Fluorescein isothiocyanate (FITC)-Con A overlays of cryostat skin sections gave a similar fluorescent pattern for both mouse strains: all the viable epidermal cell layers were labeled but not the stratum corneum. In contrast, when different populations of keratinocytes that were separated on Percoll gradients were analyzed by gel electrophoresis, and the gels then overlaid with iodinated Con A, all the epidermal layers, including the stratum corneum, were labeled. For all the epidermal cell layers there are substantial differences between the two mouse strains. We observe changes in the glycoprotein distribution with the stage of differentiation. Comparison with our earlier data for human epidermis indicates that the discrepancies between the nude mouse and the hairless mouse are much greater than those between the latter and man. The most striking difference is the absence in the stratum corneum of the nude mouse of a 40 K glycoprotein which is the dominant feature for the hairless mouse and for man. The gel patterns point to functional discrepancies in the epidermis of the nude mouse, particularly in the stratum corneum, not evident histologically or with FITC-Con A.     

Prolongation of murine skin allograft survival by immunologic manipulation with anti-interleukin 2 receptor antibody

Administration of a rat monoclonal antibody (M7/20) directed against the murine interleukin 2 (IL 2) receptor in combination with sublethal x-irradiation of the recipient significantly enhanced the survival of skin allografts, both when the grafts were MHC disparate from the hosts and when only minor histocompatibility differences were present compared with untreated controls. No prolongation in graft survival was seen with either treatment alone at the dose employed. M7/20 and x-ray-treated allograft recipients also displayed significantly decreased alloantigen-specific reactivity against donor-strain spleen cells in both delayed-type hypersensitivity and cytotoxicity assays. Thus, such combination treatment reduces expression of host immune reactivity against graft determinants by several criteria. This work provides additional evidence that monoclonal antibodies directed against the IL 2 receptor may be useful in clinical transplantation.     

Effect of Dietary Selenium and Cancer Cell Xenograft on Peripheral T and B Lymphocytes in Adult Nude Mice

Selenium (Se) is known to regulate tumorigenesis and immunity at the nutritional and supranutritional levels. Because the immune system provides critical defenses against cancer and the athymic, immune-deficient NU/J nude mice are known to gradually develop CD8⁺ and CD4⁺ T cells, we investigated whether B and T cell maturation could be modulated by dietary Se and by tumorigenesis in nude mice. Fifteen homozygous nude mice were fed a Se-deficient, Torula yeast basal diet alone (Se−) or supplemented with 0.15 (Se+) or 1.0 (Se++) mg Se/kg (as Na₂SeO₄) for 6 months, followed by a 7-week time course of PC-3 prostate cancer cell xenograft (2 × 10⁶ cells/site, 2 sites/mouse). Here, we show that peripheral B cell levels decreased in nude mice fed the Se −  or Se++ diet and the CD4⁺ T cell levels increased in mice fed the Se++ diet. During the PC-3 cell tumorigenesis, dietary Se status did not affect peripheral CD4⁺ or CD8⁺ T cells in nude mice whereas mice fed with the Se++ diet appeared to exhibit greater peripheral CD25⁺CD4⁺ T cells on day 9. Dietary Se status did not affect spleen weight in nude mice 7 weeks after the xenograft. Spleen weight was associated with frequency of peripheral CD4⁺, but not CD8⁺ T cells. Taken together, dietary Se at the nutritional and supranutritional levels regulates peripheral B and T cells in adult nude mice before and after xenograft with PC-3 prostate cancer cells.     

Indomethacin inhibition of cell proliferation induced by the phorbolester TPA is reversed by prostaglandin E2 in mouse epidermis in vivo

The proliferative response of mouse epidermis to the phorbolester TPA (10 nmoles) in vivo is completely inhibited by a single topical application of indomethacin one hour before TPA. DNA labeling in normal mouse epidermis is not significantly depressed by the drug. The inhibition can be reversed by applying prostaglandin E₂ (>3 nmoles) simultaneously with TPA, whereas prostaglandin F_2α (100 nmoles) is ineffective. The indomethacin-sensitive event is restricted to the first hour after phorbol ester treatment. TPA-induced skin inflammation is not influenced by the drug. It is proposed that prostaglandin E₂, or a closely related compound, mediates the mitogenic effect of TPA in mouse skin.     

The number and distribution of blood dendritic cells in the epidermis and dermis of healthy human subjects

Human blood dendritic cells (BDC) can be divided into three subsets: plasmacytoid DC (PDC) and two myeloid subsets--MDC1 and MDC2. Several studies revealed the presence of both MDC and PDC in blood of healthy subjects, however no precise literature data exist on the number and distribution of BDC in the skin. The aim of our study was to assess the number and distribution of BDC and their subtypes in the healthy skin. The-study included 30 healthy volunteers (age 18-51). Punch biopsies were taken from the buttock skin from each subject, and immunofluorescent staining was performed using monoclonal mouse IgG1 antibodies directed against BDCA-1, BDCA-2, BDCA-3 and BDC-4. The BDC were present both in the epidermis and dermis. PDC were detected mainly in the dermis (mean 1.2 cells per field). Myeloid subtypes were observed mainly in the middle layers of the epidermis and in the upper part of the dermis (mean 1.8 cells per field). The detection of blood dendritic cells in the skin proves their role in immune cutaneous surveillance.     

Carcinogen metabolism in human skin grafted onto athymic nude mice: a model system for the study of human skin carcinogenesis

Human skin grafted onto athymic nude mice maintains its major histological features and may provide a useful system with which to assess the carcinogen interaction with human skin. Significant differences were observed in basal levels of cytochrome P-450 and cytochrome P-448-dependent monooxygenase activities between human grafted and nude mouse epidermis. Topical application of crude coal tar (CCT) to human skin transplanted onto nude mice resulted in 3.9 & 3.5; 3.2 & 2.9 and 1.1 & 1.2 fold increases in mouse and human epidermal aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin deethylase (ERD) and ethoxycoumarin deethylase (ECD) activities, respectively. CCT applied topically to mouse skin resulted in 27.8 & 6.4; 12.8 & 3.3 and 1.7 & 2.6 fold increases in mouse and human epidermal AHH, ERD and ECD activities, respectively. Topical application of coal tar either onto human transplanted skin or to mouse skin also resulted in substantial induction of hepatic and pulmonary AHH and ERD activities. These studies indicate that human skin grafted onto nude mice preserves its metabolic capacity and offers a useful model system with which to assess the effects of polycyclic aromatic hydrocarbons and CCT on cutaneous xenobiotic metabolism in the human population.     

Localization of calmodulin positive immunoreactivity in the surface epidermis of the brown trout,Salmo trutta

Summary Calmodulin is a Ca²⁺-dependent modulatory protein which is required in the general regulation of a large number of key processes of cellular metabolism. In the present study, the localization of calmodulin positive immunoreactivity in the epidermis of the brown trout, Salmo trutta was investigated using a specific mouse monoclonal antibody to calmodulin of IgG₂ class. The immunoreaction was found only in the superficial epithelial cells that constitute the main histological site for the production of calmodulin positive substances. Because of its distribution, this protein might have a physiological significance in the activation of the microvillar skeleton and in the control of the permeability of the skin epithelium.     

Beneficial Effect of Insulin Treatment on Islet Transplantation Outcomes in Akita Mice

Islet transplantation is a promising potential therapy for patients with type 1 diabetes. The outcome of islet transplantation depends on the transplantation of a sufficient amount of β-cell mass. However, the initial loss of islets after transplantation is problematic. We hypothesized the hyperglycemic status of the recipient may negatively affect graft survival. Therefore, in the present study, we evaluated the effect of insulin treatment on islet transplantation involving a suboptimal amount of islets in Akita mice, which is a diabetes model mouse with an Insulin 2 gene missense mutation. Fifty islets were transplanted under the left kidney capsule of the recipient mouse with or without insulin treatment. For insulin treatment, sustained-release insulin implants were implanted subcutaneously into recipient mice 2 weeks before transplantation and maintained for 4 weeks. Islet transplantation without insulin treatment did not reverse hyperglycemia. In contrast, the group that received transplants in combination with insulin treatment exhibited improved fasting blood glucose levels until 18 weeks after transplantation, even after insulin treatment was discontinued. The group that underwent islet transplantation in combination with insulin treatment had better glucose tolerance than the group that did not undergo insulin treatment. Insulin treatment improved graft survival from the acute phase (i.e., 1 day after transplantation) to the chronic phase (i.e., 18 weeks after transplantation). Islet apoptosis increased with increasing glucose concentration in the medium or blood in both the in vitro culture and in vivo transplantation experiments. Expression profile analysis of grafts indicated that genes related to immune response, chemotaxis, and inflammatory response were specifically upregulated when islets were transplanted into mice with hyperglycemia compared to those with normoglycemia. Thus, the results demonstrate that insulin treatment protects islets from the initial rapid loss that is usually observed after transplantation and positively affects the outcome of islet transplantation in Akita mice.     

Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen

Summary Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG_2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 × 10⁶ cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotypematched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-M _r MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.     

Both VH and VL regions contribute to the antigenicity of anti-idiotypic antibody that mimics melanoma associated ganglioside GM3

Previously we developed a murine monoclonal anti-idiotype (antiid) antibody (4C10) that mimics the melanoma-associated ganglioside antigen GM₃, that is, it carries the internal image of GM₃. 4C10 was made against the human monoclonal antibody (HuMAb) L612, which reacts with several types of human cancer cells, including melanoma and breast cancer. To reduce mouse components of 4C10, the constant region was replaced by a human constant domain to form the murine/human chimeric anti-id antibody TVE-1. In the present study, we sought to determine which chain (V_H or V_L) of the anti-id is responsible for the antigenicity of GM₃. The TVE-1 V_H and V_L expression vectors were simultaneously transfected with either the V_H or V_L expression vector of a murine-human chimeric IgG antidansyl haptenic antibody, resulting in the construction of three different combinations of V_H and V_L chimeric antibodies. These IgG molecules were produced from the transfectomas, and their reactivity to HuMAb L612 was tested. Neither of the IgG proteins that had cross-combined the V_H-V_L pair showed positive results, suggesting that both heavy and light chains are required to express the antigenicity. The in vivo antigenicity of this chimeric anti-id was confirmed by skin tests in melanoma patients receiving active specific immuntherapy.     

Circadian rhythms of DNA synthesis in nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) occurs frequently in southern China. The circadian rhythm of DNA synthesis of a poorly differentiated NPC human cell line (CNE2) was investigated as an experimental prerequisite for designing chrono-chemotherapy schedules for patients with this disease. Twenty-two nude mice with BALB/c background were synchronized alternatively in 12 h of light and 12 h of darkness (LD12:12) for at least 3 wk prior to the transplantation of a CNE2 tumor fragment into each flank (area of ～2×2 mm²). Ten days later, a tumor sample (area of ～5 mm²) was obtained at 3, 9, 15, and 21 h after light onset (HALO) alternatively from different sites in each mouse. Single-cell suspensions were prepared and stained with propidium iodide. Cellular DNA content was measured with flow cytometry. Data were analyzed by ANOVA and cosinor methods. The average proportion of tumor cells in G₁, S or G₂-M phase varied according to circadian time with statistical significance. The maximum occurred at 9 HALO for G1, 2 HALO for S and 21 HALO for G₂-M phase cells. The approximate average distribution patterns of G₁ and G₂-M phases of cosine curve was 24 h. This was not the case for S-phase cells, which displayed a bimodal temporal pattern. Inter-individual variability in peak time was large, possibly due to relatively sparse sampling time. Nevertheless, no more than 6% of the time series displayed a maximum at 3 HALO for G₁, 21 HALO for S and 15 HALO for G₂-M. The cell cycle distribution of this human NPC cell line displayed circadian regulation following implantation into nude mice. The mechanisms involved in this rhythm and its relevance to the chrono-chemotherapy of patients deserve further investigation.     

Monoclonal antibodies to feline sarcoma virus gag and fes gene translational products

A series of hybridomas have been isolated which produce monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strains of FeSV. Within these are representatives of several immunoglobulin classes including IgG₁, IgG_2a, IgG_2b, IgG_2c, and IgM. Antibody produced by one hybridoma recognizes immunologic determinants localized within an FeLV gag gene structural component (p15) common to polyproteins encoded by all three FeSV isolates whereas antibody produced by a second is specific for p30 determinants unique to P170^gag-fms. Additional hybridomas secrete antibody directed against v-fes-encoded determinants common to the Gardner and Snyder-Theilen FeSV-encoded polyproteins. GA P110^gag-fes and ST P85^gag-fes immunoprecipitated by antibody directed against p15 exhibit tyrosine-specific protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components.