Correlation of individual papilloma latency time with DNA adducts, 8-hydroxy-2'-deoxyguanosine, and the rate of DNA synthesis in the epidermis of mice treated with 7,12-dimethylbenz[alpha]anthracene.

The question was addressed whether the risk of cancer of an individual in a heterogeneous population can be predicted on the basis of measurable biochemical and biological variables postulated to be associated with the process of chemical carcinogenesis. Using the skin tumor model with outbred male NMRI mice, the latency time for the appearance of a papilloma was used as an indicator of the individual cancer risk. Starting at 8 weeks of age, a group of 29 mice was treated twice weekly with 20 nmol of 7,12-dimethylbenz[alpha]anthracene (DMBA) applied to back skin. The individual papilloma latency time ranged from 13.5 to 25 weeks of treatment. Two weeks after the appearance of the first papilloma in each mouse, an osmotic minipump delivering 5-bromo-2'-deoxyuridine was s.c. implanted and the mouse was killed 24 hr later. Levels of DMBA-DNA adducts, of 8-hydroxy-2'-deoxyguanosine, and various measures of the kinetics of cell division were determined in the epidermis of the treated skin area. The levels of 8-hydroxy-2'-deoxyguanosine and the fraction of cells in DNA replication (labeling index for the incorporation of 5-bromo-2'-deoxyuridine) were significantly higher in those mice that showed short latency times. On the other hand, the levels of DMBA-DNA adducts were lowest in animals with short latency times. The latter finding was rather unexpected but can be explained as a consequence of the inverse correlation seen for the labeling index: with each round of cell division, the adduct concentration is reduced to 50% because the new DNA strand is free of DMBA adducts until the next treatment. Under the conditions of this bioassay, therefore, oxygen radical-related genotoxicity and the rate of cell division, rather than levels of carcinogen-DNA adducts, were found to be of predictive value as indicators of an individual cancer risk.

Platelet-derived growth factor BB induces functional vascular anastomoses in vivo.

Neovascularization that generates collateral blood flow can limit the extent of tissue damage after acute ischemia caused by occlusion of the primary blood supply. The neovascular response stimulated by the BB homodimeric form of recombinant platelet-derived growth factor (PDGF-BB) was evaluated for its capacity to protect tissue from necrosis in a rat skin flap model of acutely induced ischemia. Complete survival of the tissue ensued, when the original nutritive blood supply was occluded, as early as 5 days after local PDGF-BB application, and the presence of a patent vasculature was evident compared to control flaps. To further evaluate the vascular regenerative response, PDGF-BB was injected into the muscle/connective tissue bed between the separated ends of a divided femoral artery in rats. A patent new vessel that functionally reconnected the ends of the divided artery within the original 3- to 4-mm gap was regenerated 3 weeks later in all PDGF-BB-treated limbs. In contrast, none of the paired control limbs, which received vehicle with an inactive variant of PDGF-BB, had vessel regrowth (P < 0.001). The absence of a sustained inflammatory response and granulation tissue suggests locally delivered PDGF-BB may directly stimulate the angiogenic phenotype in endothelial cells. These findings indicate that PDGF-BB can generate functional new blood vessels and nonsurgically anastomose severed vessels in vivo. This study supports the possibility of a therapeutic modality for the salvage of ischemic tissue through exogenous cytokine-induced vascular reconnection.

A transgenic mouse model with an inducible skin blistering disease phenotype

One of the current limitations of gene transfer protocols involving mammalian genomes is the lack of spatial and temporal control over the desired gene manipulation. Starting from a human keratin gene showing a complex regulation as a template, we identified regulatory sequences that confer inducible gene expression in a subpopulation of keratinocytes in stratified epithelia of adult transgenic mice. We used this cassette to produce transgenic mice with an inducible skin blistering phenotype mimicking a form of epidermolytic hyperkeratosis, a keratin gene disorder. Upon induction by topical application of a phorbol ester, the mutant keratin transgene product accumulates in the differentiating layers of epidermis, leading to keratinocyte lysis after application of mechanical trauma. This mouse model will allow for a better understanding of the complex relationship between keratin mutation, keratinocyte cytoarchitecture, and hypersensitivity to trauma. The development of an inducible expression vector showing an exquisite cellular specificity has important implications for manipulating genes in a spatially and temporally controlled fashion in transgenic mice, and for the design of gene therapy strategies using skin as a tissue source for the controlled delivery of foreign substances.

A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice.

Previously, a hypomorphic mutation in CD18 was generated by gene targeting, with homozygous mice displaying increased circulating neutrophil counts, defects in the response to chemically induced peritonitis, and delays in transplantation rejection. When this mutation was backcrossed onto the PL/J inbred strain, virtually all homozygous mice developed a chronic inflammatory skin disease with a mean age of onset of 11 weeks after birth. The disease was characterized by erythema, hair loss, and the development of scales and crusts. The histopathology revealed hyperplasia of the epidermis, subcorneal microabscesses, orthohyperkeratosis, parakeratosis, and lymphocyte exocytosis, which are features in common with human psoriasis and other hyperproliferative inflammatory skin disorders. Repetitive cultures failed to demonstrate bacterial or fungal organisms potentially involved in the pathogenesis of this disease, and the dermatitis resolved rapidly after subcutaneous administration of dexamethasone. Homozygous mutant mice on a (PL/J x C57BL/6J)F1 background did not develop the disease and backcross experiments suggest that a small number of genes (perhaps as few as one), in addition to CD18, determine susceptibility to the disorder. This phenotype provides a model for inflammatory skin disorders, may have general relevance to polygenic human inflammatory diseases, and should help to identify genes that interact with the beta2 integrins in inflammatory processes.

A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels

P-glycoprotein (MDR-1) is a well-known transporter that mediates efflux of chemotherapeutic agents from the intracellular milieu and thereby contributes to drug resistance. MDR-1 also is expressed by nonmalignant cells, including leukocytes, but physiologic functions for MDR-1 are poorly defined. Using an initial screening assay that included >100 mAbs, we observed that neutralizing mAbs MRK16, UIC2, and 4E3 against MDR-1 specifically and potently blocked basal-to-apical transendothelial migration of mononuclear phagocytes, a process that may mimic their migration into lymphatic vessels. Antagonists of MDR-1 then were used in a model of authentic lymphatic clearance. In this model, antigen-presenting dendritic cells (DC) migrate out of explants of cultured human skin and into the culture medium via dermal lymphatic vessels. DC and T cells derived from skin expressed MDR-1 on their surfaces. Addition of anti-MDR-1 mAbs MRK16, UIC2, or the MDR-1 antagonist verapamil to skin explants at the onset of culture inhibited the appearance of DC, and accompanying T cells, in the culture medium by approximately 70%. Isotype-matched control mAbs against other DC molecules including CD18, CD31, and major histocompatibility complex I did not block. In the presence of MDR-1 antagonists, epidermal DC were retained in the epidermis, in contrast to control conditions. In summary, this work identifies a physiologic function for MDR-1 during the mobilization of DC and begins to elucidate how these critical antigen-presenting cells migrate from the periphery to lymph nodes to initiate T lymphocyte-mediated immunity.

Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1β converting enzyme cDNA

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1β converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-γ. First, we compared the levels of IFN-γ secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-γ production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-γ levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8+ T cell-mediated and partially IFN-γ-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8+ T cells and IFN-γ-dependent pathways.

Collagenase-3 Induction in Rat Lung Fibroblasts Requires the Combined Effects of Tumor Necrosis Factor-α and 12-Lipoxygenase Metabolites: A Model of Macrophage-induced, Fibroblast-driven Extracellular Matrix Remodeling during Inflammatory Lung Injury

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-α (TNF-α) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-α and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.

Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair

Nerve growth factor (NGF) is a polypeptide which, in addition to its effect on nerve cells, is believed to play a role in inflammatory responses and in tissue repair. Because fibroblasts represent the main target and effector cells in these processes, to investigate whether NGF is involved in lung and skin tissue repair, we studied the effect of NGF on fibroblast migration, proliferation, collagen metabolism, modulation into myofibroblasts, and contraction of collagen gel. Both skin and lung fibroblasts were found to produce NGF and to express tyrosine kinase receptor (trkA) under basal conditions, whereas the low-affinity p75 receptor was expressed only after prolonged NGF exposure. NGF significantly induced skin and lung fibroblast migration in an in vitro model of wounded fibroblast and skin migration in Boyden chambers. Nevertheless NGF did not influence either skin or lung fibroblast proliferation, collagen production, or metalloproteinase production or activation. In contrast, culture of both lung and skin fibroblasts with NGF modulated their phenotype into myofibroblasts. Moreover, addition of NGF to both fibroblast types embedded in collagen gel increased their contraction. Fibrotic human lung or skin tissues displayed immunoreactivity for NGF, trkA, and p75. These data show a direct pro-fibrogenic effect of NGF on skin and lung fibroblasts and therefore indicate a role for NGF in tissue repair and fibrosis.

Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro

Sun exposure has been clearly implicated in premature skin aging and neoplastic development. These features are exacerbated in patients with xeroderma pigmentosum (XP), a hereditary disease, the biochemical hallmark of which is a severe deficiency in the nucleotide excision repair of UV-induced DNA lesions. To develop an organotypic model of DNA repair deficiency, we have cultured several strains of primary XP keratinocytes and XP fibroblasts from skin biopsies of XP patients. XP skin comprising both a full-thickness epidermis and a dermal equivalent was succesfully reconstructed in vitro. Satisfactory features of stratification were obtained, but the expression of epidermal differentiation products, such as keratin K10 and loricrin, was delayed and reduced. In addition, the proliferation of XP keratinocytes was more rapid than that of normal keratinocytes. Moreover, increased deposition of cell attachment proteins, α-6 and β-1 integrins, was observed in the basement membrane zone, and β-1 integrin subunit, the expression of which is normally confined to basal keratinocytes, extended into several suprabasal cell layers. Most strikingly, the in vitro reconstructed XP skin displayed numerous proliferative epidermal invasions within dermal equivalents. Epidermal invasion and higher proliferation rate are reminiscent of early steps of neoplasia. Compared with normal skin, the DNA repair deficiency of in vitro reconstructed XP skin was documented by long-lasting persistence of UVB-induced DNA damage in all epidermal layers, including the basal layer from which carcinoma develops. The availability of in vitro reconstructed XP skin provides opportunities for research in the fields of photoaging, photocarcinogenesis, and tissue therapy.

Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors.

Particle-mediated (gene gun) in vivo delivery of the murine interleukin 12 (IL-12) gene in an expression plasmid was evaluated for antitumor activity. Transfer of IL-12 cDNA into epidermal cells overlying an implanted intradermal tumor resulted in detectable levels (266.0 +/- 27.8 pg) of the transgenic protein at the skin tissue treatment site. Despite these low levels of transgenic IL-12, complete regression of established tumors (0.4-0.8 cm in diameter) was achieved in mice bearing Renca, MethA, SA-1, or L5178Y syngeneic tumors. Only one to four treatments with IL-12 cDNA-coated particles, starting on day 7 after tumor cell implantation, were required to achieve complete tumor regression. This antitumor effect was CD8+ T cell-dependent and led to the generation of tumor-specific immunological memory. By using a metastatic P815 tumor model, we further showed that a delivery of IL-12 cDNA into the skin overlying an advanced intradermal tumor, followed by tumor excision and three additional IL-12 gene transfections, could significantly inhibit systemic metastases, resulting in extended survival of test mice. These results suggest that gene gun-mediated in vivo delivery of IL-12 cDNA should be further developed for potential clinical testing as an approach for human cancer gene therapy.

Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.

High levels of the p53 protein are immunohistochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, approximately 70% of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.

Correlation of peptide specificity and IgG subclass with pathogenic and nonpathogenic autoantibodies in pemphigus vulgaris: a model for autoimmunity.

Pemphigus vulgaris (PV) is a rare, potentially fatal, autoimmune disease that affects the skin and mucous membranes. The PV antigen (PVA) has been characterized as desmoglein 3. PV patients carry HLA-DR4- or HLA-DR6-bearing extended haplotypes. We recently demonstrated that patients with active disease have high titers of PV autoantibodies of the IgG1 and IgG4 subclasses. Patients in remission, healthy unaffected relatives, and some MHC-matched normal individuals have low levels of PV autoantibodies, which are IgG1 only. Furthermore, intraperitoneal injection of IgG from patients with active disease caused clinical disease in mice, but IgG from patients in remission, healthy relatives, or MHC-matched normal individuals did not. We prepared 12 peptides of 30 amino acids each (peptides Bos 1-12) spanning the extracellular domain of PVA. Patients with active disease recognize peptides Bos 1 and Bos 6 with high titers of IgG1 and IgG4 autoantibodies. Patients in remission have IgG1 autoantibodies to peptide Bos 1 only, in statistically significantly lower titers (P < 0.01). They no longer have IgG4 subclass autoantibodies to peptide Bos 6. Healthy relatives and normal unrelated individuals have low levels of only IgG1 autoantibodies that recognize only Bos 1. In vitro studies indicate that Bos 6-specific IgG and, to a lesser extent, Bos 1-specific IgG can cause acantholysis. Our data suggest that Bos 6-specific IgG4 is probably the main acantholytic autoantibody, while Bos 1-specific IgG4 may act as a facilitator or enhancer of the process. In this study we illustrate some of the paradigms that demonstrate the interactions between the MHC, subclass of autoantibodies, and peptide specificities of the autoantibodies in the autoimmune process. Thus, PV provides an important model to study the pathogenesis of autoimmunity.