Ultraviolet light selection assay to optimize oligonucleotide correction of mutations in endogenous xeroderma pigmentosum genes.

Various oligonucleotide (ODN)-based approaches have been proposed for their ability to correct mutated genes at the normal chromosomal locations. However, the reported gene correction frequencies of these approaches have varied markedly in different experimental settings, including when different tissues or cell types are targeted. In order to find the optimal ODN-based approach for a specific target tissue, an assay system that allows direct comparison of the different methods on that tissue is necessary. Herein, we describe an XP-UVC selection assay that can be used to evaluate and compare gene correction frequencies in different cell types obtained from a xeroderma pigmentosum (XP) patient, following treatment by different ODN-based approaches. As an experimental example, the XP-UVC selection assay was used to assess the ability of chimeric RNA/DNA ODN to correct point mutations in the XPA gene. This assay can be used to assess and evaluate other types of ODN-based approaches, and to further optimize them.

Topical colchicine selection of keratinocytes transduced with the multidrug resistance gene (MDR1) can sustain and enhance transgene expression in vivo.

For skin gene therapy, achieving prolonged high-level gene expression in a significant percentage of keratinocytes (KC) is difficult because we cannot selectively target KC stem cells. We now demonstrate that topical colchicine treatment can be used to select, in vivo, KC progenitor cells transduced with the multidrug resistance gene (MDR1). When human skin equivalents containing MDR1-transduced KC were grafted onto immunocompromised mice, topical colchicine treatments significantly increased (7-fold) the percentage of KC expressing MDR1, compared to vehicle-treated controls, for up to 24 wk. Topical colchicine treatment also significantly enhanced the amount of MDR1 protein expressed in individual KC. Furthermore, quantitative real-time PCR analysis of MDR1 transgene copy number demonstrates that topical colchicine treatment selects and enriches for KC progenitor cells in the skin that contain and express MDR1. For clinical skin gene therapy applications, this in vivo selection approach promises to enhance both the duration and expression level of a desired therapeutic gene in KC, by linking its expression to the MDR1 selectable marker gene.

Occurrence of neutrophils and activated Th1 cells in UVB-induced erythema.

We investigated the sequential changes in infiltrating inflammatory cells and several cytokine levels over a period of 48 h in human back skin exposed to 3 minimal erythematous doses of UVB. The measurement of blood flow, using a laser Doppler method, indicated that UVB-induced erythema reached a peak 12-24 h after irradiation. Immunohistochemically, an increase in the number of CD4+ T cells was observed in perivascular areas 6 h after the UVB treatment and continued for up to 48 h. CD8+ T cells were scarce until 24 h, but their numbers gradually increased thereafter. HLA-DR+ cells were detected perivascularly and interstitially in parallel with the pattern of CD4+ T-cell infiltration. In contrast, neutrophils were found 3 h after UVB exposure and reached a peak at 24 h. Using a RT-PCR method, we demonstrated that mRNAs for the Th1 cytokines (interferon-gamma and interleukin-2), together with a proinflammatory cytokine (interleukin-8), became detectable at 6 h, whereas mRNA for the Th2 cytokine (interleukin-4) was not found at all during the first 48 h. In contrast, we found an increase in mRNA levels for C3 and tumor necrosis factor-alpha even at 3 h, suggesting a relationship between complement activation and accumulating neutrophils. Our results suggest that neutrophils and CD4+ T cells in UVB-induced inflammation play different roles: neutrophils are more closely related to UVB-induced erythema, while T cells appear to be involved in subsequent dermal and epidermal inflammation accompanied by epidermal hyperproliferation.

Cytokine mRNA profiles in cultured human skin component cells exposed to various chemicals: a simulation model of epicutaneous stimuli induced by skin barrier perturbation in comparison with that due to exposure to haptens or irritant.

The skin protects our body by producing an efficient barrier membrane, the stratum corneum, from desiccation as well as from various damaging effects of environmental chemicals. Although the skin expresses various cytokines after barrier perturbation, exact cell types producing each cytokine have not been determined. Using a cell culture system, we analyzed the initial responses of various cutaneous cells to treatments simulating epicutaneous stimuli induced by a barrier perturbation of the skin in comparison with those caused by irritant or hapten exposure. We used cultured normal human epidermal keratinocytes (NHEK), human microvascular endothelial cells (HMVEC) and normal human dermal fibroblasts (NHDF). We treated them with the following chemicals and examined their cytokine mRNA levels 6 h later: high osmotic (0.5 molar) NaCl and hydrogen peroxide (H2O2), which simulate desiccation and exposure to high oxygen pressure, respectively, that may take place in vivo after perturbation of the barrier. In addition, we also studied their response to two representive haptens, nickel chloride (NiCl2) and dinitrochlorobenzene (DNCB), and an irritant, sodium dodecyl sulfate (SDS). We found that 0.5 M NaCl treatment increased mRNA levels of proinflammatory cytokines such as IL-1alpha, IL-6 and IL-8 as well as ICAM-1 in NHEK and IL-1alpha, IL-1beta and IL-6 mRNA levels in NHDF. In contrast, H2O2 treatment remarkably increased IL-10, GMCSF and ICAM-1 mRNA levels in NHEK, and IL-6 mRNA levels in HMVEC and NHDF. The exposure to haptens did not induce any remarkable increase in mRNA levels of the proinflammatory cytokines in NHEK. But NiCl2 increased IL-1alpha, IL-6 and IL-8 mRNA levels in HMVEC, while DNCB increased only their IL-6 mRNA levels. By contrast, SDS stimulated all the cell types to increase at least some of these proinflammatory cytokine mRNA levels. Our present data suggest that each skin component cell participates in inflammatory processes of the skin through its distinctive cytokine production profile when the skin barrier is compromized physically or chemically.

Ultraviolet B radiation exerts enhancing effects on the production of a complement component, C3, by interferon-gamma-stimulated cultured human epidermal keratinocytes, in contrast to photochemotherapy and ultraviolet A radiation that show suppressive effects.

The present study was designed to investigate by using enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction methods whether photochemotherapy (PUVA) or ultraviolet (UV) B treatment affects C3 production by interferon (IFN)-gamma-stimulated keratinocytes cultured in serum-free medium. The results showed that PUVA and UVA reduced C3 production by IFN-gamma-stimulated epidermal keratinocytes dose-dependently, although the effect of PUVA was stronger than that of UVA alone. Interestingly, UVB induced an enhancement of C3 production at doses ranging from 10 to 50 mJ cm-2. This phenomenon was found at both the protein and mRNA levels. In every experiment, changes in C3 mRNA levels preceded those in its protein levels. Reduced C3 production at higher doses of 75 and 100 mJ cm-2 were probably due to cytotoxic effects of UVB. In our experimental system, PUVA, UVA or UVB treatment did not affect C3 production without IFN-gamma stimulation. Our results suggest that a reduction in C3 production by PUVA treatment may in part explain the efficacy of PUVA in the treatment of inflammatory dermatoses such as psoriasis, while the results of the UVB experiments may partially explain the proinflammatory nature of UVB.

Mycobacterium avium infection of the skin associated with lichen scrofulosorum: report of three cases.

We report three Japanese children with Mycobacterium avium infection of the skin who also developed lichen scrofulosorum, a previously undescribed association. They were healthy except for the presence of several noduloulcerative lesions associated with multiple asymptomatic papules on the trunk and extremities. Histology of the ulcerative lesions showed features of mixed-cell granuloma, whereas the papular lesions showed features consistent with lichen scrofulosorum. M. avium was identified by polymerase chain reaction-aided DNA-DNA hybridization analysis in specimens obtained from the noduloulcerative lesions. Both the noduloulcerative and the papular lesions responded well to combination chemotherapy consisting of antituberculous agents and antibiotics.

Dendritic cells differently respond to haptens and irritants by their production of cytokines and expression of co-stimulatory molecules.

After application of haptens to the skin, Langerhans cells (LC), i.e. immature dendritic cells (DC) in the skin, move to secondary lymphoid organs to sensitize naive T cells. During this process, LC become mature DC with augmented expression of various co-stimulatory molecules and MHC class II antigens. In this scenario, however, critical questions remain as to what kind of chemicals can induce this maturation process through what kind of mechanisms. To clarify these questions, we used monocyte-derived CD1a+ DC instead of LC since LC maturated spontaneously in vitro culture. After we confirmed that monocyte-derived DC showed at least phenotypic characteristics and a response to TNF-alpha similar to LC, we added various chemicals, i.e., dinitrochlorobenzene (DNCB), trinitrochlorobenzene (TNCB), NiCl2, ZnCl2, sodium dodecyl sulfate (SDS), or benzalkonium chloride (BC), to a culture of purified monocyte-derived CD1a+ DC. Of these chemicals, only NiCl2 and DNCB significantly increased the surface expression of CD54, CD86, HLA-DR antigen, and interleukin (IL)-1 beta production, while SDS, BC, or ZnCl2 could not augment them, except for weak augmentation of CD86 expression by SDS. The increase in the expression of CD86 induced by NiCl2 or DNCB was most remarkable, being observed in DC from almost all the subjects we examined. TNCB could also induce responses similar to those induced with DNCB, but the number of subjects whose DC responded to it was far less than that of subjects whose DC responded to NiCl2 or DNCB. In spite of the augmented CD86 expression on DC treated with DNCB or NiCl2, these chemicals induced different responses of DC in their expression of CD54 and HLA-DR and the production of IL-6 and tumor necrosis factor (TNF)-alpha. In addition, the up-regulation of CD86 expression on DC treated with DNCB was significantly suppressed by either anti-IL-1 beta or anti-TNF-alpha antibody, while that by NiCl2 was relatively insensitive to these antibody treatments. Finally, the protein kinase C inhibitor, H7, but not staurosporine, could suppress the augmentation of CD86 expression on DC induced either by NiCl2 or by DNCB. These data suggest that DC respond to some haptens by changing their expression of several co-stimulatory molecules and their production of cytokines with a resultant change in antigen-presenting function. They also suggest that these chemicals stimulate DC by different mechanisms. By these responses, DC may modulate the final immune response to chemicals.

A novel genetic system to isolate a dominant negative effector on DNA-binding activity of Oct-2.

Recent studies have revealed that interactions between transcription factors play an important role in regulation of gene expression in eukaryotic cells. To isolate cDNA clones that dominantly inhibit the DNA-binding activity of Oct-2, chosen as a representative factor, we have developed a novel screening system. This employs an Escherichia coli tester strain carrying a modified lac operon as a reporter gene, with the lac operator sequence replaced by an octamer sequence. Oct-2 expressed in this tester strain represses the expression of the reporter gene and changes the phenotype of the cell from Lac+to Lac-. Introduction of a cDNA expression library prepared from a human T-cell line into the Oct-2-harboring tester strain allowed selection of three Lac+clones out of 1 x 10(5) transformants. One of them, hT86, encoding a putative zinc finger protein was found to derepress beta-galactosidase activity in the Oct-2-harboring tester strain at the transcriptional level. In gel mobility shift assays, hT86 attenuated the intensity of the retarded band composed of the octamer probe and Oct-2, suggesting a dominant negative effect on the DNA-binding activity of Oct-2. The strategy described here provides a new approach for studying protein-protein interactions that govern the complex regulation of gene expression.

Annular psoriasiform eruption with lymphocytic infiltration of the epidermis: a variant of acute psoriasis?

Disseminated annular psoriasiform lesions developed over a period of 2 months in a 48-year-old man with no preceding psoriatic history of drug intake, being accompanied by general dullness and arthralgia. Etretinate was effective for both skin eruption and arthralgia; only the latter recurred on its cessation 5 months later. However, histologic features examined by serial sections totally lacked those of pustular psoriasis; there were no neutrophils in the epidermis where massive T lymphocyte infiltration existed instead, in a fashion similar to that of early psoriatic lesions. We differentiated this peculiar annular psoriasiform eruption from the annular erythematous lesions noted in pityriasis rosea, erythema annulare centrifugum, subacute cutaneous lupus erythematosus, annular erythema associated with Sjögren's syndrome and erythema chronicum migrans. It is our speculation that this dermatosis represents a variant of acute psoriasis, rather than annular pustular psoriasis. The histopathologic and immunohistologic findings suggest ongoing cellular immune responses in these lesions where some unknown inhibitory mechanisms might be operative against further production of neutrophil chemotactic factors that usually takes place in psoriatic lesions.