Gene expression is differently affected by pimecrolimus and betamethasone in lesional skin of atopic dermatitis.

BACKGROUND: Topical corticosteroids and calcineurin inhibitors are well-known treatments of atopic dermatitis (AD) but differ in their efficacy and side effects. We recently showed that betamethasone valerate (BM) although clinically more efficient impaired skin barrier repair in contrast to pimecrolimus in AD. OBJECTIVE: This study elucidates the mode of action of topical BM and pimecrolimus cream in AD. METHODS: Lesional AD skin samples after topical treatment with either BM or pimecrolimus were subjected to gene expression profile analysis. RESULTS: Betamethasone valerate resulted in a significant reduction in mRNA levels of genes encoding markers of immune cells and inflammation, dendritic cells, T cells, cytokines, chemokines, and serine proteases, whereas pimecrolimus exerted minor effects only. This corroborates the clinical finding that BM reduces inflammation more effectively than pimecrolimus. Genes encoding molecules important for skin barrier function were differently affected. Both BM and pimecrolimus normalized the expression of filaggrin and loricrin. BM, but not pimecrolimus, significantly reduced the expression of rate-limiting enzymes for lipid synthesis and the expression of involucrin and small proline-rich proteins, which covalently bind ceramides. This may explain the lack of restoration of functional stratum corneum layers observed after BM treatment. CONCLUSION: The gene expression profiles are consistent with our previous findings that corticosteroids may exert a more potent anti-inflammatory effect but may impair the restoration of the skin barrier. Corticosteroids are still the main treatment for severe and acutely exacerbated AD; pimecrolimus may be preferable for long-term treatment and stabilization.

Gene expression profiling of skin and draining lymph nodes of rats affected with cutaneous contact hypersensitivity.

OBJECTIVES AND DESIGN: The present study aimed at a broad genome expression analysis of rat skin and draining lymph nodes affected with allergic contact dermatitis (ACD). METHODS: ACD was elicited in sensitized SD rats with 0.5% 2,4-dinitrofluorobenzene on skin areas distant from the sensitization sites. Involved skin and lymph nodes were dissected 8 and 24 h after challenge. RNA was hybridized on Affymetrix GeneChips. Expression data were analyzed with the GeneSpring software. RESULTS: Expression of 1,882 out of 8,799 examined genes in skin or lymph nodes was significantly (p < or = 0.01) changed compared to levels in normal tissue. After an additional 2- fold filtering, four expression patterns were selected and interpreted. Prominently up-regulated genes were IL-6, CCR-5, CCL-2, CCL-3, CXCL-1, CXCL-10, TIMP-1, OX-40, calgranulin b, ST2, beta-defensin, iNOS, STAT-1, MMP-3, MMP-9, MMP-12, and MMP-13. CONCLUSIONS: In addition to previously reported transcript changes, the present findings suggest/support that plasma cells, mast cells, and a specific IFN-gamma pathway play a substantial role in the pathogenesis of ACD.

Functional genomics in sarcoidosis--reduced or increased apoptosis?

BACKGROUND: A variety of studies have stressed the importance of the control of inflammatory cell longevity and the balance of pro-survival and pro-apoptotic signaling pathways. The aim of the study was to investigate the systemic activation of apoptosis pathways using cDNA array technology in patients with acute onset sarcoidosis. METHOD: We have performed a comprehensive genomic analysis, applying high-density human GeneChip probe arrays (HGU95A, Affymetrix) for RNA expression profiling from peripheral blood mononuclear cells from patients with acute pulmonary sarcoidosis and matched healthy controls. Twelve patients and 12 controls were assessed, mean age 36 +/- 12 and 33 +/- 10 years respectively. Results focus on apoptosis-related gene products. Group differences were assessed with the Mann-Whitney U-test. RESULTS: Seven patients had self-limited disease (all type I sarcoidosis) and 5 progressive disease requiring immunosuppression (all type II or III sarcoidosis). We found 53 of 112 (47%) apoptosis-related gene products dysregulated in sarcoidosis compared to controls. Particular growth factors, especially heparin-binding EGF-like GF, EGF, PDEGF, SISPDGF2 and VEGF, were upregulated in patients consistent with a pro-survival profile. The Bcl-2 family of genes also showed a net pro-survival profile in sarcoidosis patients. In contrast, alterations in the TNF-pathway were compatible with increased apoptosis signals in both, type I and type II/III sarcoidosis patients. Other cell death receptors were equally expressed, as were caspases and p53-associated genes. In contrast to patients with type I-sarcoidosis, patients with progressive type II or III disease showed an upregulation of NFKB and a leak of downregulation of inhibitor of apoptosis 1. CONCLUSION: Significant differences in the expression of apoptosis-related genes were found in peripheral blood of patients with acute onset sarcoidosis. Gene expression did not show a definite pattern that was suggestive of pro-survival or proapoptosis. However, the number of genes whose altered expression would be predicted to favour increased survival exceeded that of genes likely to reduce survival. Protein-based confirmation of the differences in the activity of apoptosis-pathways needs to be done in further studies.

The potential use of mutation spectra in cancer related genes in genetic toxicology: a statement of a GUM working group.

In recent years, there has been widespread interest in the relationship between carcinogenic exposure and mutation spectra in cancer-related genes. To evaluate potential benefits and/or limitations in the use of mutation spectra in genetic toxicology, a GUM working group has been established to discuss this subject. Based on methodological possibilities and limitations, the impact of mutation spectra in the interpretation of animal experiments and in the identification of etiological agents in human cancer has been considered. With respect to experimental animals, the analyses of mutation spectra within long-term rodent carcinogenicity studies may provide some additional information on the mode of action of the respective carcinogen, however, the interpretation of results should be done carefully and only in context with other toxicological data available. Regarding human exposure, the analysis of mutation spectra in p53 or ras genes supplies information on the genotoxic properties of the respective agent. Nevertheless, on the individual level, the presence or absence of defined mutations in cancer-related genes in human tumors does not permit a definite conclusion about the causative agent.

4-chloro-o-phenylenediamine induces a dose-related increase in G:C > T:A transversions and one major DNA adduct in the liver of Big Blue mice after 26 weeks in feed treatment.

The monocyclic aromatic amine 4-chloro-o-phenylenediamine (4-C-o-PDA), a known mutagen and mouse hepatocarcinogen, was tested for its in vivo mutagenic potential in the Big Blue transgenic mouse assay system. Genomic DNA was isolated from liver tissue of control and treated animals and lacI mutants were recovered. In an initial 2-week study 4-C-o-PDA was administered daily per os to groups of male and female C57BL/6 Big Blue mice at doses of 0 and 200 mg/kg for 2 weeks (on working days) followed by a treatment free expression time of 10 days. Only a weak increase in the mutant frequencies in females was observed. In a 26-week study, where 4-C-o-PDA was given to groups of male and female Big Blue mice in feed at dietary concentrations of 0, 5,000 and 10,000 ppm, 4-C-o-PDA was found to induce a pronounced dose-dependent increase in mutant frequencies in either sex. In the present work, we analyzed the mutation spectrum by automated DNA sequencing of lacI mutants from both studies. Following the 2-week administration of 4-C-oT:A transversions in both sexes. In addition, upon 26-week treatment with 4-C-o-PDA, one major DNA adduct was detected by 33P postlabelling and subsequent multidimensional thin layer chromatography. It is concluded that 4-C-oT:A transversions after 26 weeks in feed treatment. This result indicates that the sensitivity of the Big Blue transgenic assay system, in detecting a unique chemically induced mutation spectrum, is dependent on experimental parameters, such as treatment time. The data suggest that the formation of one major DNA adduct upon 4-C-o-PDA treatment may be critical for its mutagenicity.

4-Chloro-o-phenylenediamine: a 26-week oral (in feed) mutagenicity study in Big Blue mice.

4-Chloro-o-phenylenediamine (4-C-o-PDA) is a liver carcinogen in mice and was found to be weakly mutagenic in the liver of female Big Blue mice after short term treatment. In the present study the test compound was given subchronically in the diet for 26 weeks at doses of 0, 5000 and 10,000 ppm. The corresponding average test substance intake was 2166 mg kg-1 day-1 (males: 1794 mg kg-1 day-1; females: 2539 mg kg-1 day-1) and 4610 mg kg-1 day-1 (males: 3926 mg kg-1 day-1; females 5925 mg kg-1 day-1) at the low and high dose, respectively. After sacrifice, tissues were flash frozen in liquid nitrogen. The lacI mutant frequency in the liver was determined from three male and three female mice per dose group. The genomically integrated transgene was recovered by packaging into lambda phage using Transpack packaging extract (Stratagene, La Jolla, USA) followed by infection of Escherichia coli strain SCS-8. Blue mutant plaques were scored against a background of clear non-mutant plaques. Food consumption decreased initially at 10,000 ppm, while no treatment related effect on food intake was observed at 5000 ppm. Body weight gain was found to be decreased in all treated animals. Absolute and relative liver weight increased in a dose-related manner, but only the latter effect was statistically significant. A clear dose dependent increase in lacI mutant frequencies was observed in the liver of both sexes. The following mutant frequencies (x10(-5)) were observed: 2.73+/-1.01 (males, untreated), 7.24+/-1.50 (females, untreated), 18.91+/-5.30 (5000 ppm, males), 24.91+/-7.58 (5000 ppm, females), 20.47+/-6.68 (10,000 ppm, males) and 36.17+/-14.98 (10,000 ppm, females). It is therefore concluded that 4-C-o-PDA is a strong mutagen in the liver of mice treated subchronically for 26 weeks.

The cyclosporine A-induced decrease in rat renal calbindin-D28kDa protein as a consequence of a decrease in its mRNA.

Cyclosporine A (CsA) is a potent immunosuppressant with the drawback of renal side-effects. We recently reported that relatively high doses of CsA markedly decreased the calcium-binding protein calbindin-D28kDa in kidneys of male Wistar rats, and showed that this decrease could be associated with some of the drug-induced adverse renal effects. To investigate the events leading to this decrease, the calbindin-D28kDa mRNA level in kidneys of rats treated with 15 or 50 mg/kg/day CsA for 12 days was analysed by reverse transcription followed by polymerase chain reaction. At both doses, a marked dose-dependent decrease in the calbindin-D28kDa mRNA level was found, one very similar to the decrease measured in the calbindin-D28kDa protein abundance. Thus, the CsA-mediated down-regulation of the renal calbindin-D28kDa protein is most likely the result of a decrease in the calbindin-D28kDa mRNA level.

Efficient identification of point mutations by automated DNA sequencing of artificial heterozygote samples.

DNA sequencing templates of individual point mutants of the lacI target gene were amplified by polymerase chain reaction (PCR). By mixing the PCR fragments from two individual mutants in a defined ratio, samples of artificial heterozygous composition were prepared. These samples were then submitted to automated DNA sequencing. The simultaneous, visual comparison of the mixed mutant traces using a graphics program efficiently revealed all heterozygous positions. Based on the individual intensities of the heterozygous base signals the identified point mutations could be assigned to the corresponding mutants. This efficient approach doubles the sample throughput for both the sequencing reactions and the gel electrophoresis using an automated DNA sequencing system.

Evaluation of the in vivo genotoxic potential of three carcinogenic aromatic amines using the Big Blue transgenic mouse mutation assay.

Three genotoxic mouse carcinogens, 4-chloro-o-phenylenediamine (4-C-o-PDA), 2-nitro-p-phenylenediamine (2-N-p-PDA), and 2,4-diaminotoluene (2,4-DAT), were tested in the Big Blue transgenic mouse mutation assay. Each experiment consisted of a vehicle control group with ten Big Blue C57BL/6 mice, five of either sex, and an equally sized group treated with a high dose of the test chemical. In addition, four animals were treated with the vehicle and six animals with the test compound for the measurement of bromodeoxyuridine (BrdU) incorporation to determine cellular proliferation. Prior to the mutagenicity experiments, the maximally tolerated dose of each compound was determined using nontransgenic C57BL/6 mice. Based on these results the doses used in the main study were 200 mg/kg/day for 4-C-o-PDA, 150 mg/kg/ day for 2-N-p-PDA, and 80 mg/kg/day for 2,4-DAT. Animals were treated for 10 days over a 2 week period and were killed 10 days after the ast treatment. In an additional experiment with 2,4-DAT, animals were killed 28 days after treatment. Since all three chemicals are liver carcinogens in the mouse, the DNA of the liver was analyzed using the standard procedures for the Big Blue assay. Hepatocyte proliferation was assessed by immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and, in some studies, by measuring BrdU incorporation. 4-C-o-PDA and 2-N-p-PDA did not induce an increase in PCNA expression when measured 10 days after the last treatment. There was no increase in BrdU incorporation immediately after treatment with 4-C-o-PDA or with 2,4-DAT. However, 10 days after the last treatment with 2,4-DAT, a strong mitogenic effect was found with both techniques, i.e., in the PCNA and BrdU assays. 4-C-o-PDA, a liver carcinogen in both genders of mice, induced a small, statistically significant increase of the mutant frequencies in females. No increase was found in males. 2-N-p-PDA, which has been reported to induce liver tumors only in females, was found positive in males and was clearly negative in females. 2,4-DAT, a liver carcinogen in female mice, was positive in females and negative in males when the animals were killed 10 days after the last treatment. After an expression time of 28 days, 2,4-DAT induced a statistically significant increase in both sexes. The effect in females was marginally stronger than after 10 days' expression time and almost identical to the effect observed in males under these test conditions. In conclusion, the experiments showed that the Big Blue assay detects the genotoxicity of the three carcinogenic monocyclic aromatic amines tested. However, it seems that the sex specificity of the carcinogenic effects of these compounds is not reflected by the mutagenicity data in Big Blue mice.

Primary osteogenic sarcoma of the breast.

A rapidly growing tumor in the left breast of a 38-year-old woman was mammographically suggested to be a fibroadenoma because of its sharply circumscribed and quite homogeneous shadow, with coarse calcifications. Fine needle aspiration biopsy showed an unusual cytologic picture, with polymorphous and multinucleated giant tumor cells and scattered plaques of osteoid. After excision followed by mastectomy, the tumor was histologically diagnosed as an osteogenic sarcoma. Radiologically, there was no evidence of another tumor localization. The cytologic findings of this rare tumor, never previously reported, are presented.