The chemokine RANTES is a chemoattractant for eosinophils, T lymphocytes of memory phenotype and monocytes, suggesting that it plays an important part in chronic inflammatory and allergic diseases. In various types of cells, RANTES production is markedly induced by tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma in combination. Psoriasis vulgaris is a chronic cutaneous inflammatory disease. Cytokines and chemokines produced by T cells and epidermal keratinocytes, such as interleukin (IL) 8, are involved in the pathogenesis of psoriasis. T-cell clones obtained from psoriatic skin have been shown to produce the Th1 cytokine IFN-gamma. In addition, abnormal expression of proinflammatory cytokines including TNF-alpha has been observed in psoriatic lesions. These reports led us to hypothesis that psoriatic skin could provide epidermal keratinocytes with TNF-alpha and IFN-gamma, so that keratinocytes could produce RANTES. In this study, we addressed the question as to whether RANTES was involved in psoriasis vulgaris. Immunohistochemistry of skin biopsies showed RANTES was present in the intercellular spaces between epidermal keratinocytes, in the fully developed lesions from the middle to the edge of psoriatic plaques, but not in the perilesional uninvolved and healthy control skin. Further, we confirmed the production of RANTES, together with IL-8, by cultured normal human epidermal keratinocytes, using an enzyme-linked immunosorbent assay. Stimulation with TNF-alpha and IFN-gamma in combination synergistically increased the RANTES production in this system. These results clearly demonstrate the expression of RANTES in psoriatic lesions and suggest the involvement of this chemokine in the outcome of cutaneous inflammatory diseases. Tacalcitol (1 alpha,24(R)-dihydroxyvitamin D3), an active vitamin D3 analogue, inhibited RANTES and IL-8 production in cultured normal epidermal keratinocytes. This result indicates that active vitamin D3 is effective in the regulation of chemokine production by epidermal keratinocytes, which may partly account for its action as an antipsoriatic drug.

We prepared a novel recombinant tumor necrosis factor-alpha (TNF) mutant (mutant 471), in which 7 N-terminal amino-acids were deleted and Pro8Ser9Asp10 was replaced by ArgLysArg, and compared its biological activity with that of wild-type recombinant TNF. Mutant 471 had a 7-fold higher anti-tumor activity against murine L-M cells in vitro, and a higher binding activity to TNF receptors on L-M cells, than wild-type TNF. Furthermore, mutant 471 showed a higher anti-tumor effect on murine Meth A-HM tumors transplanted into BALB/c mice, with complete regression of the tumors being observed in the animals. The possible cachectin activity of mutant 471 was almost the same as that of wild-type TNF. The acute lethal toxicity of mutant 471 in beta-D-galactosamine-sensitized C3H/HeJ mice was 18 times lower than that of wild-type TNF. These results suggest that mutant 471 might be a more promising anti-cancer agent than wild-type TNF.

The human epidermal keratinocyte cell line K-TL-1, developed from a benign epidermal tumor, was cultured in the presence of the synthetic vitamin D₃ analogue tacalcitol [1α,24(R)-dihydroxyvitamin D₃] to assess the effects on the production of nerve growth factor (NGF). Confluent K-TL-1 cells were cultured with 10^–8M of tacalcitol. Supernatants and cell homogenates were collected and NGF concentrations were determined by enzyme-linked immunosorbent assay. The concentration of NGF in the supernatants of cultures treated with tacalcitol peaked within 24 h after the start of tacalcitol treatment and remained stable for 96 h. This NGF induction caused by tacalcitol was dose-dependent, showing an ED₅₀ between 10^–10 and 10^–9M. Induction of NGF mRNA expression by tacalcitol was also observed by RT-PCR, indicating that tacalcitol induced NGF expression through transcriptional activation. These results suggest that active vitamin D₃ could treat peripheral neuropathy by inducing NGF production in the skin.

1 The production of chemokines, RANTES and IL-8 in cultured human dermal ®broblasts and the eects of tacalcitol (1a,24(R)-dihydroxyvitamin D 3 ) were studied using an enzyme-linked immunosorbent assay. 2 In the unstimulated condition, RANTES and IL-8 were at a trace level in the culture supernatant. On stimulation with TNF-a alone for 24 h, RANTES and IL-8 production were induced. Tacalcitol suppressed RANTES and IL-8 production dose-dependently at concentrations between 10 712 M and 10 77 M.3 When the cells were treated with TNF-a and IFN-g in combination, RANTES production was enhanced, but IL-8 production was not changed, compared to TNF-a-treated cells. Tacalcitol decreased IL-8 production dose-dependently as observed in the TNF-a-treated cells. On the other hand, RANTES production was enhanced by 10 711 M and 10 710 M of tacalcitol, and dose-dependently suppressed by tacalcitol concentrations higher than 10 79 M.4 Active vitamin D 3 compounds, betamethasone valerate and cyclosporin A were compared with respect to their eects on chemokine production. Three active vitamin D 3 compounds, tacalcitol, 1a,25-dihydroxyvitamin D 3 and MC903 (calcipotriol), inhibited the production of RANTES and IL-8, with very similar potencies. Betamethasone valerate also inhibited these chemokine productions, but with greater potency than active vitamin D 3 compounds. Cyclosporin A signi®cantly stimulated RANTES production at 10 76 M and IL-8 production at 10 77 M and 105 The results of this study suggest that active vitamin D 3 compounds exert some bene®cial eects in the treatment of in¯ammatory skin diseases via regulation of the production of chemokines by dermal ®broblasts.

Cytochrome a1c1 (nitrite-cytochrome c oxidoreductase) purified from Nitrobacter winogradskyi (formerly N. agilis) contained molybdenum, non-heme iron, and acid-labile sulfur in addition to hemes a and c; it contained 1 mol of heme a, 4-5 g atoms of non-heme iron, 2-5 g atoms of acid-labile sulfur, and 1-2 g atoms of molybdenum per mol of heme c, but did not contain copper. The fluorescence spectra of the molybdenum cofactor derivative prepared from cytochrome a1c1 were very similar to those of the cofactor derivative from xanthine oxidase, and the aponitrate reductase of nit-1 mutant of Neurospora crassa was complemented by addition of the molybdenum cofactor derived from the cytochrome. Further, the ESR spectrum of cytochrome a1c1 was similar to that of liver sulfite oxidase. The content of cytochrome a1 in the cells cultivated with the medium in which tungsten was substituted for molybdenum markedly decreased as compared with that in the cells cultivated in the molybdenum-supplemented medium. These results indicate that cytochrome a1c1 is an iron-sulfur molybdoenzyme which contains hemes a and c.