No abstract

SDZ ASM 981, a novel ascomycin macrolactam derivative, has high anti-inflammatory activity in animal models of allergic contact dermatitis and shows clinical efficacy in atopic dermatitis, allergic contact dermatitis and psoriasis, after topical application. Here we report on the in vitro activities of this promising new drug. SDZ ASM 981 inhibits the proliferation of human T cells after antigen-specific or non-specific stimulation. It downregulates the production of Th1 [interleukin (IL)-2, interferon-gamma] and Th2 (IL-4, IL-10) type cytokines after antigen-specific stimulation of a human T-helper cell clone isolated from the skin of an atopic dermatitis patient. SDZ ASM 981 inhibits the phorbol myristate acetate/phytohaemagglutinin-stimulated transcription of a reporter gene coupled to the human IL-2 promoter in the human T-cell line Jurkat and the IgE/antigen-mediated transcription of a reporter gene coupled to the human tumour necrosis factor (TNF)-alpha promoter in the murine mast-cell line CPII. It does not, however, affect the human TNF-alpha promoter controlled transcription of a reporter gene in a murine dendritic cell line (DC18 RGA) after stimulation via the FcgammaRIII receptor. SDZ ASM 981 also prevents the release of preformed pro-inflammatory mediators from mast cells, as shown in the murine cell line CPII after stimulation with IgE/antigen. In summary, these results demonstrate that SDZ ASM 981 is a specific inhibitor of the production of pro-inflammatory cytokines from T cells and mast cells in vitro.

Screening of one-bead one-compound libraries by incubating beads with fluorescently labeled target protein requires isolation and structure elucidation of a large number of primary hit beads. However, the potency of the identified ligands is only revealed after time consuming and expensive larger scale resynthesis and testing in solution. Often, many of the resynthesized compounds turn out to be weak target binders in solution due to large differences between surface and solution binding affinities. For an industry style high-throughput screening (HTS) process a high false positive rate is detrimental. We have therefore combined single bead and single molecule/single cell techniques into an integrated HTS process in which the picomole amount of substance contained on one isolated hit bead is sufficient for quality control, structure determination, and precise affinity determination to the target protein in solution.

The NK gene complex is a region on human chromosome 12 containing several families of lectin‐like genes including the CD94 and NKG2 NK receptor genes. We report here that the region telomeric of CD94 contains in addition to the LOX‐1 gene the novel human DECTIN‐1 and the CLEC‐1 and CLEC‐2 genes within about 100 kb. Sequence similarities and chromosomal arrangement suggest that these genes form a separate subfamily of lectin‐like genes within the NK gene complex. DECTIN‐1 is selectively expressed in dendritic cells and to a lowerextent in monocytes and macrophages. mRNA forms with and without a stalk exon are observed. During functional maturation of dendritic cells the level of DECTIN‐1 mRNA is down‐regulated several‐fold. CLEC‐1 is found to be not only expressed in dendritic cells, but also in endothelial cells and in the latter aspect resembles the LOX‐1 gene. Whereas recombinant full‐length DECTIN‐1 and LOX‐1 are transported to the cell surface, CLEC‐1 proteins accumulate in perinuclear compartments. We propose that this family of lectin‐like genes encodes receptors with important immune and/or scavenger functions in monocytic, dendritic and endothelial cells.

Basal cell carcinoma (BCC) is a distinctive manifestation in nevoid basal cell carcinoma syndrome (NBCCS) patients. Both inherited and acquired mutations of patched 1 (PTCH1), a tumor-suppressor gene controlling the activity of Smoothened (SMO), are the primary cause of the constitutive activation of the Hedgehog (HH) pathway, leading to the emergence of BCCs in NBCCS. LDE225, a distinct, selective antagonist of SMO, showed potent inhibition of basaloid tumor nest formation and mediated regression of preformed basaloid tumors in organ cultures of skin derived from Ptch1 heterozygous knockout mice. In a double-blind, randomized, vehicle-controlled, intraindividual study, a total of 8 NBCCS patients presenting 27 BCCs were treated twice daily with 0.75% LDE225 cream or vehicle for 4 weeks. Application of 0.75% LDE225 cream was well tolerated and showed no skin irritation. Of 13 LDE225-treated BCCs, 3 showed a complete, 9 a partial, and only 1 no clinical response. Except for one partial response, the vehicle produced no clinical response in any of the 14 treated BCCs. Treatment with 0.75% LDE225 cream in NBCCS patients was very well tolerated and caused BCC regression, thus potentially offering an attractive therapeutic alternative to currently available therapies for this indication.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub.

Cutaneous lymphocyte antigen (CLA), defined by the HECA-452 antibody, is a cell surface glycoprotein found on a subset of T cells in peripheral blood that binds specifically to E-selectin. This marker is present on the majority of T cells at sites of cutaneous inflammation and immune responses. Based upon such evidence, an association between T cell CLA expression and skin homing has been proposed. To understand better this relationship, we asked whether putative disease-related, antigen-specific T cells expressed CLA. In this study, we employed T helper type 2 (TH2) T cell clones specific for house dust mite (Dermatophagoides pteronyssinus) antigens. These cells were derived from challenged skin of an individual known to react positively to epicutaneous challenge with this agent. In this study, we show that these cloned T cells showed very high homogeneous expression of CLA (nearly 500-fold higher than T cell clones derived from peripheral blood) and bound specifically to recombinant E-selectin. The CLA molecule on these cells was identified not only by HECA-452, but also by CSLEX-1, indicating that it contained sialyl-Le(x) (S-Le(x)) determinants. T cells cloned under similar conditions from peripheral blood were CLA negative or low and bound poorly to E-selectin. Surprisingly, both skin and blood clones bound comparably to P-selectin. This binding was independent of S-Le(x) or CLA expression. We conclude that in sensitized individuals, antigen-specific T cells expressing high levels of CLA localize in skin promptly after epicutaneous challenge. This localization is likely to involve the interaction of S-Le(x) determinants on the CLA molecule with E-selectin on the dermal microvasculature. We further conclude that T cells can interest with P-selectin on endothelium and that S-Le(x) does not appear to be necessary for this interaction.

The tyrosine kinases JAK1 and JAK3 have been shown to undergo tyrosine phosphorylation in response to interleukin-2 (IL), IL4, IL7, and IL9, cytokines which share the common IL2 receptor gamma-chain (IL2R gamma), and evidence has been found for a preferential coupling of JAK3 to IL2R gamma and JAK1 to IL2R beta. Here we show, using human premyeloid TF-1 cells, that IL4 stimulates JAK3 to a larger extent than JAK1, based upon three different evaluation criteria. These include a more vigorous tyrosine phosphorylation of JAK3 as measured by anti-phosphotyrosine immunoblotting, a more marked activation of JAK3 as determined by in vitro tyrosine kinase assays and a more manifest presence of JAK3 in activated IL4-receptor complexes. These observations suggest that IL4 receptor signal transduction does not depend on equimolar heterodimerization of JAK1 and JAK3 following IL4-induced heterodimerization of IL4R alpha and IL2R gamma. Indeed, when human IL4R alpha was stably expressed in mouse BA/F3 cells, robust IL4-induced proliferation and JAK3 activation occurred without detectable involvement of JAK1, JAK2, or TYK2. The present study suggests that JAK1 plays a subordinate role in IL4 receptor signaling, and that in certain cells exclusive JAK3 activation may mediate IL4-induced cell growth. Moreover, mutational analysis of human IL4R alpha showed that a membrane-proximal cytoplasmic region was critical for JAK3 activation, while the I4R motif was not, which is compatible with a role of JAK3 upstream of the recruitment of the insulin receptor substrate-1/4PS signaling proteins by IL4 receptors.

VAF347 represents a novel type of immunomodulator by affecting two major pathways in allergic airway pathogenesis: dendritic cell-mediated T-helper-cell activation and induction of IgE production by human B lymphocytes.