Heinrich Aschauer

Purification and amino acid sequencing of NAF, a novel neutrophil-activating factor produced by monocytes

Human blood mononuclear cells were cultured for 24 h in the presence of LPS (100 ng per 5 x 10(6) cells), and a monocyte-derived neutrophil-activating factor (NAF) was purified to apparent homogeneity from the conditioned media. The purification consisted of ammonium sulphate precipitation, gel filtration, chromatography on phosphocellulose followed by hydroxylapatite, and reversed-phase HPLC on C4 and CN-propyl columns. Amino acid sequence analysis (32 of 50 presumed residues) shows that NAF is a novel peptide with little homology to known ones. Crude and pure NAF stimulated human neutrophils to release granule enzymes and to produce superoxide and H2O2.

Topical Treatment of Basal Cell Carcinomas in Nevoid Basal Cell Carcinoma Syndrome with a Smoothened Inhibitor

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.

THE SOLUBLE HYALURONIDASE FROM BULL TESTES IS A FRAGMENT OF THE MEMBRANE-BOUND PH-20 ENZYME

The membrane‐bound PH‐20 hyaluronidase is known to be essential for fertilization. Here we addressed the question whether the soluble hyaluronidase from bull teste is related to the PH‐20 polypeptide. The sequence of the membrane‐bound PH‐20 hyaluronidase from bovine sperm was determined via cDNA cloning. In parallel, from a commercial preparation of bovine hyaluronidase the major 60‐kDa form was purified to apparent homogeneity. The soluble enzyme was digested with two different proteases and with cyanogen bromide and the amino acid sequence of 44 different fragments was determined. All the peptide sequences could be aligned to the sequence deduced from the cloned cDNAs. Our results thus show that the soluble 60‐kDa hyaluronidase from bovine testes is a glycoprotein derived from the sperm PH‐20 enzyme. As compared to the primary translation product of the PH‐20 mRNA, it lacks the signal peptide at the amino terminus and 56 amino acids at the carboxyl end. These results demonstrate that the soluble 60‐kDa enzyme is a fragment of the PH‐20 hyaluronidase. It is currently not known whether the soluble testes hyaluronidase has a distinct biological function.

Pimecrolimus permeates less than tacrolimus through normal, inflamed, or corticosteroid-pretreated skin

Abstract:  The permeabilities of normal human and normal, inflamed, or corticosteroid (CS) pretreated skin of young domestic pigs for pimecrolimus and tacrolimus were compared in vitro, using Franz‐type diffusion cells. The test articles were either used as 1.0% solutions or as the marketed formulations (Elidel 1% cream, Protopic 0.1%, and 0.03% ointment). In normal human skin, the permeation rate of pimecrolimus from the 1% cream was about sixfold lower than that of tacrolimus from 0.1% ointment and by a factor of 4.3 lower compared with tacrolimus from Protopic 0.03%. In pigs, sodium laurylsulfate‐induced irritant contact dermatitis resulted in significantly faster skin permeation of both drugs from applied solutions. The permeation rate for pimecrolimus was lower than that for tacrolimus. Thus, at 24 h, pimecrolimus concentrations in the receptor fluid were 2.8‐fold lower than the tacrolimus levels. Compared with normal porcine skin, permeation of drugs through hydrocortisone (1.0%)‐, mometasone (0.1%)‐, or clobetasol‐17‐butyrate (0.05%)‐pretreated skin was increased by factors of 3.6 (pimecrolimus, applied as 1% cream) and 1.7 (tacrolimus, applied as 0.1% ointment). In normal pig skin, the permeation rate of tacrolimus was found to be 11.2 times higher than that of pimecrolimus and 3.5‐ to 7.1‐fold higher in CS‐pretreated skin, independent of the potency of the CSs. The present in vitro data suggest that in patients with acute skin inflammation or after therapy with topical CSs, percutaneous absorption and, as a consequence, systemic drug exposure will be lower with Elidel 1% cream as compared with Protopic 0.1% and 0.03% ointment.

The membrane-proximal part of FcϵRIα contributes to human IgE and antibody binding – implications for a general structural motif in Fc receptors

The high affinity receptor for human IgE (FcϵRI) on tissue mast cells and blood basophils is responsible for immediate hypersensitivity reactions. Binding of human IgE (hIgE) to FcϵRI has been shown to be mediated via three independent regions in the extracellular part of the α‐subunit of FcϵRI (ecFcϵRIα). By site‐directed mutagenesis we investigated the contribution of amino acids within the ecFcϵRIα FG loop (residues Lys154–Leu165) to binding to hIgE and two monoclonal anti‐FcϵRIα antibodies (15/1, 5H5/F8). The mutated receptors were expressed and secreted from eukaryotic cells as amino‐terminal fusion to HSA. We show that the proposed loop region contributes partly to hIgE binding and that the epitope of mAb 15/1, which inhibits hIgE/FcϵRIα interaction, maps to this region whereby a single W156A mutation results in complete loss of mAb 15/1 binding. In contrast, hIgE binding is not affected by the W156A mutation indicating that different amino acid residues within the loop are recognized by the mAbs 15/1 and hIgE. MAb 5H5/F8 does not recognize a receptor mutant truncated to Ile170. By screening a random dodecapeptide library displayed on bacterial flagella the epitope for mAb 5H5/F8 was mapped to P173REKY177 whereas one of the 15/1 binding clones displayed a peptide with an amino acid sequence homologous to Leu158–Ile167. Based on the epitopes identified for the inhibitory mAb 15/1 and the non‐inhibitory mAb 5H5F8 and on binding data obtained with polyclonal antisera raised against two ecFcϵRIα peptides, we propose a structural element in the membrane proximal part of ecFcϵRIα which forms a 3D structure which might facilitate specific and efficient attachment of hIgE.

Pharmacological Inhibition of Stearoyl CoA Desaturase in the Skin Induces Atrophy of the Sebaceous Glands

Stearoyl-CoA desaturase-1 (SCD-1) catalyzes the formation of delta9-monounsaturated fatty acids from saturated precursors. Deficiency of SCD-1 in mice causes atrophy of sebaceous glands. We asked whether local pharmacological inhibition of this enzyme in the skin would lead to a similar effect. To this end, we characterized the low-molecular-weight compound XEN103 as a potent and selective inhibitor of SCD-1 activity. The compound blocks microsomal and cellular SCD-1 activity across species with IC50 values in the range of 10-15 nM. Upon topical application to the skin of mice as a 1% solution, XEN103 induces pronounced sebaceous gland atrophy with a rapid onset after a few days of dosing, both sebaceous gland numbers and size being reduced by 50 to 75%, and without any signs of skin irritation. We show that the effect is due to the local action of the compound on SCD-1 in the skin and occurs in the presence of only minimal plasma exposure. Based on these observations, SCD-1 inhibitors such as XEN103 may provide an opportunity to develop a novel topical therapy for acne, as a disease characterized by overproduction of sebum from hypertrophic sebaceous glands.

Discovery and characterization of NVP-QAV680, a potent and selective CRTh2 receptor antagonist suitable for clinical testing in allergic diseases

Optimization of a 7-azaindole-3-acetic acid CRTh2 receptor antagonist chemotype derived from high throughput screening furnished a highly selective compound NVP-QAV680 with low nM functional potency for inhibition of CRTh2 driven human eosinophil and Th2 lymphocyte activation in vitro. The molecule exhibited good oral bioavailability in the rat, combined with efficacy in rodent CRTh2-dependent mechanistic and allergic disease models and was suitable for clinical development.