Histamine 2 Receptor Agonism and Histamine 4 Receptor Antagonism Ameliorate Inflammation in a Model of Psoriasis

Psoriasis is a chronic inflammatory skin disorder characterized by hyperproliferative keratinocytes and immune cell infiltration into the skin, often accompanied by itch. Histamine, acting via histamine 1–4 receptors, is known to modulate immune responses in the skin and to induce itch. The aim of this study was to test the role of histamine 2 receptors and histamine 4 receptors in the imiquimod-induced psoriasis-like skin inflammation model. BALB/c mice were treated intraperitoneally with amthamine (histamine 2 receptor agonist), JNJ-39758979 (histamine 4 receptor antagonist), a combination of both, or vehicle twice daily in a preventive manner. Imiquimod was applied once daily onto the back skin for 10 consecutive days. Stimulation of histamine 2 receptors and blockade of histamine 4 receptors ameliorated imiquimod-induced skin inflammation. The combination of amthamine and JNJ-39758979 reduced skin inflammation even more pronounced, diminished epidermal hyperproliferation, and inhibited spontaneous scratching behaviour. A combination of histamine 2 receptor agonist and histamine 4 receptor antagonists could represent a new strategy for the treatment of psoriasis

MALDI-TOF High Mass Calibration up to 200 kDa Using Human Recombinant 16 kDa Protein Histidine Phosphatase Aggregates

Background: Protein histidine phosphatase (PHP) is an enzyme which removes phosphate groups from histidine residues. It was described for vertebrates in the year 2002. The recombinant human 16 kDa protein forms multimeric complexes in physiological buffer and in the gas phase. High-mass calibration in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has remained a problem due to the lack of suitable standards. Large proteins can hardly be freed of their substructural microheterogeneity by classical purification procedures so that their use as calibrants is limited. A small adduct-forming protein of validated quality is a valuable alternative for that purpose. Methodology/Principal Findings: Three major PHP clusters of,113, 209 and.600 kDa were observed in gel filtration analysis. Re-chromatography of the monomer peak showed the same cluster distribution. The tendency to associate was detected also in MALDI-TOF MS measuring regular adducts up to 200 kDa. Conclusions/Significance: PHP forms multimers consisting of up to more than 35 protein molecules. In MALDI-TOF MS it generates adduct ions every 16 kDa. The protein can be produced with high quality so that its use as calibration compoun

Sphingosine 1-phosphate modulates antigen capture by murine langerhans cells via the S1P2 receptor subtype

Dendritic cells (DCs) play a pivotal role in the development of cutaneous contact hypersensitivity (CHS) and atopic dermatitis as they capture and process antigen and present it to T lymphocytes in the lymphoid organs. Recently, it has been indicated that a topical application of the sphingolipid sphingosine 1-phosphate (S1P) prevents the inflammatory response in CHS, but the molecular mechanism is not fully elucidated. Here we indicate that treatment of mice with S1P is connected with an impaired antigen uptake by Langerhans cells (LCs), the initial step of CHS. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Our results indicate that S1P inhibits macropinocytosis of the murine LC line XS52 via S1P2 receptor stimulation followed by a reduced phosphatidylinositol 3-kinase (PI3K) activity. As down-regulation of S1P2 not only diminished S1P-mediated action but also enhanced the basal activity of LCs on antigen capture, an autocrine action of S1P has been assumed. Actually, S1P is continuously produced by LCs and secreted via the ATP binding cassette transporter ABCC1 to the extracellular environment. Consequently, inhibition of ABCC1, which decreased extracellular S1P levels, markedly increased the antigen uptake by LCs. Moreover, stimulation of sphingosine kinase activity, the crucial enzyme for S1P formation, is connected not only with enhanced S1P levels but also with diminished antigen capture. These results indicate that S1P is essential in LC homeostasis and influences skin immunity. This is of importance as previous reports suggested an alteration of S1P levels in atopic skin lesions

Large-Scale Screening: Phenotypic and Mutational Spectrum in Isolated and Combined Dystonia Genes

© 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.[Background] Pathogenic variants in several genes have been linked to genetic forms of isolated or combined dystonia. The phenotypic and genetic spectrum and the frequency of pathogenic variants in these genes have not yet been fully elucidated, neither in patients with dystonia nor with other, sometimes co-occurring movement disorders such as Parkinson's disease (PD).[Objectives] To screen >2000 patients with dystonia or PD for rare variants in known dystonia-causing genes.[Methods] We screened 1207 dystonia patients from Germany (DysTract consortium), Spain, and South Korea, and 1036 PD patients from Germany for pathogenic variants using a next-generation sequencing gene panel. The impact on DNA methylation of KMT2B variants was evaluated by analyzing the gene's characteristic episignature.[Results] We identified 171 carriers (109 with dystonia [9.0%]; 62 with PD [6.0%]) of 131 rare variants (minor allele frequency <0.005). A total of 52 patients (48 dystonia [4.0%]; four PD [0.4%, all with GCH1 variants]) carried 33 different (likely) pathogenic variants, of which 17 were not previously reported. Pathogenic biallelic variants in PRKRA were not found. Episignature analysis of 48 KMT2B variants revealed that only two of these should be considered (likely) pathogenic.[Conclusion] This study confirms pathogenic variants in GCH1, GNAL, KMT2B, SGCE, THAP1, and TOR1A as relevant causes in dystonia and expands the mutational spectrum. Of note, likely pathogenic variants only in GCH1 were also found among PD patients. For DYT-KMT2B, the recently described episignature served as a reliable readout to determine the functional effect of newly identified variants.This work was supported by the German Ministry of Education and Research (BMBF, DYSTRACT consortium, 01GM1514B, to A.A.K., T.B., C.Klein and K.L.) and the German Research Foundation (DFG, LO1555/10-1 to H.B., C.Klein, and K.L. and Project-ID 424778381-TRR 295 to A.A.K). The DysTract registry was further supported by the Arbeitskreis Botulinumtoxin der DGN e.V., Merz Therapeutics, AbbVie/Allergan, and Ipsen Pharma. The Korean DNA samples for this study were provided by the Seoul National University Hospital Human Biobank, a member of the National Biobank of Korea, which is supported by the Ministry of Health and Welfare. All samples derived from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols. Several authors are members of the European Reference Network for Rare Neurological Diseases (Project ID No. 739510). Open Access funding enabled and organized by Projekt DEAL.Peer reviewe

Plasma pharmacokinetics of tigolaner, emodepside, and praziquantel following topical administration of a combination product (Felpreva®) and of intravenous administration of the individual active ingredients in cats

Felpreva® for cats contains the new acaricidal/insecticidal active ingredient tigolaner in a fixed combination with the nematocidal and cestocidal compounds emodepside and praziquantel, respectively. The plasma pharmacokinetics of tigolaner, emodepside, and praziquantel were evaluated in clinically healthy cats following topical (spot-on) treatment as fixed combination Felpreva®. For the determination of bioavailability intravenous administration of single active ingredients was also performed. After a single topical administration of Felpreva® using the target dose volume of 0.148 ​ml/kg to cats, tigolaner reached mean peak concentrations of 1352 ​μg/l with a Tmax of 12 days and a mean half-life of 24 days. Simulation of repetitive topical administration every 91 days indicates only a low risk of accumulation after reaching steady state within two to three administrations. The volume of distribution calculated after intravenous dosing was 4 ​l/kg and plasma clearance was low with 0.005 ​l/h/kg. Overall plasma exposure was 1566 ​mg∗h/l after topical administration, providing an absolute bioavailability of 57%. Tigolaner was mainly cleared via the faeces (54% within 28 days), renal clearance was neglectable (< 0.5% within 28 days). Emodepside and praziquantel showed mean peak concentrations of 44 ​μg/l and 48 ​μg/l (reached after 1.5 days and 5 ​h, respectively). Overall plasma exposures were 20.6 and 3.69 ​mg∗h/l, respectively. The elimination half-life was 14.5 days for emodepside and 10 days for praziquantel after topical administration. After topical administration of Felpreva® using 2.5× and 5× dose multiples an almost proportional increase of plasma exposure was observed for all three active ingredients. With the addition of tigolaner, Felpreva® combines the established pharmacokinetic (PK) characteristics of emodepside and praziquantel contained in Profender® spot-on for cats with the favourable PK of tigolaner suitable for a 3-months protection against fleas and ticks

S1P inhibits macropinocytosis of FITC-labeled dextran via the S1P₂ receptor subtype.

<p>XS52 cells were transfected with siRNA against S1P₂ or control siRNA and silencing was detected by quantitative real time PCR (A). Transfected and control cells were incubated with FITC-labeled dextran in the presence or absence of S1P (5 µM) for 15 min and macropinocytosis was detected by flow cytometry. Relative endocytosis are expressed as the mean ± SEM of results from at least three independent experiments. **P < 0.01 indicate a statistically significant difference vs. control experiments (B). Transfected or control cells were treated with S1P (5 µM) for 15 min followed by the detection of Akt activity (C). Values of the densitometric analysis are expressed as x-fold decrease of p-Akt formation compared to untreated cells ± SEM from three experiments. **P<0.01 indicates a statistically significant difference versus control (C).</p

S1P is generated by XS52 cells and released to the extracellular environment.

<p>XS52 cells were cultivated over a time period of 6 h and S1P levels in the extracellular environment was detected (A). Quantitative real time PCR analysis of ABCB1, ABCC1, and ABCG2 of three different sets of cells was performed using HPRT1 and GADPH as reference genes (B). Cells were preincubated with Reversin 121 (10 µM), Fumitremorgin C (10 µM), and Probenecid (2.5 mM) for 6 h (C). Cells were transfected with siRNA against ABCC1 or control siRNA and silencing was detected by quantitative real time PCR (D). Then, cells were incubated with FITC-labeled dextran for 15 min. Fluorescence intensity of cells was analyzed by flow cytometry and relative endocytosis was calculated. Data are expressed as the mean ± SEM of results from at least three independent experiments. **P < 0.01 indicates a statistically significant difference vs. control experiments.</p

Effects of S1P receptor modulators on uptake of FITC-labeled dextran and PI3K activity.

<p>XS52 cells were incubated with FITC-labeled dextran in the presence or absence of S1P (5 µM), FTY720-P (1 µM), VPC24191 (10 µM), and SEW2871 (1 µM) for 15 min. Fluorescence intensity of cells was analyzed by flow cytometry and relative endocytosis was calculated. Data are expressed as the mean ± SEM of results from at least three independent experiments. **P < 0.01 indicate a statistically significant difference vs. control experiments (A). Cells were treated with similar concentrations of S1P, FTY720-P, VPC24191, and SEW2871 for 15 min followed by the detection of Akt activity (B). Values of the densitometric analysis are expressed as x-fold decrease of p-Akt formation compared to untreated cells ± SEM from three experiments. **P<0.01 indicates a statistically significant difference versus control (B).</p

The effect of S1P on the endocytosis of FITC-labeled dextran by epidermal DCs in skin explant cultures.

<p>Mice were topically treated for 3 days with 100 µg S1P/daily. Skin explant cultures were performed and the dorsal ear halves were incubated with FITC-labeled dextran (1 mg/ml) for 2 h at 37°C. Epidermal DCs were detected via MHC class II staining and endocytosis was visualized in epidermal sheets using confocal microscopy. One out of three experiments with similar results is shown (A). The percentage of dextran uptake was calculated by counting of MHC class II- and FITC-labeled positive cells. Data are expressed as the mean ± SEM of results from at least three experiments (B). **P < 0.01 indicates a statistically significant difference vs. control experiments.</p

Uptake of FITC-labeled dextran by XS52 cells via macropinocytosis in a PI3K dependent manner.

<p>Cells were preincubated with Rottlerin (3 µM), Mannan (1 mg/mL), and LY294002 (10 µM) for 30 min. Then, cells were incubated with FITC-labeled dextran for 15 min. S1P (5 µM) was used as positive control. Fluorescence intensity of cells was analyzed by flow cytometry and relative endocytosis was calculated. Data are expressed as the mean ± SEM of results from at least three independent experiments. **P < 0.01 indicate a statistically significant difference vs. control experiments (A). Cells were treated with the indicated concentrations of S1P or LY294002 (10 µM) for 15 min followed by the detection of Akt activity using Western blot analysis (B). Values of the densitometric analysis are expressed as x-fold decrease of phosphorylated Akt (p-Akt) formation compared to untreated cells ± SEM from three experiments. **P<0.01 indicates a statistically significant difference versus control (B).</p