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IL-22 is an immunoregulatory cytokine displaying pathological functions in models of autoimmunity like experimental psoriasis. Understanding molecular mechanisms driving IL-22, together with knowledge on the capacity of current immunosuppressive drugs to target this process, may open an avenue to novel therapeutic options. Here, we sought to characterize regulation of human IL22 gene expression with focus on the established model of Jurkat T cells. Moreover, effects of the prototypic immunosuppressant cyclosporin A (CsA) were investigated. We report that IL-22 induction by TPA/A23187 (T/A) or αCD3 is inhibited by CsA or related FK506. Similar data were obtained with peripheral blood mononuclear cells or purified CD3(+) T cells. IL22 promoter analysis (-1074 to +156 bp) revealed a role of an NF-AT (-95/-91 nt) and a CREB (-194/-190 nt) binding site for gene induction. Indeed, binding of CREB and NF-ATc2, but not c-Rel, under the influence of T/A to those elements could be proven by ChIP. Because CsA has the capability to impair IκB kinase (IKK) complex activation, the IKKα/β inhibitor IKKVII was evaluated. IKKVII likewise reduced IL-22 induction in Jurkat cells and peripheral blood mononuclear cells. Interestingly, transfection of Jurkat cells with siRNA directed against IKKα impaired IL22 gene expression. Data presented suggest that NF-AT, CREB, and IKKα contribute to rapid IL22 gene induction. In particular the crucial role of NF-AT detected herein may form the basis of direct action of CsA on IL-22 expression by T cells, which may contribute to therapeutic efficacy of the drug in autoimmunity.
Meeting Abstract : 10. Deutscher Kongress für Versorgungsforschung, 18. GAA-Jahrestagung. Deutsches Netzwerk Versorgungsforschung e. V. ; Gesellschaft für Arzneimittelanwendungsforschung und Arzneimittelepidemiologie e. V. 20.-22.10.2011, Köln
Hintergrund: Multimedikation als Folge von Multimorbidität ist ein zentrales Problem der Hausarztpraxis und erhöht das Risiko für unangemessene Arzneimittel-Verordnungen (VO). Um die Medikation bei älteren, multimorbiden Patienten zu optimieren und zu priorisieren, wurde eine computergestützte, durch Medizinische Fachangestellte (MFA) assistierte, komplexe Intervention (checklistengestütztes Vorbereitungsgespräch sowie Überprüfung eingenommener Medikamente durch MFA, Einsatz des web-basierten ArzneimittelinformationsDienstes AiD, spezifisches Arzt-Patienten-Gespräch) entwickelt und in einer 12-monatigen Pilotstudie auf Machbarkeit getestet. Ein auf 9 Items reduzierter MAI [1] wurde eingesetzt, um dessen Eignung als potentielles primäres Outcome der Hauptstudie zu prüfen.
Material und Methoden: In die Pilotstudie in 20 Hausarztpraxen mit Cluster-Randomisation auf Praxisebene in Kontrollgruppe (Regelversorgung b. empfohlenem Standard) vs. Interventionsgruppe (komplexe Intervention b. empfohlenem Standard) wurden 5 Pat./Praxis eingeschlossen (≥65 Jahre, ≥3 chron. Erkrankungen, ≥5 Dauermedikamente, MMSE ≥26, Lebenserwartung ≥6 Monate). Zur Bewertung des MAI wurden an Baseline (T0), 6 Wo. (T1) & 3 Mon. (T2) nach Intervention erhoben: VO, Diagnosen, Natrium, Kalium & Kreatinin i.S., Größe, Gewicht, Geschlecht, Cumulative Illness Rating Scale (CIRS) [2] durch die Hausarztpraxis; Symptome für unerwünschte Arzneimittelwirkungen im Patienten-Telefoninterview.
Für den MAI wurde die Angemessenheit jeder VO in den 9 Kategorien Indikation, Effektivität, Dosierung, korrekter & praktikabler Applikationsweg, Arzneimittelwechselwirkung, Drug-disease-Interaktion, Doppelverordnung, Anwendungsdauer 3-stufig bewertet (1 = korrekt - 3 = unkorrekt) und für die Auswertung auf Patientenebene summiert. Die Bewertung erfolgte ohne Kenntnis der Gruppenzugehörigkeit. Deskriptive Statistiken und Reliabilitätsanalysen, ungewichtete Auswertung und Gewichtung n. Bregnhoj [3].
Ergebnisse: Es wurden N=100 Patienten in die Studie eingeschlossen, im Mittel 76 Jahre (Standardabweichung, SD 6; Range, R: 64-93) , 52% Frauen, durchschnittlich 9 VO/Pat. (SD 2; R 4-16), mittlerer CIRS-Score 10 (SD 4; R 0-23). Basierend auf N=851 VO (100 Pat.) zu T0 betrug der Reliabilitätskoeffizient (RK, Cronbachs Alpha) der ungewichteten 9 Items 0,70. Items 1-5 wiesen akzeptable Trennschärfen auf (0,52-0,64), die der Items 6, 7 & 9 fielen mit 0,21-0,29 niedriger aus, die des Item 8 betrug 0,06. Auf der Basis der 9 gewichteten Items fiel die interne Konsistenz des MAI erwartet höher aus (0,75). Die Reliabilitätsanalysen auf VO-Ebene zeigten einen RK von 0,67 (ungewichtet) vs. 0,75 (gewichtet), die Trennschärfen waren vergleichbar. Zur Zwischenauswertung betrug der MAI (T1-T0) in der Interventionsgruppe (5 Praxen, 24 Pat.) -0,9 (SD 5,6), in der Kontrollgruppe (7 Praxen, 35 Pat.) -0,5 (SD 4,9); die Differenz zwischen beiden Gruppen Mi–Mk -0,4 [95% Konfidenzintervall: -3,4;2,6].
Schlussfolgerung: Der MAI ist als potentielles primäres Outcome in der Hauptstudie geeignet: wenige fehlende Werte, Darstellung von Unterschieden prä-post und zwischen den Gruppen, akzeptable interne Konsistenz. Der niedrige Trennschärfekoeffizient des Items 8 weist darauf hin, dass dieses Item nicht mit dem Gesamt-Skalenwert korreliert, auch die Items 6, 7 und 9 korrelieren wesentlich schwächer mit dem Gesamt-Skalenwert als die Items 1 bis 5. Eine Wichtung z.B. der Items 2, 5, 6 und 9 könnte erwogen werden, um den Fokus der Intervention in der Hauptzielgröße angemessen abzubilden.
Accumulating evidence indicates that increased generation of reactive oxygen species (ROS) contributes to the development of exaggerated pain hypersensitivity during persistent pain. In the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin C and vitamin E in mouse models of inflammatory and neuropathic pain. We show that systemic administration of a combination of vitamins C and E inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by Complete Freund's Adjuvant. In contrast, vitamin C or vitamin E given alone failed to affect the nociceptive behavior in all tested models. The attenuated neuropathic pain behavior induced by the vitamin C and E combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. Moreover, the vitamin C and E combination ameliorated the allodynia induced by an intrathecally delivered ROS donor. Our results suggest that administration of vitamins C and E in combination may exert synergistic antinociceptive effects, and further indicate that ROS essentially contribute to nociceptive processing in special pain states.
Background: MicroRNA-21 (miR-21) is up-regulated in tumor tissue of patients with malignant diseases, including hepatocellular carcinoma (HCC). Elevated concentrations of miR-21 have also been found in sera or plasma from patients with malignancies, rendering it an interesting candidate as serum/plasma marker for malignancies. Here we correlated serum miR-21 levels with clinical parameters in patients with different stages of chronic hepatitis C virus infection (CHC) and CHC-associated HCC.
Methodology/Principal Findings: 62 CHC patients, 29 patients with CHC and HCC and 19 healthy controls were prospectively enrolled. RNA was extracted from the sera and miR-21 as well as miR-16 levels were analyzed by quantitative real-time PCR; miR-21 levels (normalized by miR-16) were correlated with standard liver parameters, histological grading and staging of CHC. The data show that serum levels of miR-21 were elevated in patients with CHC compared to healthy controls (P<0.001); there was no difference between serum miR-21 in patients with CHC and CHC-associated HCC. Serum miR-21 levels correlated with histological activity index (HAI) in the liver (r = −0.494, P = 0.00002), alanine aminotransferase (ALT) (r = −0.309, P = 0.007), aspartate aminotransferase (r = −0.495, P = 0.000007), bilirubin (r = −0.362, P = 0.002), international normalized ratio (r = −0.338, P = 0.034) and γ-glutamyltransferase (r = −0.244, P = 0.034). Multivariate analysis revealed that ALT and miR-21 serum levels were independently associated with HAI. At a cut-off dCT of 1.96, miR-21 discriminated between minimal and mild-severe necroinflammation (AUC = 0.758) with a sensitivity of 53.3% and a specificity of 95.2%.
Conclusions/Significance: The serum miR-21 level is a marker for necroinflammatory activity, but does not differ between patients with HCV and HCV-induced HCC.
Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamideand 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.
Correlations between personality traits and a wide range of sensory thresholds were examined. Participants (N = 124) completed a personality inventory (NEO-FFI) and underwent assessment of olfactory, trigeminal, tactile and gustatory detection thresholds, as well as examination of trigeminal and tactile pain thresholds. Significantly enhanced odor sensitivity in socially agreeable people, significantly enhanced trigeminal sensitivity in neurotic subjects, and a tendency for enhanced pain tolerance in highly conscientious participants was revealed. It is postulated that varied sensory processing may influence an individual's perception of the environment; particularly their perception of socially relevant or potentially dangerous stimuli and thus, varied with personality.
Nerve injury leads to sensitization mechanisms in the peripheral and central nervous system which involve transcriptional and post-transcriptional modifications in sensory nerves. To assess protein regulations in the spinal cord after injury of the sciatic nerve in the Spared Nerve Injury model (SNI) we performed a proteomic analysis using 2D-difference gel electrophoresis (DIGE) technology. Among approximately 2300 protein spots separated on each gel we detected 55 significantly regulated proteins after SNI whereof 41 were successfully identified by MALDI-TOF MS. Out of the proteins which were regulated in the DIGE analyses after SNI we focused on the carboxypeptidase A inhibitor latexin because protease dysfunctions contribute to the development of neuropathic pain. Latexin protein expression was reduced after SNI which could be confirmed by Western Blot analysis, quantitative RT-PCR and in-situ hybridisation. The decrease of latexin was associated with an increase of the activity of carboxypeptidase A indicating that the balance between latexin and carboxypeptidase A was impaired in the spinal cord after peripheral nerve injury due to a loss of latexin expression in spinal cord neurons. This may contribute to the development of cold allodynia because normalization of neuronal latexin expression in the spinal cord by AAV-mediated latexin transduction or administration of a small molecule carboxypeptidase A inhibitor significantly reduced acetone-evoked nociceptive behavior after SNI. Our results show the usefulness of proteomics as a screening tool to identify novel mechanisms of nerve injury evoked hypernociception and suggest that carboxypeptidase A inhibition might be useful to reduce cold allodynia.
Effect sizes in experimental pain produced by gender, genetic variants and sensitization procedures
(2011)
Background: Various effects on pain have been reported with respect to their statistical significance, but a standardized measure of effect size has been rarely added. Such a measure would ease comparison of the magnitude of the effects across studies, for example the effect of gender on heat pain with the effect of a genetic variant on pressure pain. Methodology/Principal Findings: Effect sizes on pain thresholds to stimuli consisting of heat, cold, blunt pressure, punctuate pressure and electrical current, administered to 125 subjects, were analyzed for 29 common variants in eight human genes reportedly modulating pain, gender and sensitization procedures using capsaicin or menthol. The genotype explained 0–5.9% of the total interindividual variance in pain thresholds to various stimuli and produced mainly small effects (Cohen's d 0–1.8). The largest effect had the TRPA1 rs13255063T/rs11988795G haplotype explaining >5% of the variance in electrical pain thresholds and conferring lower pain sensitivity to homozygous carriers. Gender produced larger effect sizes than most variant alleles (1–14.8% explained variance, Cohen's d 0.2–0.8), with higher pain sensitivity in women than in men. Sensitization by capsaicin or menthol explained up to 63% of the total variance (4.7–62.8%) and produced largest effects according to Cohen's d (0.4–2.6), especially heat sensitization by capsaicin (Cohen's d = 2.6). Conclusions: Sensitization, gender and genetic variants produce effects on pain in the mentioned order of effect sizes. The present report may provide a basis for comparative discussions of factors influencing pain.