Untersuchung zur Rolle des G-Protein gekoppelten Rezeptors (GPR) 43 im intestinalen Entzündungsmodell

Morbus Crohn und Colitis ulcerosa sind die wichtigsten Entitäten der chronisch entzündlichen Darmkrankheiten (CED), deren Ursachen und Therapie derzeitig Gegenstand der Forschung sind. Eine Kombination aus genetischer Prädisposition und Umweltfaktoren wird aktuell diskutiert. Diese resultiert in einer Barrierefunktionsstörung der Darmmukosa, die Grundlage eines chronisch entzündlichen Geschehens ist. Der G-Protein gekoppelte Rezeptor (GPR) 43 wird in entzündeten Arealen auf neutrophilen Granulozyten (PMN) verstärkt exprimiert. Er detektiert kurzkettige Fettsäuren (SCFA), die von anaeroben Bakterien der Darmflora produziert werden, und aktiviert Migrationsprozesse. Für die vorliegende Arbeit wurden Gpr43-Knockout-Mäuse gezüchtet, um ihr Verhalten im Rahmen einer experimentellen Colitis mit Wildtypmäusen zu vergleichen. Die chemisch induzierte Natriumdextransulfat (DSS)-Colitis stellt ein etabliertes Mausmodell der Darmentzündung dar. Mittels klinischer Parameter, Histologie, Mini-Endoskopie und ELISA wurde das Ausmaß der Entzündung in einem chronischen und einem akuten Ansatz bestimmt. Zusätzlich wurde mit isolierten PMN Migrations-Untersuchungen in einer Transmigrationskammer (Boyden-Kammer) durchgeführt um die funktionellen und molekularen Auswirkungen der Gpr43-Defizienz zu untersuchen. In der akuten DSS-Colitis wurde für die Gpr43-/--Mäuse eine stärkere Entzündung, gestützt durch den klinischen Verlauf und die erhöhten Zytokin-Parameter, festgestellt. Der Gesamteindruck deutete auf eine systemische Infektion hin, die einherging mit dem Tod aller Versuchstiere während der Experimentalphase. Histologische und endoskopische Untersuchungen zeigten jedoch weniger entzündliche Merkmale als bei den Vergleichstieren. In der chronischen DSS-Colitis wirkte die Gpr43-Defizienz protektiv auf den Verlauf der Erkrankung. Die erhobenen Parameter und Untersuchungen zeigten geringere Entzündungsparameter als bei den Wildtypmäusen. In beiden Modellen war gleichermaßen auffällig, dass sich in den submukosalen Blutgefäßen Granulozytenkonglomerate ansammelten, während in der Submukosa selbst kaum PMNs zu finden waren. Ob eine geminderte Evasionsfähigkeit mit der Rezeptordefizienz in einen kausalen Zusammenhang gebracht werden kann, wurde anschließend in Chemotaxisexperimenten untersucht: Die Migration per se war nicht beeinträchtigt, lediglich mit SCFA stimulierte Zellen zeigten ein reduziertes Migrationsvermögen. Des Weiteren wurde die Proteinkinase p38 als Gpr43-abhängiger Mediator der Aktivierungskaskade identifiziert und erstmals zeigt diese Arbeit, dass L-Selektin-shedding GPR43- und p38-abhängig induziert wird. Damit erscheint das Ausbleiben dieses Vorgangs ursächlich für die verminderten Evasion von PMN und stellt damit ein mögliches therapeutisches Ziel zur Durchbrechung der chronischen Entzündung dar

Role of NOD2/CARD15 in coronary heart disease

<p>Abstract</p> <p>Background:</p> <p>Bacterial DNA has been repeatedly detected in atheromatous lesions of coronary heart disease (CHD) patients. Phylogenetic signatures in the atheroma lesions that are similar to those of bacterial biofilms on human barrier organs, including the respiratory or gastrointestinal tract, raise the question of a defective barrier function in CHD. NOD2 plays a major role in defense against bacterial invasion. Genetic variation in the <it>CARD15 </it>gene, which encodes NOD2, was previously shown to result in a barrier defect that causes chronic inflammatory disorders (e.g. Crohn disease). In the present study, we investigated the possible involvement of NOD2/<it>CARD15 </it>in the pathology of CHD by <it>i) </it>analyzing the local expression of NOD2 in atherectomy versus healthy tissue (n = 5 each) using histochemical immunofluorescence and <it>ii) </it>by testing the three major functional <it>CARD15 </it>variants (R702W, G908R and 1007fs) for association with early-onset CHD in 900 German patients and 632 healthy controls.</p> <p>Results:</p> <p>In atherectomy tissue of CHD patients, NOD2 was detected in inflammatory cells at the luminal sides of the lesions. However, the allele and genotype frequencies of the three major <it>CARD15 </it>polymorphisms did not differ between CHD patients and controls.</p> <p>Conclusion:</p> <p>The NOD2 up-regulation in atheroma lesions indicates an involvement of this protein in the pathology of CHD. Although NOD2 could be important in local immune response mechanisms, none of the analyzed <it>CARD15 </it>variants seem to play a significant role in the etiology of CHD.</p

Axial low back pain: one painful area--many perceptions and mechanisms.

Axial low back pain can be considered as a syndrome with both nociceptive and neuropathic pain components (mixed-pain). Especially neuropathic pain comprises a therapeutic challenge in practical experience and may explain why pharmacotherapy in back pain is often disappointing for both the patient and the therapist. This survey uses epidemiological and clinical data on the symptomatology of 1083 patients with axial low back pain from a cross sectional survey (painDETECT). Objectives were (1) to estimate whether neuropathic pain contributes to axial low back pain and if so to what extent. (2) To detect subgroups of patients with typical sensory symptom profiles and to analyse their demographic data and co-morbidities. (3) To compare patients with and without prior intervertebral disc surgery (IVD). Neuropathic pain components could be detected in 12% of the entire cohort. Cluster analyses of these patients revealed five distinct subgroups of patients showing a characteristic sensory profile, i.e. a typical constellation and combination of symptoms. All subgroups occurred in relevant numbers and some showed distinct neuropathic characteristics while others showed nociceptive features. Post-IVD-surgery patients showed a tendency to score more "neuropathic" than patients without surgery (not statistically significant). Axial low back pain has a high prevalence of co-morbidities with implication on therapeutic aspects. From these data it can be concluded that sensory profiles based on descriptor severity may serve as a better predictor for therapy assessment than pain intensity or sole diagnosis alone. Standardized phenotyping of pain symptoms with easy tools may help to develop an individualized therapy leading to a higher success rate in pharmacotherapy of axial low back pain

Short Report: TRPV1-polymorphism 1911 A>G alters capsaicin-induced sensory changes in healthy subjects

<div><p>Background</p><p>C-fibers express transient receptor potential (TRP) channels. These high-voltage gated channels function as integrators of different physical stresses (e.g. heat, protons, ATP). Additionally channel activation can be induced by capsaicin. Topically applied, capsaicin elicits burning pain, heat and mechanical hyperalgesia and serves as a human surrogate model for pain. It was suggested that the TRPV1-variant rs8065080 (1911A>G) plays a pivotal role in patients with neuropathic pain syndromes. We investigated the effect of this TRPV1-SNP on thermal sensitivity and superficial skin perfusion in 25 healthy subjects.</p><p>Methods and findings</p><p>Nine subjects being homozygous TRPV1 wild type (AA), 8 heterozygous (AG) and 8 homozygous variant (GG) carriers were selected out of a pool of genotyped healthy individuals. Under physiological conditions (no capsaicin application), there was no statistical significant difference in thermal thresholds or skin perfusion between carriers of different TRPV1 1199A>G genotypes. However, intra-individual calculations (Δ% pre vs. post capsaicin) revealed (1) less warm-detection in AA/AG (-82.1%) compared to GG (-13.1%) and (2) a gain of heat pain sensitivity in AA/AG (+22.2%) compared to GG carriers (+15.6%) after adjustment for perfusion measurements ((1)p = 0.009, (2)p = 0.021).</p><p>Conclusion</p><p>Presence of homozygous variant TRPV1 genotype (GG) demonstrated less capsaicin-induced warm hypoesthesia in warm-detection and less capsaicin-induced heat pain sensitivity suggesting an altered channel function. This demonstrates not only the functional influence of TRPV1 rs8065080 polymorphism itself; it further more underpins the relevance of genotyping-based approaches in both patients and surrogate models of neuropathic pain in healthy volunteers.</p></div

Subgroups of patients based on their sensory symptoms.

<p>To identify relevant subgroups of patients who are characterized by a characteristic symptom constellation a hierarchical cluster analysis was performed. The clusters are represented by the patterns of questionnaire scores (A: adjusted individual mean; B: non-adjusted values), thus showing the typical pathological structure of the respecting group. By using this approach five clusters with distinct symptom profiles could be detected in the cohort. Sensory profiles show remarkable differences in the expression of the symptoms. Subgroup 5 does not show any outstanding symptoms and low prevalence of symptoms in general.</p

Differences in PD-Q scores after IVD-surgery.

<p>The pie-chart depicts the proportion of patients with and without IVD-surgery scoring “positive”, “unclear” or “negative” in the PD-Q. There are no significant differences between the respective groups (χ²-Test, p = 0.2215).</p

Differences in PD-Q scores in the subgroups.

<p>The different scores calculated from the PD-Q are shown, revealing the proportion of positive, i.e. neuropathic and negative, i.e. non-neuropathic as well as unclear results. Patients from clusters 3 and 4 showed the tendency to score more neuropathic than those from clusters 1, 2 and 5.</p

Pain and perceived sensory symptoms in patients with axial low back pain.

*<p>mean ± standard deviation:</p>**<p>score >3 (strongly, very strongly).</p

Laser Doppler measurement.

<p><b>a)</b> Sample image of Laser Doppler measurement setup. Regions of interest (ROI) were created around the capsaicin patch stimulation area for further calculations. The intensity image depicts the color-coded perfusion magnitude. <b>b)</b> Area of elevated skin perfusion (flare) was measured in mm². There was no significant difference between the genotypes, however a trend towards smaller flare areas in 1911 GG carriers can be observed. <b>c-d</b>) Perfusion changes during capsaicin application were measured via Laser Doppler flowmetry. There was no significant difference between the genotypes, but a trend towards a decreased perfusion gain in 1911 GG carriers.</p

Thermal quantitative sensory testing of TRPV1 variants.

<p>Comparison of intra-individual data (Δ%change prior vs. after capsaicin) of homozygous G (1911GG) against homozygous and heterozygous A carriers (1911AA+1911AG) reveals lower changes (1) towards loss of warm detection in GG (13.1%) compared to AA/AG (82.1%) and (2) towards gain of heat pain sensitivity in GG (15.6%) compared to AA/AG (22.2%) ((1) p = 0.009; (2) p = 0.021). Note: Data are displayed as means (±SEM) for all analyzed patients. P<0.05 considered significance. ((1)Mann-Whitney U test, (2)ANCOVA). *p<0.05; **p<0.01.</p