Einfluss einer schmerzhaften Entzündung auf die Expression des Proopiomelanocortin-Gens und die β-Endorphin Produktion in Lymphozyten

1\. Titel, Contents, and Abbreviations 1 2\. Introduction 7 3\. Objective 23 4\. Materials and Methods 24 5\. Results 46 6\. Discussion 70 7\. Summary 87 8\. Zusammenfassung 89 9\. References 91 10\. Publications 106 11\. Appendix 109POMC is the precursor of different biologically active peptides like ACTH and END and is produced in the pituitary and in non-pituitary tissues such as immune cells. END is released from immune cells migrating into inflamed tissue which can attenuate inflammatory pain by activating opioid receptors on peripheral terminals of sensory neurons. While all biologically active POMC- derived peptides such as END are encoded by exon 3, the signal peptide required for the entry of the nascent polypeptide into the regulated secretory pathway, is encoded by exon 2 of the POMC gene. However, the expression of signal sequence-encoding POMC mRNA in lymphocytes has long been a matter of debate which is related to the predominant expression of 5`-end truncated transcripts that do not encode the signal sequence. Hypothesizing that the transcription of such POMC mRNA is enhanced in lymphocytes under pathological situations the present study investigated POMC mRNA expression and END levels in the draining LN during the development of a local painful paw inflammation in rats. Using RT- and RACE-PCR several POMC mRNA molecules were identified. Besides detection of low levels of full-length POMC mRNA comprising exons 1, 2, and 3, various 5`-end truncated transcripts were expressed in lymphocytes. In accordance with previous studies none of these truncated POMC mRNAs encoded the signal sequence. While levels of truncated POMC mRNA remained unchanged, qRT-PCR analysis showed that exon 2-3 spanning POMC mRNA levels increased in lymphocytes 2 h after induction of paw inflammation. In the pituitary, the expression level of exon 2-3 spanning POMC transcripts was unaltered, indicating that POMC gene expression was not systemically enhanced. Immunofluorescence showed co-localization of POMC and END in LN cells and flow cytometry analysis showed that opioid peptides were expressed in T helper and cytotoxic T-cells, in B-cells and in monocytes/macrophages. By cell separation signal sequence-encoding POMC transcripts were identified in both T- and B-cells. The mRNAs of the POMC processing enzymes PC1/3 cleaving POMC into ACTH and beta-LPH and PC2 that converts beta-LPH into END were differentially expressed in T- and B-cells. T lymphocytes expressed only PC2 while the expression of both prohormone convertases was induced in B-cells after induction of paw inflammation. Radioimmunoassay analysis revealed that the amount of END increased more than 2-fold on a cellular base within the first 12 - 24 h of inflammation. Together these findings indicate that newly synthesized lymphocytic END can be attributed to an enhanced transcription of signal sequence-encoding POMC mRNA during inflammation. This provides an important missing link in the demonstration of a classical processing of POMC into biologically active peptides in immune cells. In accordance with findings in pituitary cells, signal sequence-encoding POMC mRNA seems to be translated and processed into END within 6 - 12 h in lymphocytes.POMC ist der Precursor verschiedener biologisch aktiver Peptide wie ACTH und END. Neben der Hypophyse produzieren auch nicht-hypophysäre Gewebe wie Immunzellen POMC und die davon abgeleiteten Peptide. Immunzellen setzen END beispielsweise in entzündetem Gewebe frei und durch die Bindung dieses Opioids an Opioidrezeptoren auf peripheren Nervenendigungen kann Entzündungsschmerz vermindert werden. Während alle Sequenzen der biologisch aktiven Peptide auf dem dritten Exon des POMC-Gens kodiert sind, befindet sich die Sequenz zur Translation des Signalpeptids, welches für den Eintritt des Vorläufermoleküls in den sekretorischen Weg unverzichtbar ist, auf dem zweiten Exon. In Lymphozyten wird die Expression von Signalsequenz-kodierender POMC mRNA seit längerem kontrovers diskutiert, da diese Immunzellen vorwiegend am 5`-Ende verkürzte Moleküle ohne Signalsequenz exprimieren. Unter der Annahme, dass die Transkription Signalsequenz-kodierender POMC mRNA unter pathologischen Bedingungen in Lymphozyten induziert wird, untersuchte die vorliegende Studie die Expression von POMC mRNA und den END-Gehalt im drainierenden Lymphknoten im Verlauf einer schmerzhaften, lokalen Pfotenentzündung an Ratten. Mittels RT- und RACE-PCR konnten eine Vielzahl an verschiedenen POMC mRNA-Molekülen identifiziert werden, darunter das schwer detektierbare, Exon 1, 2 und 3 einschließende Full-length POMC und diverse am 5`-Ende verkürzte Moleküle. In Übereinstimmung mit anderen Studien wiesen die verkürzten POMC mRNAs keine Signalsequenz auf. Die Quantifizierung von Exon 2-3 überspannenden POMC mRNA Transkripten mittels qRT-PCR zeigte, dass diese nach 2 Stunden Entzündungszeit um ein Vielfaches hochreguliert wurde, dagegen zeigten sich bei verkürzter POMC mRNA keine Expressionsunterschiede. Da das Expressionsniveau von Exon 2-3 überspannender POMC mRNA nach Induktion der Pfotenentzündung in der Hypophyse unverändert blieb, scheint kein systemischer Effekt an der Erhöhung dieser POMC mRNA in den Lymphozyten beteiligt zu sein. Die Analyse von Lymphknoten mittels Immunfluoreszenz zeigte, dass POMC und END in Lymphknotenzellen co- lokalisiert sind und mittels Durchflusszytometrie und Immunohistochemie konnten Opioidpeptide wie END in T-Helferzellen, in zytotoxischen T-Zellen, in B-Zellen und Monozyten/Makrophagen identifiziert werden. Entsprechend konnte die Expression von Signalsequenz-kodierender POMC mRNA in T- und B-Zellfraktionen separierter Lymphknoten gezeigt werden. T-Lymphozyten exprimierten zudem die mRNA des POMC Prozessierungsenzyms PC2, das für die Konvertierung von beta-LPH in END zuständig ist. In B-Zellen war die Expression von PC2 erst nach Induktion der Entzündung detektierbar. Die Prohormonkonvertase PC1/3, die POMC in ACTH und beta-LPH konvertiert, war ausschließlich in B-Zellen entzündeter Lymphknoten nachweisbar. Die kinetische Peptidanalyse ergab, dass sich der zelluläre END-Gehalt von Lymphozyten innerhalb von 12 - 24 h Entzündungsdauer mehr als verdoppelte. Diese Befunde deuten darauf hin, dass die Neusynthese von END im Entzündungsverlauf auf eine verstärkte Transkription von Signalsequenz-kodierender POMC mRNA zurückgeführt werden kann. Diese Ergebnisse erbringen zudem den Nachweis eines wichtigen fehlenden Bindegliedes bei der Demonstration der klassischen Prozessierung von POMC in biologisch aktive und sezernierbare Peptide in Immunzellen. Im Einklang mit ähnlichen Befunden aus der Hypophyse scheinen in Lymphozyten zwischen erhöhter Transkription und Peptidanstieg unter Entzündungsbedingungen etwa 6 - 12 Stunden zu vergehen

The Effect of Metronidazole versus a Synbiotic on Clinical Course and Core Intestinal Microbiota in Dogs with Acute Diarrhea

The usefulness of antibiotics in dogs with acute diarrhea (AD) is controversial. It is also unclear what effect metronidazole has on potential enteropathogens such as Clostridium perfringens and Escherichia coli. Thus, the aim of this study was to evaluate the effect of metronidazole vs. a synbiotic on the clinical course and core intestinal bacteria of dogs with AD. Twenty-seven dogs with AD were enrolled in this prospective, randomized, blinded clinical trial and treated with either metronidazole (METg) or a synbiotic (SYNg; E. faecium DSM 10663; NCIMB 10415/4b170). The Canine Acute Diarrhea Severity (CADS) index was recorded daily for eleven days. Bacteria were quantified using qPCR. Data were analyzed using mixed models with repeated measures. A higher concentration of E. coli was observed in the METg group vs. the SYNg group on Day 6 (p < 0.0001) and Day 30 (p = 0.01). Metronidazole had no effect on C. perfringens. C. hiranonis was significantly lower in the METg group than in the SYNg group on Days 6 and 30 (p < 0.0001; p = 0.0015). No significant differences were observed in CADS index, fecal consistency, or defecation frequency between treatment groups (except for the CADS index on one single day). In conclusion, metronidazole negatively impacts the microbiome without affecting clinical outcomes. Thus, synbiotics might be a preferred treatment option for dogs with AD

In Silico Approaches and the Role of Ontologies in Aging Research

The 2013 Rostock Symposium on Systems Biology and Bioinformatics in Aging Research was again dedicated to dissecting the aging process using in silico means. A particular focus was on ontologies, as these are a key technology to systematically integrate heterogeneous information about the aging process. Related topics were databases and data integration. Other talks tackled modeling issues and applications, the latter including talks focussed on marker development and cellular stress as well as on diseases, in particular on diseases of kidney and skin

A low-luminosity type-1 QSO sample: II. Tracing circumnuclear star formation in HE 1029-1831 with SINFONI

Circumnuclear star formation and AGN feedback is believed to play a critical role in the context of galaxy evolution. The low-luminosity QSO (LLQSO) sample that contains 99 of the closest AGN with redshift z<=0.06 fills the gap between the local AGN population and high-redshift QSOs that is essential to understand the AGN evolution with redshift. In this paper, we present the results of near-infrared H+K-integral field spectroscopy of the inner kiloparsecs of the LLQSO HE 1029-1831 with SINFONI. Line maps show that ionized hydrogen gas is located in spiral arms within the stellar bar and in a circumnuclear ring. Line fluxes and diagnostic line ratios indicate recent or ongoing star formation in the circumnuclear region and the presence of young and intermediate-age stellar populations in the bulge. In particular, we find traces of an intense starburst in the circumnuclear region that has begun around 100 Myr ago but has declined to a fraction of the maximum intensity now. We estimate the dynamical bulge mass and find that the galaxy follows published M_BH-M_bulge relations. However, bulge-disk decomposition of the K-band image with BUDDA reveals that HE 1029-1831 does not follow the M_BH-L_bulge relations of inactive galaxies. We conclude that the deviation from M_BH-L_bulge relations of inactive galaxies in this source is rather caused by young stellar populations and not by an undermassive black hole.Comment: 16 pages, 11 figures, submitted to A&A, comments welcom

The use of urinary proteomics in the assessment of suitability of mouse models for ageing

Ageing is a complex process characterised by a systemic and progressive deterioration of biological functions. As ageing is associated with an increased prevalence of age-related chronic disorders, understanding its underlying molecular mechanisms can pave the way for therapeutic interventions and managing complications. Animal models such as mice are commonly used in ageing research as they have a shorter lifespan in comparison to humans and are also genetically close to humans. To assess the translatability of mouse ageing to human ageing, the urinary proteome in 89 wild-type (C57BL/6) mice aged between 8–96 weeks was investigated using capillary electrophoresis coupled to mass spectrometry (CE-MS). Using age as a continuous variable, 295 peptides significantly correlated with age in mice were identified. To investigate the relevance of using mouse models in human ageing studies, a comparison was performed with a previous correlation analysis using 1227 healthy subjects. In mice and humans, a decrease in urinary excretion of fibrillar collagens and an increase of uromodulin fragments was observed with advanced age. Of the 295 peptides correlating with age, 49 had a strong homology to the respective human age-related peptides. These ortholog peptides including several collagen (N = 44) and uromodulin (N = 5) fragments were used to generate an ageing classifier that was able to discriminate the age among both wild-type mice and healthy subjects. Additionally, the ageing classifier depicted that telomerase knock-out mice were older than their chronological age. Hence, with a focus on ortholog urinary peptides mouse ageing can be translated to human ageing

Prevalence of Clostridioides difficile in Canine Feces and Its Association with Intestinal Dysbiosis

The role of Clostridioides (C.) difficile as an enteropathogen in dogs is controversial. In humans, intestinal bile acid-dysmetabolism is associated with C. difficile prevalence. The relationship between fecal qPCR-based dysbiosis index (DI) and especially the abundance of bile acid-converting Clostridium hiranonis with the presence of C. difficile in dogs was explored across the following 4 cohorts: 358 fecal samples submitted for routine diagnostic work-up, 33 dogs with chronic enteropathy, 14 dogs with acute diarrhea, and 116 healthy dogs. Dogs that tested positive for C. difficile had significantly higher DI (median, 4.4 (range from 0.4 to 8.6)) and lower C. hiranonis (median, 0.1 (range from 0.0 to 7.5) logDNA/g) than dogs that tested negative for C. difficile (median DI, −1 (range from −7.2 to 8.9); median C. hiranonis abundance, 6.2 (range from 0.1 to 7.5) logDNA/g; p < 0.0001, respectively). In 33 dogs with CE and 14 dogs with acute diarrhea, the treatment response did not differ between C. difficile-positive and -negative dogs. In the group of clinically healthy dogs, 9/116 tested positive for C. difficile, and 6/9 of these had also an abnormal DI. In conclusion, C. difficile is strongly linked to intestinal dysbiosis and lower C. hiranonis levels in dogs, but its presence does not necessitate targeted treatment

Impact of Study Skills and Parent Education on First-Year GPA Among College Students With and Without ADHD: A Moderated Mediation Model

Objective: To test if the relationship between ADHD and academic achievement is mediated by service utilization and/or study skills, and if these mediation effects are moderated by parental education level. Method: A bootstrapping method within structural equation modeling was used with data from 355 first year college students meeting strict criteria for ADHD or clearly without ADHD to test the mediation and moderation effects. Results: Study skills, but not service utilization, significantly mediated the relationship between ADHD status and GPA; however, this relationship was not significant among students with at least one parent holding a master’s degree or higher. Conclusion: Among first year college students study skills may be a more salient predictor of educational outcomes relative to ADHD status. Additional research into support services for college students with ADHD is needed, however, results suggest interventions targeting study skills may hold particular promise for these students