Post-Translational Inhibition of IP-10 Secretion in IEC by Probiotic Bacteria: Impact on Chronic Inflammation

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Reduced physiological plasticity in a fish adapted to stable temperatures

Publisher Copyright: Copyright © 2022 the Author(s).Plasticity can allow organisms to maintain consistent performance across a wide range of environmental conditions. However, it remains largely unknown how costly plasticity is and whether a trade-off exists between plasticity and performance under optimal conditions. Biological rates generally increase with temperature, and to counter that effect, fish use physiological plasticity to adjust their biochemical and physiological functions. Zebrafish in the wild encounter large daily and seasonal temperature fluctuations, suggesting they should display high physiological plasticity. Conversely, laboratory zebrafish have been at optimal temperatures with low thermal fluctuations for over 150 generations. We treated this domestication as an evolution experiment and asked whether this has reduced the physiological plasticity of laboratory fish compared to their wild counterparts. We measured a diverse range of phenotypic traits, from gene expression through physiology to behavior, in wild and laboratory zebrafish acclimated to 15 temperatures from 10 °C to 38 °C. We show that adaptation to the laboratory environment has had major effects on all levels of biology. Laboratory fish show reduced plasticity and are thus less able to counter the direct effects of temperature on key traits like metabolic rates and thermal tolerance, and this difference is detectable down to gene expression level. Rapid selection for faster growth in stable laboratory environments appears to have carried with it a trade-off against physiological plasticity in captive zebrafish compared with their wild counterparts.Peer reviewe

Ex vivo drug response profiling detects recurrent sensitivity patterns in drug-resistant acute lymphoblastic leukemia

Drug sensitivity and resistance testing on diagnostic leukemia samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof of concept data by profiling 60 drugs on 68 acute lymphoblastic leukemia (ALL) samples mostly from resistant disease in cocultures of bone marrow stromal cells. Patient-derived xenografts retained the original pattern of mutations found in the matched patient material. Stromal coculture did not prevent leukemia cell cycle activity, but a specific sensitivity profile to cell cycle-related drugs identified samples with higher cell proliferation both in vitro and in vivo as leukemia xenografts. In patients with refractory relapses, individual patterns of marked drug resistance and exceptional responses to new agents of immediate clinical relevance were detected. The BCL2inhibitor venetoclax was highly active below 10 nM in B-cell precursor ALL (BCP-ALL) subsets, including MLL-AF4 and TCF3-HLF ALL, and in some T-cell ALLs (T-ALLs), predicting in vivo activity as a single agent and in combination with dexamethasone and vincristine. Unexpected sensitivity to dasatinib with half maximal inhibitory concentration values below 20 nM was detected in 2 independent T-ALL cohorts, which correlated with similar cytotoxic activity of the SRC inhibitor KX2-391 and inhibition of SRC phosphorylation. A patient with refractory T-ALL was treated with dasatinib on the basis of drug profiling information and achieved a 5-month remission. Thus, drug profiling captures disease-relevant features and unexpected sensitivity to relevant drugs, which warrants further exploration of this functional assay in the context of clinical trials to develop drug repurposing strategies for patients with urgent medical needs.Peer reviewe

Post-Translational Inhibition of IP-10 Secretion in IEC by Probiotic Bacteria: Impact on Chronic Inflammation

Peer reviewedPublisher PD

Cancer effects of formaldehyde: a proposal for an indoor air guideline value

Formaldehyde is a ubiquitous indoor air pollutant that is classified as “Carcinogenic to humans (Group 1)” (IARC, Formaldehyde, 2-butoxyethanol and 1-tert-butoxypropanol-2-ol. IARC monographs on the evaluation of carcinogenic risks to humans, vol 88. World Health Organization, Lyon, pp 39–325, 2006). For nasal cancer in rats, the exposure–response relationship is highly non-linear, supporting a no-observed-adverse-effect level (NOAEL) that allows setting a guideline value. Epidemiological studies reported no increased incidence of nasopharyngeal cancer in humans below a mean level of 1 ppm and peak levels below 4 ppm, consistent with results from rat studies. Rat studies indicate that cytotoxicity-induced cell proliferation (NOAEL at 1 ppm) is a key mechanism in development of nasal cancer. However, the linear unit risk approach that is based on conservative (“worst-case”) considerations is also used for risk characterization of formaldehyde exposures. Lymphohematopoietic malignancies are not observed consistently in animal studies and if caused by formaldehyde in humans, they are high-dose phenomenons with non-linear exposure–response relationships. Apparently, these diseases are not reported in epidemiological studies at peak exposures below 2 ppm and average exposures below 0.5 ppm. At the similar airborne exposure levels in rodents, the nasal cancer effect is much more prominent than lymphohematopoietic malignancies. Thus, prevention of nasal cancer is considered to prevent lymphohematopoietic malignancies. Departing from the rat studies, the guideline value of the WHO (Air quality guidelines for Europe, 2nd edn. World Health Organization, Regional Office for Europe, Copenhagen, pp 87–91, 2000), 0.08 ppm (0.1 mg m−3) formaldehyde, is considered preventive of carcinogenic effects in compliance with epidemiological findings

Association of respiratory symptoms and lung function with occupation in the multinational Burden of Obstructive Lung Disease (BOLD) study

Background Chronic obstructive pulmonary disease has been associated with exposures in the workplace. We aimed to assess the association of respiratory symptoms and lung function with occupation in the Burden of Obstructive Lung Disease study. Methods We analysed cross-sectional data from 28 823 adults (≥40 years) in 34 countries. We considered 11 occupations and grouped them by likelihood of exposure to organic dusts, inorganic dusts and fumes. The association of chronic cough, chronic phlegm, wheeze, dyspnoea, forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1)/FVC with occupation was assessed, per study site, using multivariable regression. These estimates were then meta-analysed. Sensitivity analyses explored differences between sexes and gross national income. Results Overall, working in settings with potentially high exposure to dusts or fumes was associated with respiratory symptoms but not lung function differences. The most common occupation was farming. Compared to people not working in any of the 11 considered occupations, those who were farmers for ≥20 years were more likely to have chronic cough (OR 1.52, 95% CI 1.19–1.94), wheeze (OR 1.37, 95% CI 1.16–1.63) and dyspnoea (OR 1.83, 95% CI 1.53–2.20), but not lower FVC (β=0.02 L, 95% CI −0.02–0.06 L) or lower FEV1/FVC (β=0.04%, 95% CI −0.49–0.58%). Some findings differed by sex and gross national income. Conclusion At a population level, the occupational exposures considered in this study do not appear to be major determinants of differences in lung function, although they are associated with more respiratory symptoms. Because not all work settings were included in this study, respiratory surveillance should still be encouraged among high-risk dusty and fume job workers, especially in low- and middle-income countries.publishedVersio

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Cohort Profile: Burden of Obstructive Lung Disease (BOLD) study

The Burden of Obstructive Lung Disease (BOLD) study was established to assess the prevalence of chronic airflow obstruction, a key characteristic of chronic obstructive pulmonary disease, and its risk factors in adults (≥40 years) from general populations across the world. The baseline study was conducted between 2003 and 2016, in 41 sites across Africa, Asia, Europe, North America, the Caribbean and Oceania, and collected high-quality pre- and post-bronchodilator spirometry from 28 828 participants. The follow-up study was conducted between 2019 and 2021, in 18 sites across Africa, Asia, Europe and the Caribbean. At baseline, there were in these sites 12 502 participants with high-quality spirometry. A total of 6452 were followed up, with 5936 completing the study core questionnaire. Of these, 4044 also provided high-quality pre- and post-bronchodilator spirometry. On both occasions, the core questionnaire covered information on respiratory symptoms, doctor diagnoses, health care use, medication use and ealth status, as well as potential risk factors. Information on occupation, environmental exposures and diet was also collected

Application of rRNA oligonucleotide probes for the detection of nutritional influences on bacterial metabolic activities in the gastrointestinal tract of broilers

Titelblatt, Inhaltsverzeichnis, Lebenslauf 1\. Einleitung 2\. Literaturýbersicht 3\. Zielsetzung 4\. Material und Methoden 5\. Ergebnisse 6\. Diskussion 7\. Schlussfolgerung 8\. Zusammenfassung / Summary 9\. Anhang LiteraturverzeichnisDer Einsatz Nicht-Stärke-Polysaccharide (NSP)-hydrolysierender Enzyme gehört seit langer Zeit zur Praxis in der Broilermast. Obwohl eine ganze Kaskade von antinutritiven Effekten der löslichen Nicht-Stärke-Polysaccharide Gegenstand umfangreicher Untersuchungen war und ist, bleibt für einige Aspekte noch immer ein hoher Aufklärungsbedarf. Insbesondere auf die Rolle veränderter mikrobieller Populationen im Verdauungstrakt wird immer wieder verwiesen, ohne dass diese umfassend bekannt ist. Die vorliegende Arbeit sollte einen Beitrag zum Verständnis von Einflüssen unterschiedlich gestalteter Diäten und einer Xylanase auf mikrobielle Gemeinschaften im Verdauungstrakt von Broilern leisten. Es wurde ein Tierversuch durchgeführt, bei dem durch unterschiedlichen Gehalt der Versuchsdiäten an löslichen NSP und durch den Einsatz eines Xylanasepräparates Veränderungen mikrobieller Gemeinschaften im vorderen Verdauungstrakt provoziert wurden. Es kamen eine auf Mais und Soja basierende Diät mit geringem Gehalt an löslichen NSP und zwei Diäten mit Weizen und Roggen als Hauptkomponenten, welche einen hohen Gehalt an löslichen NSP aufwiesen, zum Einsatz. Eine der beiden Weizen-Roggen-Diäten war mit einer Xylanase ergänzt worden. Alle Diäten enthielten Talg als Fettquelle und waren nahezu isoenergetisch und isonitrogen gestaltet. Im Rahmen dieses Versuches wurden die zootechnischen Leistungen der Versuchstiere erfasst. Am 7., 14., 21. und 28. Lebenstag wurden Versuchstiere geschlachtet und Digesta aus den Darmabschnitten Duodenum, Jejunum, vorderes und hinteres Ileum, sowie den Caeca gewonnen. Die Proben aus Duodenum, Jejunum, vorderem und hinterem Ileum vom 14., 21. und 28. Lebenstag der Tiere wurden zur Bestimmung intestinaler Enzymaktivitäten verwendet. Für molekularbiologische Untersuchungen wurden die Proben aus Jejunum, vorderem und hinterem Ileum verwendet. Durch Zentrifugation der Proben wurden Überstände gewonnen und damit Agardiffusionsassays zur Bestimmung verschiedener Enzymaktivitäten durchgeführt. Zur molekularbiologischen Untersuchung von RNA-Extrakten aus den Digestaproben musste die Spezifität und Sensitivität von Digoxygenin-markierten Oligonukleotidsonden, welche teilweise der Literatur entnommen und teilweise am Institut für Tierernährung der FU Berlin entwickelt worden waren, überprüft werden. Dabei wurden mögliche Abhängigkeiten der Signalstärken von der jeweils eingesetzten RNA-Menge geprüft. Nur Sonden mit reproduzierbar spezifischen Ergebnissen wurden in weiteren Untersuchungen eingesetzt. Die Gewinnung der Nukleinsäuren aus den Digestaproben erfolgte mittels modifizierter Phenol:Chloroform-Extraktion. Die Rohextrakte wurden säulenchromatographisch in DNA und RNA aufgetrennt und gereinigt. Durch Hybridisierung der aus den Proben extrahierten Gesamt-RNA mit 16S und 23S rRNA Oligonukleotidsonden wurde die Menge ribosomaler RNA ausgewählter Bakteriengruppen und -arten in den Extrakten bestimmt. Sofern möglich, wurde eine Umrechnung der jeweiligen Lichtintensitäten in ng spezifische RNA je µg Gesamt-RNA vorgenommen. Zur Auswertung der Ergebnisse wurde das arithmetische Mittel und die Standardabweichung der parallel angelegten Proben berechnet. Zur statistischen Bewertung wurden die Daten aus den einzelnen Darmabschnitten über den gesamten Versuchszeitraum zu einer Datengrundlage zusammengefasst. Die zootechnischen Leistungen der Versuchstiere lassen die in der Literatur gut belegten antinutritiven Effekte von Getreidesorten mit stark viskositätsbildenden Eigenschaften erkennen. Mittels Agardiffusionsassays wurden die Aktivitäten der Enzyme zum Abbau von NSP (Xylanase- und 1,3-1,4-β-Glucanaseaktivitäten) bestimmt. Es zeigte sich, dass durch die höhere Konzentration löslicher NSP in der Diät vor allem solche Mikroorganismen gefördert wurden, zu deren Enzymausstattung β-Glucanasen gehören. Die partielle Hydrolyse der Pentosane durch die zugesetzte Xylanase führte zu einer zusätzlichen Förderung solcher Bakterien. Ein stimulierender Einfluss von löslichen NSP und/oder der Xylanase auf xylanspaltende Mikroorganismen konnte in der vorliegenden Untersuchung nicht festgestellt werden. Ebenfalls durch Agardiffusionsassays sollte Hinweisen aus der Literatur auf eine schlechtere Verdauung der Fette bei Diäten mit hohen Gehalten an löslichen NSP nachgegangen werden. Die Ergebnisse der vergleichenden Messung der Gallensäurehydrolase- und Lipaseaktivität in diesem Versuch lassen jedoch eindeutige Relationen zwischen den Aktivitäten dieser Enzyme und der in der Literatur gut belegten schlechteren Verwertung der Nahrungsfette nicht zu. Bei den Lipaseaktivitäten wurden höhere Aktivitäten in der Gruppe mit pentosanarmer Diät erwartet. Der Umstand, dass bei den Gruppen mit pentosanreicher Diät höhere Aktivitäten gemessen wurden, könnte auf eine kompensatorische Regulation und so auf negative Einflüsse löslicher NSP auf die Fettverdauung hindeuten. Höhere Aktivitäten im Duodenum bei der Enzymgruppe gegenüber der Weizen-Roggen-Gruppe weisen auf eine bessere Durchmischung der Digesta mit Verdauungsenzymen hin. Die in der Literatur beschriebene Erhöhung mikrobieller Gallensäurehydrolaseaktivitäten bei hochviskösen Diäten konnte nicht beobachtet werden. Es wurde versucht, die mikrobiellen Gemeinschaften im Verdauungstrakt mittels Hybridisierung der aus Digestaproben der Versuchstiere gewonnenen Gesamt-RNA mit spezifischen Oligonukleotidsonden auf der Ebene von Bakteriengruppen und -arten zu charakterisieren. Die Überprüfung von Oligonukleotidsonden, welche der Literatur entnommen oder am Institut für Tierernährung der FU Berlin entwickelt wurden, hinsichtlich ihrer Spezifität zeigte, dass für gruppenspezifische Oligonukleotidsonden durchweg spezifische Hybridisierungsbedingungen zu schaffen waren. Entsprechend konnten die Oligonukleotidsonden S-D-Bact-0338-a-A-18 (bakterielle 16S rRNA), S-G-Bact-0303-a-A-17 (Bacteroides-Pretovella-Cluster), S-*-Chis-0150-a-A-23 (Clostridium histolyticum-Gruppe), 16E1 (E.coli und Shigella spp.), S-G-Enc-038-a-A-18 (Enterococcus spp.), S-F-Lact-0770-a-A-24 (Lactobacillus spp.) und S-G-Bif-1432-a-A-21 (Bifidobacterium spp.) zu Untersuchungen der RNA-Extrakte eingesetzt werden. Unter den am Institut für Tierernährung entwickelten Oligonukleotidsonden zum Nachweis von Vertretern der Enterococcus spp. konnten lediglich für S-S-Ecaec-0181-a-A-24 (E.caecorum), S-S-Efaes-1237-b-A-17 und S-S-Efeas-203-a-A-20 (E.faecalis, detektierten auch E.faecium), sowie S-S-casflaga-0185-a-A-21 (E.casseliflavus, E.flavescens, E.gallinarum, war nur für E.gallinarum spezifisch) spezifische Hybridisierungsbedingungen ermittelt werden. Sondenkanditaten zum Nachweis von E.asini, E.avium/E.raffinosus, E.columbae, E.faecium und E.hirae konnten wegen mangelnder Spezifität nicht zur Untersuchung der ENA-Extrakte eingesetzt werden. 23S rRNA-Oligonukleotidsonden zum Nachweis von E.faecium (DB6), E.avium, E.malodoratus, E.pseudoavium und E.raffinosus (DB9), von E.durans und E.hirae (Eduhi9b), sowie von E.gallinarum (Ega9b) zeigten bei Untersuchungen zur Spezifität keinerlei Signale. Die zum Nachweis von Vertretern der Lactobacillus spp. eingesetzten Oligonukleotidsonden S-S-Lacet-0061-a-A-25 (L.acetolerans), S-S-Lacid-2519-a-A-20 (L.acidophilus), S-S-Lamy-0499-a-A-24 (L.amylophilus), S-S-Lfer-061-a-A-26 (L.fermentum), S-S-Lgas-0054-a-A-24 (L.gasseri) und S-S-Lreu-0485-a-A-23 (L.reuteri) waren bereits erfolgreich in vorangegangenen Untersuchungen am Institut für Tierernährung der FU Berlin eingesetzt worden. Bei fast allen Oligonukleotidsonden konnte durch Auftrag der jeweiligen Lichtintensitäten gegen die Mengen der eingesetzten Gesamt-RNA ein Bereich linearer Abhängigkeit dieser beiden Parameter ermittelt werden. Dieser Umstand ermöglichte eine Umrechnung von Signalstärken in den Gehalt der Proben an spezifischer 16S bzw. 23S rRNA/µg Gesamt-RNA. Bei der Hybridisierung der RNA-Extrakte wies S-S-Lfer-061-a-A-26 eine exponentielle Beziehung zwischen RNA-Menge und Signalstärke auf. Bei Einsatz der Oligonukleotidsonde (16E1) war keine Abhängigkeit zu beobachten. Die Einordnung der Ergebnisse der Hybridisierungen in die in der Literatur veröffentlichten Erkenntnisse erwies sich als schwierig, da eindeutige Angaben über den Gehalt einzelner Bakterienzellen an ribosomaler RNA in Masseeinheiten bislang fehlen und Literaturangaben zu mikrobiellen Gemeinschaften im Verdauungstrakt von Broilern durch Kultivierung erzeugt wurden. Vergleiche mit solchen Untersuchungen können folglich nur unter Vorbehalten durchgeführt werden. Der Einsatz von Diäten, welche bezüglich ihres Gehaltes an löslichen NSP unterschiedlich gestaltet waren, und der Einsatz einer Xylanase führte zu unterschiedlichen Mengen von ribosomaler RNA der ausgewählten Bakteriengruppen und -arten in den Proben. Während die bakteriellen Gesamtstoffwechselaktivitäten und die Aktivitäten von Enterobakterien und Enterokokken bei der Weizen-Roggen-Diät im Vergleich zur Maisgruppe nach distal verschoben wurden, konnte insgesamt eine Verringerung der Aktivitäten von Laktobazillen beobachtet werden. Der Xylanasezusatz führt zu einer Senkung der Stoffwechselaktivitäten von Enterobakterien gegenüber der nicht supplementierten Gruppe, die Aktivitäten von Laktobazillen stiegen an. Eine Verschiebung der bakteriellen Gesamtaktivitäten gegenüber der Mais-Diät nach distal ist geringer ausgeprägt. Die Untersuchung ausgewählter Arten der Enterokokken und Laktobazillen zeigten, dass qualitative Änderungen innerhalb von Bakteriengruppen bei unterschiedlich gestalteten Diäten stattfinden. So werden vermutlich Enterokokken und Laktobazillen mit der Fähigkeit zum Abbau von β-Glucanen gefördert, wenn NSP in der Diät vermehrt enthalten sind. Der Zusatz einer Xylanase könnte diese Mikroorganismen zusätzlich fördern. Andere Vertreter dieser Bakteriengruppen scheinen durch einen hohen NSP-Gehalt der Diät im Wachstum gehemmt zu werden. Hier kann der Zusatz einer Xylanase zu einer weiteren Hemmung führen. Weiterführende Untersuchungen zur Diversität der mikrobiellen Gemeinschaften im Verdauungstrakt von Broilern und deren Charakterisierung sind wünschenswert.Non-starch-polysaccharide(NSP)-hydrolyzing enzymes are commonly used in broiler fattening. The possible mode of action of soluble non-starch- polysaccharides on growth depressing effects and the effect of NSP-hydrolyzing enzymes has been investigated for a long time. However, there is still a lack of knowledge concerning the role of intestinal microbial communities, which is often pointed out even though its role is not known in detail yet. The aim of this thesis was to contribute to a better knowledge and understanding of microbial com-munities in the gastrointestinal tract of broiler chickens and their changes due to different diets. A feeding trial was carried out to provoke changes in intestinal microbial communities by modifica-tion of the NSP content in different diets and the use of a xylanase preparation. Birds were fed ei-ther a maize based diet with low content of soluble NSP or diets based on wheat and rye with high contents of soluble NSP. One of the wheat and rye based diets was supplemented with a xylanase. Tallow was used as fat source in all diets. Performance of birds was recorded and animals were killed on day 7, 14, 21, and 28. Digesta were sampled from the duodenum, jejunum, upper and lower part of the ileum and caeca. Samples from the duodenum, jejunum, upper and lower ileum from day 14 to day 28 were used for further exami-nations. Supernatants of digesta samples were collected by centrifugation and used for agar diffu-sion assays to examine selected enzyme activities. For molecular studies digoxygenin labeled oli-gonucleotide probes were tested for specific and sensitive results in hybridization experiments and relations between optical density and amount of used RNA were examined. Only oligonucleotide probes with reproducible specific results were used for further examinations. Oligonucleotide probes were taken from literature references or developed at the Institute of Animal Nutrition, Free University Berlin. Nucleid acids were extracted from digesta samples using a modified phe-nol: chloroform protocol and raw extractions were divided in RNA and DNA and purified using silica gel columns. The amount of ribosomal RNA of selected bacterial groups and species was de-termined by hybridization of samples with specific 16S/23S oligonucleotide probes and the achieved optical density was converted in specific RNA in ng per ýg total RNA. For statistical in- terpretation, mean values and standard deviations of triplicate samples were calculated. Statistical analysis was performed by using a data pool based on the total trial period. The performance of the birds showed antinutritive effects of soluble NSP in broiler diets, which have been well documented in literature. Xylanase and β -glucanase activities in digesta supernatants were determined by agar diffusion as-says. The assays showed that an increasing content of NSP in broiler diets promotes the activity of those bacteria which are able to hydrolyze β -glucans. Partial hydrolysis of NSP by xylanase sup- plementation led to a further increase. An influence of soluble NSP and/or xylanase supplementa-Non-starch-polysaccharide(NSP)-hydrolyzing enzymes are commonly used in broiler fattening. The possible mode of action of soluble non-starch-polysaccharides on growth depressing effects and the effect of NSP- hydrolyzing enzymes has been investigated for a long time. However, there is still a lack of knowledge concerning the role of intestinal microbial communities, which is often pointed out even though its role is not known in detail yet. The aim of this thesis was to contribute to a better knowledge and understanding of microbial com-munities in the gastrointestinal tract of broiler chickens and their changes due to different diets. A feeding trial was carried out to provoke changes in intestinal microbial communities by modifica-tion of the NSP content in different diets and the use of a xylanase preparation. Birds were fed ei-ther a maize based diet with low content of soluble NSP or diets based on wheat and rye with high contents of soluble NSP. One of the wheat and rye based diets was supplemented with a xylanase. Tallow was used as fat source in all diets. Performance of birds was recorded and animals were killed on day 7, 14, 21, and 28. Digesta were sampled from the duodenum, jejunum, upper and lower part of the ileum and caeca. Samples from the duodenum, jejunum, upper and lower ileum from day 14 to day 28 were used for further exami-nations. Supernatants of digesta samples were collected by centrifugation and used for agar diffu-sion assays to examine selected enzyme activities. For molecular studies digoxygenin labeled oli-gonucleotide probes were tested for specific and sensitive results in hybridization experiments and relations between optical density and amount of used RNA were examined. Only oligonucleotide probes with reproducible specific results were used for further examinations. Oligonucleotide probes were taken from literature references or developed at the Institute of Animal Nutrition, Free University Berlin. Nucleid acids were extracted from digesta samples using a modified phe-nol: chloroform protocol and raw extractions were divided in RNA and DNA and purified using silica gel columns. The amount of ribosomal RNA of selected bacterial groups and species was de-termined by hybridization of samples with specific 16S/23S oligonucleotide probes and the achieved optical density was converted in specific RNA in ng per ýg total RNA. For statistical in- terpretation, mean values and standard deviations of triplicate samples were calculated. Statistical analysis was performed by using a data pool based on the total trial period. The performance of the birds showed antinutritive effects of soluble NSP in broiler diets, which have been well documented in literature. Xylanase and β -glucanase activities in digesta supernatants were determined by agar diffusion as-says. The assays showed that an increasing content of NSP in broiler diets promotes the activity of those bacteria which are able to hydrolyze β -glucans. Partial hydrolysis of NSP by xylanase sup- plementation led to a further increase. An influence of soluble NSP and/or xylanase supplementa- tion on xylan degrading microorganisms could not be shown. Suggestions to the negative effect of NSP on digestibility of fat were also elaborated by agar diffusion assays. However, the obtained results of lipase and bile salt hydrolase activity gave no evidence that a relation exists between the activity of these enzymes and the well documented depression in fat digestibility when NSP are added to broiler diets. Higher activities of lipases were expected in samples from birds fed the maize based diet. However, higher activities were observed in animals fed the wheat and rye based diets. This could indicate regulatory processes to compensate inferior fat digestion. Higher activities of lipases in the duodenum of the enzyme supplemented group in comparison to the group with wheat and rye diet indicate a better antiperistaltic movement of digesta. Higher activities of bacte-rial bile salt hydrolases due to high intestinal viscosity as described in literature could not be ob-served. Microbial communities of the digestive tract of broilers were characterized by hybridization of RNA from digesta samples with specific oligonucleotide probes on the level of bacterial groups and species. Hybridization results of oligonucleotide probes have shown that specific hybridization parameters could be achieved for all of the group specific oligonucleotide probes. According to these results the oligonucleotide probes S-D-Bact-0338-a-A-18 (total bacterial 16S rRNA), S-G-Bact-0303-a-A-17 (Bacteroides-Pretovella-cluster), S-*-Chis-0150-a-A-23 (Clostridium histolyticum-group), 16E1 (E.coli and Shigella spp.), S-G-Enc-038-a-A-18 (Enterococcus spp.), S-F-Lact-0770-a-A-24 (Lac-tobacillus spp.) and S-G-Bif-1432-a-A-21 (Bifidobacterium spp.) were used to screen RNA extracts of digesta samples. However, only S-S-Ecaec-0181-a-A-24 (E.caecorum), S-S-Efaes-1237-b-A-17, S-S-Efeas-203-a-A-20 (E.faecalis and E.faecium), and S-S-casflaga-0185-a-A-21 (comprising E.casseliflavus, E.flavescens, E.gallinarum, but specific only for E.gallinarum) that were developed at the Institute of Animal Nutrition, Free University Berlin, have shown specific results on species level. Sequence candidates tested for their applicability as oligonucleotide probes to detect E.asini, E.avium/E.raffinosus, E.columbae, E.faecium and E.hirae could not be used to screen RNA extracts due to their lack of specificity. 23S rRNA oligonucleotide probes taken from literature references to detect E.faecium (DB6), E.avium, E.malodoratus, E.pseudoavium and E.raffinosus (DB9), E.durans and E.hirae (Eduhi9b), and E.gallinarum (Ega9b) have shown no signals with the tested conditions. S-S-Lacet-0061-a-A-25 (L.acetolerans), S-S-Lacid-2519-a-A-20 (L.acidophilus), S-S-Lamy-0499- a-A-24 (L.amylophilus), S-S-Lfer-061-a-A-26 (L.fermentum), S-S-Lgas-0054-a-A-24 (L.gasseri) and S-S-Lreu-0485-a-A-23 (L.reuteri) had been used successfully in earlier experiments at the Insti-tute of Animal Nutrition, Free University Berlin. Most of the probes presented a linear relationship between chemiluminescence and amount of RNA. This could be used to calculate the amount of specific RNA in the samples per ýg total RNA. In hybridization experiments using RNA extracts from digesta samples S-S-Lfer-061-a-A-26 showed an exponential relationship between amount of RNA and intensity of signals. No relation-ship was observed for the enterobacterial probe 16E1. The lack of information about the content of ribosomal RNA of single bacterial cells made it diffi-cult to compare results of hybridization experiments with data from cultivation methods published in literature. Diets with different contents of soluble NSP and supplementation of a wheat and rye based diet led to different amounts of ribosomal RNA of selected bacterial groups and species. In comparison to the maize fed broilers total bacterial metabolic activities and metabolic activities of enterobacteria and enterococci were shifted distal parts of small intestine in birds fed the wheat and rye based diet. Furthermore, metabolic activities of lactobacilli decreased. Xylanase supplementation led to lower metabolic activities of enterobacteria and higher activities of lactobacilli than in the unsupple-mented group with wheat and rye based diet. The shift of total bacterial activities to the lower parts of the intestine was less pronounced. Hybridization of selected species of enterococci and lactoba-cilli showed qualitative modifications within bacterial groups according to different diets. Further examinations are necessary to evaluate and enumerate the microbial diversity in the intes-tine of broilers

Interaktionen zwischen Bakterien, Wirt und Ernährung im Darm

The intestine is colonized by a vast number of bacteria. In addition, archaea, eukaryotic microorganisms and viruses are present in the digestive tract. The entity of microorganisms that inhabit the gut are referred to as intestinal microbiota. Although this definition includes all microbes, the term microbiota will be used synonymously for the bacterial members throughout this thesis. In a physiological state, gut bacteria beneficially influence host physiology and health. In contrast, alterations in composition and function of the intestinal microbiota have been implicated in adverse effects. Endogenous host factors and nutrition are considered important modulators of intestinal microbiota and may influence the nature of bacterial effects on the host. However, the interactions between gut bacteria, the host and diet are highly complex and far from being completely understood. This thesis is based on ten peer-reviewed publications that focus on (i) endogenous factors that shape the composition of bacterial communities in a given host, (ii) selected dietary components that affect gut microbiota composition and function, and (iii) the targeted modulation by dietary supplements of intestinal microbiota composition and function. In the Introduction, microbiota composition and important functions of gut bacteria are described. Chapter 1 “Host factors influencing intestinal microbiota composition” addresses endogenous factors that may influence intestinal microbiota composition and function including the host genotype and health status. In Chapter 2 “Interactions between gut bacteria and diet”, bacterial responses towards dietary intervention in animals with a complex microbiota and in gnotobiotic rodents with a simplified model microbiota are described. In addition, the bacterial transformation of selected dietary ingredients has been studied and effects on host physiology are presented. The efficacy of microbiota manipulation by the use of prebiotics and probiotics, respectively, is the topic of Chapter 3 “Modulation of intestinal microbiota by dietary intervention”. Finally, in the General Discussion the most significant findings of the studies that are presented in this thesis are discussed in a more general context. In addition, future perspectives for studies on open questions are presented.Der Darm wird von einer großen Zahl von Bakterien besiedelt. Hinzu kommen Vertreter der Eukaryoten und der Archaeen. Die Gemeinschaft dieser Mikroorganismen wird als intestinale Mikrobiota bezeichnet, allerdings stellen die Bakterien die wichtigste Gruppe im Ökosystem Darm dar. Unter physiologischen Bedingungen tragen Darmbakterien zur Aufrechterhaltung der Gesundheit ihres Wirtes bei. Unter bestimmten Umständen können sie jedoch auch negative Effekte ausüben. Es wird angenommen, dass endogene Wirtsfaktoren und die Ernährung die Zusammensetzung und Funktion der intestinalen Mikrobiota maßgeblich beeinflussen. Allerdings sind die Interaktionen zwischen Darmbakterien, Wirt und Ernährung hoch komplex und bislang noch nicht ausreichend verstanden. Die vorliegende Arbeit basiert auf zehn Publikationen. Themen dieser Publikationen sind (i) endogene Wirtsfaktoren, die die Zusammensetzung der intestinalen Mikrobiota beeinflussen, (ii) ausgewählte Nahrungsfaktoren, welche die Zusammensetzung oder Funktion der intestinalen Mikrobiota modulieren können und (iii) Möglichkeiten zur gezielten Beeinflussung der Mikrobiota durch Nahrungs- und Futtermittelzusätze. Der Arbeit ist eine Einleitung vorangestellt, in welcher die Zusammensetzung und wichtige Eigenschaften der intestinalen Mikrobiota dargestellt werden. In den folgenden drei Kapiteln werden die oben genannten Aspekte anhand publizierter Befunde behandelt. Abschließend werden die wichtigsten Erkenntnisse dieser Arbeit in einem breiteren wissenschaftlichen Kontext diskutiert