Institut für Ernährungswissenschaften

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Antimikrobielle Aktivität der Histone bei chronisch entzündlichen Darmerkrankungen
(2017) Kunkel, Yasmin; Stange, Eduard F.
The human intestine harbours a multitude of microorganisms. In addition to its func-tion as a protective layer against pathogens, it has to prevent an excessive immune answer against commensales simultaneously. Antimicrobial Peptides (AMPs) with their cationic character are playing an essential role in protection, because they are able to form voltage dependent channels on the surface of microorganism, which kill pathogens. In addition to the classical AMPs more antimicrobial active polypeptides, such as members of the histone family, were isolated. Histones are alkaline proteins, which are components of the chromatine. They are foremost responsible for packaging the DNA and for posttranslational modifications. Five different families can be differentiated: the core histones H2A, H2B, H3 and H4, as well as the linker histone H1. While extracellular histones show strong antimicrobial activity against a broad spectrum of microorganisms, the mechanism of their toxicity has not yet been sufficiently determined. If the antimicrobial protection layer of the intestine is weakened, due to a diminished expression of AMPs for instance, microorganisms can penetrate the mucosa and trigger inflammations. These findings have been confirmed in different tissues of patients with inflammatory bowel diseases (IBD), such as Crohn’s disease (CD) and ulcerative colitis (UC). The aim of this work is to determine whether histones play a role in IBD. In the first part a systematic analysis of the transcriptome (Q-PCR) and the transla-tome (Western Blotting) of the core histones of colonic tissue was performed. In tis-sues of patients with CD gene expression data showed generally an increase of his-tones. In the cases of H2A and H2B the increase was significant. The quantification on the protein level offered an extreme variance of the expression of all core histones, irrespective of the analysed group. Significant differences were not detected. However, in trend H2B was lower in inflammation. After the systematic analysis, histones were then isolated of human colonic tissue. Before the extracted histones were fractionated via RP-HPLC and screened via MALDI-TOF-MS, different methods for the isolation of histones had been compared. The antimicrobial activity of the isolated histones of different intestinal tissues and mucus showed no differences between healthy controls and patients with IBD in flow cytometric tests. A significant increase of the histone activity in inflamed tissues of UC was only detected against S. aureus. The impact of the extracted histones seems to be strain-specific and higher against gram-positive species. As expected, extracel-lular histones could be detected in the mucus by immunehistological staining. Through ELISAs, protein concentrations of H2A and H2B were determined in the mucus and thus a slight increase of the histone proteins in UC was observed. In the last part of this work, the interactions of recombinant histones among them-selves and with other AMPs were analysed by flow cytometric viability assays. A strain-specific increase of the antimicrobial activity of histones among themselves and with AMPs was found. Thereby synergistic effects occurred frequently. The in-teractions of histones against several bacteria were visualised by electron microscope images and furthermore an agglutination of the microorganisms as well as a massive loss of cell integrity were detected. Variantions of the histones’ transcriptome and the translatome, as well as variations of the antimicrobial activity of histones in CED would have been evaluated as patho-logic defects. However, in this work such effects could not been confirmed. Because of their enormous antimicrobial activity histones still play an important role in the protection against microorganisms in the colon. Further studies have to show, if his-tones possess a therapeutic potential, and if they can be used as new antibiotics. This work was able to verify the strong potential of histones against different pathogens, which is absolutely comparable with the potential of classic AMPs, and could pro-mote inspiration for subsequent studies.
Entwicklung eines computergestützten Assessment-Tools zur Erfassung des Ernährungszustandes von Senioren
(2011) Ott-Renzer, Cornelia; Biesalski, Hans-Konrad
Introduction: Worldwide a demographic change of population is to be observed, thus also in Germany. Special expertise in geriatric diagnostics and therapy will gain in importance. Thereby, a special relevance comes up to determination of nutritional status by corresponding screening and assessment. Conventional assessment mostly equates to questionnaire methods. Purpose/Question: Focus was on developing a software (geroMAT-Malnutrition Assessment and Therapy for gerontologic patients) for identification Senior´s nutritional state, a kind of "management tool" for diagnostics and intervention. Methods: Investigation was open, cooperative and multi-centric, as well as clinical-experimentally invested. In three partial studies (I: Suitability of the MNA® as a reference method; II: Anthropo-metry, biochemistry, body composition; III: Food patterns and intake, not-nutritive factors) indicators of malnutrition were initially selected. In a final analysis Model I (prognosis of malnutritional risk) and accordingly Model II (prognosis of the MNA®) have been developed for their use in geroMAT. Results: Prevalence of malnutrition was (in this random sample) 5%, 44% were at risk and 51% were well nourished. Due to inhomogeneity in class range by assessment with the MNA®, modelling of a dichotomic risk variable ("RiskMal", homogeneous) occurred. All up 25 features and 12 (optional) additional items from the partial studies I-III could be generated and attached to further analysis. Model I prognosticated "RiskMal" reliably (auROC=.739). Although firstly Model II predicted MNA® well (r=0,5167), model quality could be improved even further by the well-chosen parametres of a feature subset selection for Models I and II (Í: r=0,822;II: r=0,6634). Discussion: The Models I/II reached the requirements made on developing geroMAT. According to the features, geroMAT would be multi-centric usable, simple to learn and operable, documentable and reproduceable, interprofessionally and without high expense, as well as modern. Advantages of geroMAT, compared with the MNA® lay in its detailedness and its choice of further options, its capture of documented information off the normal anamnesis process and the initiation or monitoring of individual interventions. Conclusions: Mean aim of the study, the identification of indicators and model development, was reached. Other model validation studies should follow before the final clinical practice of geroMAT.
Entwicklung und Charakterisierung verschiedener humaner 3D Fettgewebemodelle mit und ohne Hydrogelmatrix als in vivo nahe Alternative
(2023) Albrecht, Franziska Brigitte; Kluger, Petra
Humanes Fettgewebe sekretiert hunderte regulatorisch aktive Hormone, die bei der Entstehung und Manifestierung schwerwiegender Krankheiten wie Diabetes oder kardiovaskulären Erkrankungen beteiligt sind. Um die Vorhersagekraft von in vitro Modellen zu verbessern und die Komplexität des nativen Fettgewebes besser nachzubilden, werden dringend dreidimensionale (3D) in vivo nahe Fettgewebemodelle benötigt. Um solch ein flexibel anwendbares möglichst physiologisches Modell aufzubauen wurde in dieser Arbeit der Aufbau und die Evaluierung verschiedener 3D Fettgewebemodelle teils mit artifizieller Matrix angestrebt und im Anschluss mit dem nativen Zustand vergleichen. Beginnend wurden aus primärem humanem Fett Lobuli in verschiedenen Größenbereichen isoliert, kultiviert und der Zelltod sowie Zeichen der Lipolyse bestimmt. Es hat sich gezeigt, dass Lobuli mit einem Gewicht von 27 – 70 mg über 15 Tagen die stabilsten Ergebnisse geliefert haben und wurden somit tiefergehend analysiert. Bei der Analyse der Viabilität und Zellfunktionalität in Form der Lipidakkumulation und der Expression von Perilipin A konnte bewiesen werden, dass Lobuli als Fettgewebemodell genutzt werden können. Als weiteres Modell erfolgte die Etablierung von Sphäroiden aus humanen primären Stammzellen aus dem Fettgewebe (adipose-derived stem cells, ASC). Die Sphäroide wurden für 14 Tage adipogen differenziert und währenddessen die Veränderung des Volumens und der Rundheit sowie die Expression von Zelltodmarkern und das adipogene Differenzierungspotenzial betrachtet. Dabei haben Sphäroide mit mehr als 100.000 Zellen ihr Volumen über die Zeit verkleinert und Apoptose- und Nekrosemarker an den Tagen 0 und 14 gezeigt. Sphäroide bis 100.000 Zellen haben ihr Volumen während der Differenzierung leicht vergrößert und haben die meisten Lipide akkumuliert, Perilipin A und Kollagen Ⅵ exprimiert. Somit konnten ASC-basierte Sphäroide aus 10.000 Zellen, mit signifikanter Lipidzunahme, als weiteres Fettgewebemodell mit hohem Potenzial identifiziert werden. Im Anschluss erfolgte die Etablierung von methacrylierter Gelatine (GelMA) als Biomaterial für die Verkapselung von primären ASCs und reifen Adipozyten (adipocyte, AC) mit anschließendem extrusions-basierten 3D Druck. Es konnte gezeigt werden, dass die homogene Verteilung der lipidgefüllten ACs in eine wässrige Biotintenlösung nur durch schnelles Herunterkühlen auf Eis möglich war. Der anschließende 3D Druck hatte, weder auf die Viabilität noch Funktionalität bzw. adipogene Differenzierung der Zellen signifikante Einflüsse. Die additiv aufgebauten Modelle wiesen an Tag 1 und 8 bzw. 15 in der Lebend-Tot-Färbung keinen vermehrten Zelltod im Vergleich zu den manuellen Modellen auf. Die Färbung der intrazellulären Lipide und Perilipin A sowie die Glycerolfreisetzung als Funktionalitätsmarker, haben dies bestätigt. Im Vergleich zu nativem Gewebe haben differenzierte ASCs (diffASC) nach der Differenzierung signifikant weniger lipidpositive Zellen und freigesetztes Glycerol gezeigt. Auch morphologisch betrachtet, haben ACs in GelMA mehr Ähnlichkeiten zum nativen Gewebe als diffASCs. Aufgrund der begrenzten Langzeitstabilität von GelMA, des Ursprungs und der Vernetzung wurden Biomaterialien ohne tierischen Ursprung evaluiert. Dabei hat sich das niedrig acetylierte Polysaccharid Gellan Gum (GG) als vielversprechendes Material erwiesen. Es konnten stabile und transparente Hydrogele durch die Vernetzung mit divalenten Ionen im Zellkulturmedium aufgebaut werden. Die 1 %-igen Hydrogele waren weder bei indirekter noch direkter Testung zytotoxisch oder haben Monozyten aktiviert. Bestätigt wurde es durch die Laktatdehydrogenase (LDH)-Freisetzung, den Resazurinumsatz und eine Lebend-Tot-Färbung. Azelluläre Gele wurden über 98 Tagen ohne signifikante Veränderungen erhalten und zeigten für Fettgewebe geeignete Materialeigenschaften. Aufgrund dieser Langzeitstabilität konnten diffASCs für 98 Tage in GG kultiviert werden und zu univakuolären Fettzellen reifen. Dies wurde durch die Färbung von intrazellulären Lipiden und Perilipin A sowie der Leptinsekretion und Glycerolfreisetzung bewiesen. Verglichen mit nativem Gewebe wiesen die diffASCs eine vergleichbare, wenn auch kleinere Morphologie, ähnliche Anteile an lipidpositiven und univakuolären Zellen auf. Auf Basis von GG konnten ACs erfolgreich verkapselt und aufgebaut werden. Hierbei erwies sich eine GG Konzentration von 0,5 % als geeignet, da homogen gemischte Hydrogele mit hohem Resazurinumsatz und geringer Glycerolfreisetzung kultiviert werden konnten. Für den additive Aufbau der GG-basierten Modelle, fand eine Optimierung zur Biotinte statt, was durch die initiale Zugabe divalenter Ionen zur Erhöhung der Viskosität und damit Druckbarkeit ermöglicht wurde. Die additiv gefertigten Modelle zeigten keine signifikanten Unterschiede in Bezug auf Viabilität oder Funktionalität. Nach 32 Tagen in Kultur konnten univakuoläre und funktionale Zellen gezeigt werden. Durch die Erhöhung der GG Konzentration auf 1,5 % wurde ein erfolgreiches 6D Bioprintingverfahren etabliert. Auch hier zeigten die in GG verkapselten ASCs keine Viabilitäts-, Morphologie-, oder Differenzierungsunterschiede nach dem Druckprozess. Abschließend erfolgte ein Vergleich der etablierten Modelle wobei die Adipogenese der ASC-basierten Modelle und zum anderen der adipozytenspezifische Phänotyp evaluiert wurde. Hinsichtlich der Viabilität konnten keine Unterschiede festgestellt werden. Das adipogene Differenzierungspotenzial zeigte sich am stärksten im diffASC Hydrogel. Sowohl hier als auch in den Sphäroiden konnten eingelagerte Lipide ohne hormonelle Induktion mit Medium nachgewiesen werden. Die Betrachtung der adipozytenspezifischen Morphologie der ausgereiften Modelle erlaubte den Rückschluss, dass die Zellen in den Monolayern den unausgereiftesten Zustand in Form von multivakuolären, elongierten diffASCs mit Aktin-Stressfasern aufweisen. Die Zellen der anderen Modelle zeigen deutlich größere Lipidvakuolen mit einem abgerundeten Zytoskelett. Die quantitative Bestimmung der Lipid-, Leptin- und Glycerolmenge legt nahe, dass sich die Zellen, außer die im Monolayer, in einem Steady-State befinden, was die relative Genexpression adipozytenspezifischer Gene untermauert. Zusammengefasst wurden in dieser Arbeit sechs unterschiedliche Fettgewebemodelle entwickelt und mit nativem Gewebe verglichen. Jedes Modell weist individuelle Stärken auf, wodurch sich der Anwendungsbereich und die Forschungsfragen unterscheiden. Um ein möglichst in vivo-nahes Modell zu erreichen, sind diffASCs in GG Hydrogelen ein vielversprechendes, langzeitstabiles, ausgereiftes Fettgewebemodell und können daher als flexible Plattform für in vitro Testungen genutzt werden.
Intrazelluläres Trafficking des intestinalen Anionenaustauschers Down-Regulated in Adenoma (DRA;SLC26A3)
(2011) Lissner, Simone; Graeve, Lutz
Electroneutral NaCl absorption occurs from the small intestine to the distal colon. This ion exchange is preferentially mediated by DRA and NHE3. Knockout mice, which suffer from chronic diarrhea, as well as the human genetic disorder congenital chloride diarrhea, in which a nonfunctional DRA leads to life-threatening diarrhea emphasize the importance of these two transporters. To elucidate this defective NaCl absorption it is necessary to understand the physiological regulation of these two transport proteins within enterocytes as well as the responsible extra- and intracellular signal transduction pathways. Both transport proteins interact with PDZ adaptor proteins of the NHERF family. Furthermore, both exchangers are partially localized within lipid rafts. The situation for NHE3 is complex in that its lipid raft localization is not only necessary for its normal activity but also for its basal and stimulated trafficking. Lipid rafts are involved in PI3-kinase dependent exocytosis of NHE3. Since the function of NHE3 and DRA appears to be regulated in parallel the function of DRA maybe depends on its rafts association as well. Thus the first objective of this thesis was to investigate whether the lipid raft association of DRA is essential for the surface expression and transport activity of DRA and also to analyze whether DRA is inserted into the plasma membrane in a PI3-kinase and lipid raft dependent manner. The present data show that: (A) Disruption of lipid raft integrity leads to functional inhibition and decreased cell surface expression of DRA. In HEK cells the inhibition of DRA activity as well as the decreased cell surface expression are entirely dependent on the presence of the PDZ interaction motif of DRA. In Caco-2/BBE cells on the other hand only part of the inhibition of DRA activity by disruption of raft integrity depends on the ability of DRA to interact with PDZ adaptor proteins. (B) Basal activity as well as basal surface expression of DRA depend on PI3-kinase activity in a way that requires the ability of DRA to interact with PDZ adaptor proteins. (C) Lipid rafts and PI3-kinase are situated along the same pathway, where DRA is present in lipid rafts before it is inserted into the plasma membrane. However, the inhibition of PI3-kinase has no influence on the raft association of DRA. Furthermore, the disruption of raft integrity does not inhibit the PI3-kinase activity. Based on these findings a model can be established as follows: DRA is present in lipid rafts in an intracellular fraction. Insertion into the plasma membrane from this intracellular compartment requires the interaction with one (or several) PDZ adaptor proteins, raft integrity and the action of PI3-kinase. To characterize the interplay between PI3-kinase, raft association and PDZ interaction of DRA with its insertion into the plasma membrane the recycling pathway of DRA was then investigated. The generated data show that the proteolytic degradation of DRA-ETKFminus occurs faster than the degradation of wild type DRA. Endosomal distribution of DRA depends on its PDZ-binding motif. The sorting process from early to recycling endosomes depends on the interaction of DRA with one or several PDZ adaptor proteins. Expression of dominant negative Rab11a leads to a decreased surface expression and transport activity of DRA. In conclusion, it was shown in this thesis that an intense interplay between PDZ interaction, lipid raft association, PI3-kinase and the activity and surface expression of DRA exists. It was also shown that the endosomal distribution of DRA depends on its PDZ-binding motif. Finally, it was demonstrated that DRA is recycled to the plasma membrane by Rab11a-enriched recycling endosomes.
Nutrition and tuberculosis in Ethiopia : the role of vitamin D2 derived from sun exposed oyster mushroom on the treatment outcomes of tuberculosis
(2019) Keflie, Tibebeselassie Seyoum; Biesalski, Hans-Konrad
Tuberculosis (TB) is an old infectious disease which causes ill-health among millions of people each year. Effective anti-TB drugs are available since 1950’s, but still the global burden of TB remains enormous. The disease is very complex and there is a need to look for supportive treatment to the standard anti-TB drugs. Cognizant of this, the present doctoral study was undertaken by giving emphasis on nutrition and TB in Ethiopia. The aim of this doctoral dissertation thesis was to deal with the nutritional situation of people with and without TB and come-up with solutions that could support the effort of combating TB. In this thesis, five papers (four published and one submitted) were included. The first paper encompassed the study of dietary and nutritional assessment. In this study, dietary inadequacy, poor nutritional quality and high risk of micro nutrient deficiencies were identified. The main dietary pattern included cereals, vegetables and legumes. About one-third of the population consumed animal source food (ASF). Malnutrition was the common problem in people with and without TB. This suggested that malnutrition may pave the way for TB. The case-control study in the second paper revealed that more than one-half of TB patients had vitamin A and zinc deficiencies. More than three-fourth of TB patients had below half of the energy fulfillment. The protein intake was above the average fulfillment, but most TB patients relied on cereal-based diets. Patients with TB used a larger proportion of proteins from oral feeding for oxidation and hence for energy production. About half of the patients were undernourished. Thus, vitamin A and zinc deficiencies along with protein-energy malnutrition need to be addressed in the management program of TB. The third paper included systematic review which explored the existence of vitamin D deficiency (VDD). Sunshine, which is very important for the synthesis of vitamin D under the skin is widely available in Africa throughout the year. Surprisingly, more than three-fourth of TB patients in Africa had VDD and vitamin D insufficiency (VDI). Statistically significant variables such as use of sun protection (lack of sun-exposure), inadequate dietary intake, low body mass index (BMI), high skin pigmentation, use of drugs (anti-retro viral and /or anti-TB), low socioeconomic status, rainy season, covering body skin with clothes, old age and co-morbidity were identified as the main predictor variables that hampered the status of vitamin D. Vitamin D can be obtained from dietary intakes apart from endogenous synthesis after sun exposure. Mushroom as such, is a potential non-animal source of vitamin D. The experimental study in the fourth paper revealed that sun-exposure significantly increased the content of vitamin D2 in oyster mushroom. Increasing the surface area for sun-exposure enhanced the production of vitamin D2. Other factors such as duration of sun-exposure and moisture content determined the production of vitamin D2. Exposing slices of oyster mushroom to direct sun for brief period provided enough vitamin D2 that could satisfy the current recommended dietary allowance (RDA) of vitamin D without any visible changes in color and texture. The study in the fifth paper was a randomized controlled trial and demonstrated for the first time the role of mushroom-derived vitamin D2 on the treatment outcomes of TB. Intervention with vitamin D2 derived from sun-exposed oyster mushrooms brought significant improvement in vitamin D status, clinical outcomes and immunological responses, but not in sputum smear and culture conversion. The intervention corrected VDD in more than one-third of TB patients. About one-third of the variability in TB score in the intervention group was accounted for by the change in the serum 25 hydroxy (OH) vitamin D level. There were also significant improvements in the serum IFN-gamma and cathelicidin LL-37 peptide levels after intervention. The balance of cytokines was skewed to TH1 responses due to high level of IFN-gamma. Thus, mushroom-derived vitamin D2 could serve as potential, safe, easily available and cost-effective adjunctive therapy for TB. Taken collectively, foods enriched with vitamin D need to be included in the national TB control program to support the first line anti-TB drugs, increase the cure rate and reduce the infectiousness of TB.
Promoting dietary diversification in the ASEAN region : exposing food taboos, and exploring the nutrient profiles of underutilized, indigenous food resources
(2020) Köhler, Realm; Biesalski, Hans-Konrad
The Association of Southeast Asian Nations (ASEAN) is composed of Brunei Darussalam, Cambodia, Indonesia, Lao PDR, Malaysia, Myanmar, Philippines, Singapore, Thailand, and Vietnam. The ASEAN region is ailing from moderate to serious incidence of malnutrition. Among the member countries, Brunei Darussalam has the highest prevalence of child obesity (17.8%), while Malaysia has the highest prevalence of adult obesity (15.6%). Indonesia has the highest percentage of young children suffering from wasting at 13.5%. Lao PDR has the highest percentage of the undernourished in the population (16.5%), at the same time, having the highest percentage of stunted children under five years of age (43.85%). It also has the highest HHI score with 38.7, which corresponds to having a severe case of micronutrient deficiencies, and the highest death rates for both children under five years of age (63 deaths per 1,000 live births) and mothers (197 deaths per 100,000 live births). To fight malnutrition, nutrition-specific interventions address the immediate determinants of nutrition of specific vulnerable groups – young children, pregnant and lactating women, and others. Dietary diversification is an example of a nutrition-specific intervention. This dissertation was conducted to turn the spotlight towards the ASEAN region, its triple burden of malnutrition, and to dietary diversification as a sustainable way to lighten the load. It tackled one of the stumbling blocks to the acceptance of dietary diversification – food taboos, and one of the stepping stones towards its successful implementation – nutrient profiling of underutilized, indigenous resources in the region. This dissertation postulated that plant- and animal-based food taboos adhered to by pregnant, post-partum, and lactating Southeast Asian women can hinder dietary diversification in the most vulnerable and crucial moment of the first 1,000 days of life. The two review papers generated were the first to consolidate and showcase researches on food taboos covering the region. They highlighted the need for culture-sensitive health interventions to address maternal and child health problems that could lead to the attainment of the sustainable development goals of reducing the maternal and under-five mortality ratios and empowering women in Southeast Asia, as well as the priority health goals of the ASEAN. The underutilized, indigenous resources in the ASEAN region have the potential to be valuable components of a diversified diet. To prove this statement and to further promote dietary diversification, the dissertation tackled the nutrient profiling of the edible insects – Bombay locust (Patanga succincta), scarab beetle (Holotrichia sp.), house cricket (Acheta domesticus), and mulberry silkworm (Bombyx mori) from Thailand, and the sago grub (Rhynchophorus bilineatus) from Indonesia. For the pigmented rice varieties, the Camoros (red), Tinta (purple) and Malinao black rice from the Philippines were analyzed, while a review of pigmented rice varieties from Thailand was also conducted. The results of the analysis showed, and based on the Codex Alimentarius on food labelling, that the edible insects are “high in” protein and can be “sources of” or “high in” minerals. Also, data showed that the pigmented rice varieties from the Philippines and the pigmented Thai rice varieties have higher mineral and vitamin contents in comparison with white Jasmine rice. The findings in this dissertation have shown that edible insects and pigmented rice varieties can be added to diversify and improve the nutritional quality of people’s diets and to fight malnutrition from the household level. The novel research into indigenous food resources contributes to the advancement of knowledge in the field of entomology and biodiversity conservation, and of course, in food science and nutrition. Most importantly, the dissertation’s contribution to the promotion of dietary diversification in the hope of attaining improved human health and nutrition will benefit the whole ASEAN region.

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