Institut für Ernährungswissenschaften

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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.
Epidemiology and social determinants of chronic diseases attributed adult mortality and its influence on maternal and young child nutrition in Tigray, 2009-2015: evidence from Kilte Awlaelo- Health and Demographic Surveillance Site
(2023) Abera, Semaw Ferede; Scherbaum, Veronika
In Ethiopia, the burden of disease related to communicable diseases has recently decreased significantly, while morbidity and mortality due to non-communicable diseases (NCDs) have increased. At the same, maternal and child malnutrition remained a major public health problem of Ethiopia. In developing countries, where health insurance is largely unavailable, individual medical conditions can also affect the overall and nutritional well-being of household members. In particular, the occurrence of disease and adult mortality in households can affect the nutritional well-being of the most vulnerable household members, especially lactating mothers and their young children. If the diseases are of chronic nature, which usually are costly and adult household members die from it in the long-term, this can be devastating for the family. The aim of this Ph.D. project was to investigate the epidemiology and social determinants of NCDs-attributed adult mortality, and to examine the association of chronic diseases attributed adult mortality with undernutrition of lactating mothers and their young children in rural population of Kilte Awlaelo-Health and Demographic Surveillance Site (KA-HDSS), Eastern Zone of Tigray, Ethiopia. During the data analysis, causes of death in adults were classified into chronic and non-chronic causes. The category of adult mortality due to chronic diseases refers to all causes that may be characterized by a long duration of illness. This group includes all deaths caused by NCDs and chronic communicable diseases such as tuberculosis and HIV/AIDS. The thesis has three articles, all published in peer-reviewed journals. The first article reports findings on the epidemiology and social-determinants of adult mortality caused by NCDs among 45,982 adult residents of KA-HDSS using population-based longitudinal data collected from 2009 to 2015. The second article tested whether the burden of undernutrition was higher among lactating mothers who were living in households with adult mortality from chronic diseases than among lactating mothers living in households with no adult mortality from chronic diseases. The third article examined whether there was an association between undernutrition of children and adult mortality from chronic diseases. Both longitudinal and cross-sectional data were used in the second and third articles. To our knowledge, this study showed for the first time that adult mortality caused by NCDs varied according household members’ relationship to their household head: extended family and non-family members of the household head had higher hazard of mortality compared to the household heads. In addition, this work can be considered as the first study from a low-income setting to examine whether mortality of an adult household member from chronic diseases is associated with undernutrition of lactating mothers and their young children. The results of the first study indicate a double mortality burden from both communicable diseases and NCDs in the study population. Between 2009 and 2015, the leading causes of NCDs-attributed adult mortality were cardiovascular diseases, cancer and renal failure. Compared to heads of households, extended family and non-family co-residents had an increased hazard of mortality from NCDs. Literacy and younger age were protective factors against adult mortality caused by NCDs. However, the protective role of literacy against adult mortality from NCDs decreased with increasing age. Next, we assessed the level of undernutrition among the lactating mothers and examined its association with household-level occurrence of adult mortality from chronic diseases by controlling the effect of a wide range of epi-demographic and agro-ecological variables. Nearly two-fifths (38%; 95% CI: 36.1, 40.1%) of the mothers were undernourished. We found an increased risk of maternal undernutrition for lactating mothers who were living in households which experienced adult mortality from chronic diseases. In addition, maternal undernutrition was strongly associated with recent history of household-level morbidity, poor health-seeking practice, lack of diverse food crops, and a low index score for housing and environmental factors. In the third article, we determined the burden of undernutrition among children of complementary feeding age (6 to 23 months) and its factors within the context of nutrition-specific and -sensitive drivers of young child undernutrition. Here, mortality from chronic diseases were constructed as a nutrition-specific factor. We found high prevalence of wasting (13.7%; 95% CI: 12.1, 15.5%) and inadequate child dietary diversity (81.3%; 95%CI: 79.2, 83.1%). Adult mortality history from chronic diseases was not associated with young child undernutrition and child dietary diversity. However, child undernutrition was strongly associated with recent history of household-level morbidity, maternal undernutrition, low child dietary diversity, poverty, larger family size, insecure employment of household heads, and living in highland areas. Poor household wealth status and lack of diverse food crops production, particularly in highland areas, were also strongly associated with lower child dietary diversity. Overall, this thesis has shown that an epidemiological transition is ongoing in the surveillance population. Population-based intervention measures are recommended that aim to reduce NCD-related adult mortality by targeting the leading causes of death and focusing on vulnerable population subgroups, such as the extended family and nonfamily household members. In this study, there was no association between the occurrence of chronic diseases attributed adult mortality and young child undernutrition. However, adult mortality from chronic diseases was associated with maternal undernutrition. Our findings appear to call for multi-sectoral interventions, mainly by the agriculture, nutrition and health sectors, to promote nutritional well-being of lactating mothers and their dyads in the long-term.
Nutritional and hemoglobin status in relation to dietary micronutrient intake: studies in female and male small-scale farmers from Lindi region, Tanzania, and Gurué district, Mozambique
(2024) Eleraky, Laila; Frank, Jan
Inadequate consumption of micronutrient-dense and protein-rich foods, such as vegetables, legumes and meat, are important contributing causes for malnutrition, anemia and micronutrient deficiencies in rural communities of Tanzania and Mozambique. The increasing public health concern of the malnutrition form of overweight has repeatedly been reported in urban as well as rural areas of Sub-Sahara Africa and may have already reached farmers in Tanzania and Mozambique. Nutritional status is assessed by anthropometry, dietary intake and hemoglobin. Compared to the often-used body mass index (BMI) and traditional 24-hour recall, the mid-upper-arm-circumference (MUAC), as well as a food group-based algorithm (CIMI) can be suitable additional assessment tools, especially in resource poor environments. Cross-sectional studies within the framework of the Vegi-Leg project were conducted to assess the nutritional status (anthropometrics and hemoglobin measurements), and the dietary behaviours (Household Dietary Diversity Scores (HDDS), Food Frequency Questionnaires (FFQ) and 24-hour recalls) of female and male farmers from rural areas of Tanzania and Mozambique. Data were analysed by region, sex, age, partly season (Tanzania)and correlates. Additional data from similar projects, namely Scale-N and Trans-SEC in rural villages of Tanzania were included in MUAC and CIMI analysis. MUAC as an additional and easy-to-handle anthropometric marker for underweight, as well as overweight was evaluated using data from Vegi-Leg and Scale-N surveys. MUAC cut-offs, calculated via BMI cut-offs and multiple linear regression (MLR), compared to those selected by highest Youden’s index (YI) value, were assessed. The CIMI algorithm included 23 food groups and was tested in comparison to NutriSurvey (detailed quantitative 24 hour recalls) with data from Scale-N and Trans-SEC.A total of 1526 farmers from the Vegi-Leg project (669 from Tanzania, 857 from Mozambique) were studied, of whom 19% were overweight and 35% were anemic. The study showed an overall higher prevalence of overweight (19%) than underweight (10%), mainly due to the high prevalence of overweight female farmers (up to 35%) in southern Tanzania. The highest prevalence of overweight and anemia, at 35% and 48%, was observed in Tanzanian and Mozambican women, respectively. Regarding HDDS and FFQ data, pigeon pea farmers in Lindi and Gurué reported high consumption frequencies of cereals, legumes, vegetables and oil, while meat, fish and eggs were only consumed rarely. Overall, only a small proportion of enrolled women and men reached the recommended daily dietary intake of vitamin A (10%), iron (51%) and zinc (44%) according to the 24-hour recalls. Multiple regression models revealed that dark green leafy vegetables (DGLVs) highly predicted vitamin A intake, whereas legumes in Tanzania and starchy plants in Mozambique were the dominant sources of vitamin A. Cereals contributed to over half of the iron and the zinc intake in both countries. Seasonal analysis revealed high fluctuations for the consumption frequency of food items from the food groups ‘legumes and pulses’, ‘green leafy vegetables’, ‘other vegetables’ and ‘fruits’, including tomatoes, pigeon peas, mangoes and oranges. The results from Lindi Tanzania revealed, that in seasons, when the availability of food groups like fruits, legumes or vegetables was low, the consumption frequency decreased significantly. BMI, which correlated positively and strongly with MUAC, was higher in Tanzania than in Mozambique and higher among female than male farmers, and decreased significantly from the age of 65 years. MUAC cut-offs of <24 cm and ≥30.5 cm, calculated by multiple linear regression, detected 55% of farmers being underweight and 74% being overweight, with a specificity of 96%; the higher cut-off <25 cm and lower cut-off ≥29 cm, each selected according to Youden’s Index, consequently detected more underweight (80%) and overweight farmers (91%), but on the basis of a lower specificity (87–88%). The results of the algorithm CIMI and NutriSurvey were similar with regard to the average intake and range of data distribution. The correlation coefficients of NutriSurvey and CIMI with regards to energy (0.931), protein (0.898), iron (0.775) and zinc (0.838) intake, supported the matching of both calculations. An increased consumption of micronutrient rich DGLVs and legumes, while reducing the high amounts of refined sugar, maize and polished rice, is suggested to counteract the high prevalence of anemia and overweight among smallholder farmers in rural Tanzania and Mozambique. MUAC cut-offs to detect malnutrition whether defined via linear regression or Youden’s Index, proved to be easy-to-use tools for large-scale rural screenings of both underweight and overweight. The food group based CIMI algorithm is a valid instrument that calculates energy and nutrient intake in agreement with the preferred nutrition software NutriSurvey.
Food and nutrition security and associated health aspects of Syrian refugee mothers and their offspring : a mixed-method study in Greater Beirut, Lebanon
(2023) Abou-Rizk, Joana; Scherbaum, Veronika
Nutrition during the first 1000 days of life – from conception to two years of age – lays the foundation for optimal health, cognitive and social development, and well-being of children and mothers. Malnutrition and hidden hunger in mothers and their offspring during the perinatal period can have long-lasting and irreversible consequences on human capital and increase the risk to non-communicable diseases among adulthood through intergenerational effects. Since the Arab Spring in 2011, the Syrian humanitarian crisis has escalated to become the world’s largest by the end of 2018, forcing 6.7 million to flee, mainly to neighboring countries. In 2015, Lebanon was the host of more than 1.5 million refugees and displaced persons from Syria, reaching the highest per capita concentration of refugees worldwide. The protracted crisis has increasingly weakened Lebanon, in turn exposing vulnerable groups, especially women of reproductive age and children below five years, to increased risks of inadequate nutrition and health status. To date, there has been a lack of studies among refugees that examine the nutritional and health situation and investigate the underlying and immediate determinants of malnutrition among women of reproductive age and children below five years. Therefore, knowledge gaps regarding the prevalence of anemia and nutritional status of Syrian refugee mothers and children were identified as important research areas in this dissertation. Assessing the food security and mental health of Syrian refugee mothers, examining the dietary intake and infant feeding practices, and investigating their respective determinants and associated risk factors were also important areas of focus. This research project was conducted between July and October 2018 in collaboration with the American University of Beirut in primary healthcare centers in the Greater Beirut area in Lebanon using quantitative and qualitative surveys (mixed-methods survey) among Syrian refugee mothers and their child aged below five years of age. This work comprises a total of three research articles that have been published in peer-reviewed international journals. The first article (Chapter 3) describes the prevalence of anemia and the nutritional status of mothers and children below five years of age and indicates a moderate public heath significance among Syrian refugees in the Greater Beirut area, Lebanon. The existence of the so-called double burden of malnutrition and hidden hunger (micronutrient deficiencies) has been documented, as mothers and children from the same household were affected by undernutrition, overnutrition, and / or anemia. A strong association between maternal and child anemia in the same household and an association between anemia and overweight / obesity among mothers has also been demonstrated. Analysis of the 24-hr dietary recalls of pregnant, lactating, non-pregnant non-lactating mothers revealed inadequate dietary intake and nutrient deficiencies during pregnancy and lactation. The second article (Chapter 4) presents data on the prevalence of food insecurity, low dietary diversity, and poor mental health among Syrian refugee mothers in Greater Beirut, Lebanon. A significant association between food insecurity and low dietary diversity as well as poor mental health status was found. Low household monthly income and a high household crowding index were significantly associated with low dietary diversity and food insecurity. In addition, the results showed that low-income households consumed monotonous diets, characterized by a high consumption of refined starchy staples and grains. The third paper (Chapter 5) focused on the nutritional and health status of infants under six months. Overall, the public heath significance for anemia and wasting was moderate. Inadequate infant feeding practices, such as the intake of pre-lacteal feeding and liquids, were demonstrated in newborns, which in turn contributed to delayed initiation of breastfeeding and low rates of exclusive breastfeeding. Factors supporting infant feeding practices included proper knowledge on maternal nutrition and exclusive breastfeeding and support from healthcare professionals or family during breastfeeding. Identified barriers included cultural practices such as the early introduction of foods, breastmilk substitutes, and giving water or herbal tea to infants under six months of age. Maternal health issues and social factors such as misinformation from family members and violations of the Code of Marketing of Breastmilk Substitutes also affected breastfeeding. A conceptual framework was also developed in this dissertation to provide a comprehensive understanding of the linkages and pathways between the enabling, underlying, and immediate determinants and the nutritional and health outcomes in the context of humanitarian crises. Based on this conceptual framework, three principal pathways were identified to explain the following associations: (1) from the enabling determinants to anemia and nutritional status, (2) from socio-economic status and host country resources to food consumption and diets, and (3) from pre-resettlement experiences and dietary acculturation to food consumption and diets. In addition, the concluding discussion (Chapter 6) presented the role of maternal mental health status and health services and their impact on maternal nutrition and anemia status as well as infant feeding. In summary, this dissertation contributes to the knowledge about the anemia and nutritional status of Syrian refugee women and children as well as on the food security and mental health status of mothers living in an urban setting of a protracted humanitarian crisis in Lebanon. It also provides insights into the emergence of malnutrition among women and children in the context of refugees and shows the pathways between different levels of determinants of nutrition and health. Implications for practice can be drawn from this dissertation, such as the development or strengthening of evidence-based culture-specific nutritional interventions that are targeted to the refugee context. Priorities for future research were also identified, including the need to improve the effectiveness of nutritional interventions in conflict settings.
Toll-like Receptor 9 (TLR9) activation and the innate immune response to microbial and human DNA
(2023) Hsu, Emily; Fricke, Florian W.
The human Toll-like Receptor 9 (TLR9) is an endosomal Pattern Recognition Receptor (PRR) that recognizes DNA sequences containing the unmethylated Cytosine-Guanine (CpG) dimers, which are present in greater abundance in most bacterial genomes compared to those of vertebrates. Specific CpG-containing sequences are strongly stimulatory of human TLR9, as shown in published studies using synthetic oligonucleotides (ODN) and DNA from bacterial species of varying genomic CpG concentration. Human TLR9 activation was experimentally examined in this thesis using DNA extracted from different bacterial sources, human DNA from Caco-2 cells, known immunostimulatory ODN, and short ODN. In vitro assays using fragment length-standardized microbial genomic DNA on HEK-Dual TLR9 Cells and human peripheral blood mononuclear cells (PBMCs) revealed that TLR9 activation strongly correlated to CpG concentration of the input DNA, with an additional influence of CpG-containing 5-mer TCGTT concentration. When DNA of varying origins and fragment lengths were used together, however, complex dynamics of TLR9 activation, co-activation, and repression were observed, which were less predictable than expected from genomic CpG concentration alone. DNase I-treated microbial DNA fragments of less than 15 bp of length were non-activating on their own, but co-activated human TLR9 together with ODN-2006 in Ramos Blue (B) cells. Similarly, human DNA fragments at the length of 50-200 bp co-activated human TLR9 with both ODN-2006 and Escherichia coli DNA in HEK-dual TLR9 cells. In contrast, large human DNA fragments at over 10000 bp of length repressed TLR9 activation by ODN-2006 in Ramos Blue cells. Finally, a preliminary study was conducted in HT-29 cells on the effect of TLR9 activation on the invasion of Fusobacterium nucleatum, an opportunistic gut pathogen with a very low genomic CpG concentration at 0.296%, using ODN-2006 and human DNA as TLR9 activators. While increased presence of intracellular Fusobacterium nucleatum upon treatment with both ODN-2006 and human DNA was noted, more studies are needed to confirm TLR9 activation as a cause of greater bacterial invasion. The human colon is the location of the largest microbial population of the human body, which provides a rich source of non-human DNA in contact with human TLR9 present in intestinal epithelial cells, plasmacytoid dendritic cells (pDCs), and B lymphocytes. Additionally, the daily mass shedding and death of human intestinal epithelial cells provide large amounts of human DNA, which when combined with microbial DNA could result in co-activation and possible autoimmunity. The thesis thus provided an in vitro model of TLR9 activation by complex DNA of varying origins and fragment lengths likely to present in the human gut environment, and prepared a working basis for future studies of TLR9 activation by human fecal metagenomic DNA.
Strain-resolved analysis of the human intestinal microbiota
(2022) Podlesny, Daniel; Fricke, Florian W.
The gut microbiota is ascribed a crucial role in human health, particularly in regulating immune and inflammatory responses, which is why it is being associated with a wide range of diseases, including obesity, diabetes, and cancer. Nonetheless, fundamental ecological questions of microbiome establishment, stability and resilience, as well as its transmission across hosts and generations remain incompletely understood, partly due to the lack of methods for high-resolution microbiome profiling. New insights in this field can therefore directly contribute to the development of bacterial and microbiota-based therapies. This work introduces SameStr, a novel bioinformatic program for strain-resolved metagenomics that allows for the specific tracking of microbes across samples, enabling the detection and quantification of microbial transmission and persistence, as well as the observation of direct strain competition. Deployed across cohorts to process over 4200 metagenomes, SameStr enabled analysis of the microbiome with unprecedented phylogenetic resolution. The data included both publicly available metagenomes and sequence data generated in collaboration with our research partners, and was examined using multivariate statistics and machine learning frameworks. First, the establishment and development of the neonatal microbiota was studied, revealing a birth mode-dependent vertical transmission of the maternal microbiota. The microbiota of neonates born by cesarean section was characterized by increased relative abundance of oxygen-tolerant and atypical organisms and showed signs of a delayed establishment of a strictly anaerobic gut environment in these children. Such birth mode-dependent differences diminished over time, yet were measurable within the first two years of life. Furthermore, strain analysis verified the transmission and colonization of parental microbes, which indicated a possible lifelong colonization by microbes from selected species. The temporal persistence of microbes was also characterized in healthy adults, revealing similar taxonomy-dependent patterns of stability. For some species, persistence has been demonstrated both in children and in adults over a period of at least two years. These species are known for their capability to metabolize host-derived glycans found both in breastmilk and intestinal mucus, pointing to a potential strategy for effective cross-generational microbiota transmission, and warranting additional research to assess the implications of their disturbed transfer for long-term health. Since their specificity allows assignment to individual hosts, fingerprints of individual microbial strains offer the potential to be used in forensics and data quality control applications. Finally, to gain new insights into the microbiota dynamics during Fecal Microbiota Transplantation (FMT), microbial strain transmission was analyzed in the context of a diverse set of patient, microbiome, and clinical conditions. In the analyzed studies, FMT was used for the experimental treatment of a variety of diseases, including colonization with drug-resistant and pathogenic microbes, metabolic and inflammatory bowel diseases, and as an adjunct to the immunotherapeutic treatment of cancer. Analyses uncovered what appear to be the universal drivers of post-FMT microbiota assembly, including clinical and ecological factors that are important for successful transplantation of donor strains. In particular, the relevance of the microbiota dysbiosis of the recipient was emphasized, which was inducible by pre-treating the patient with antibiotics or laxatives. Presumably, this can open up ecological niches in the patients intestines, which favors colonization with donor strains. Colonization rates did not play a role for the treatment success of recurrent C. difficile infections and inflammatory bowel disease, but indicated a trend associated with an improved immune response in cancer patients. Concerningly, the transfer of an atypical and potentially pro-inflammatory microbial community from one donor was also observed, calling for further investigations into the immediate and long-term clinical consequences of FMT. These analyses demonstrate the advantages of a strain-based microbiome analysis. Due to the achieved methodological accuracy, strain-resolved microbial dynamics could be precisely disentangled when comparing longitudinal samples from healthy adults as well as parent-child and patient-donor pairs. This revealed taxonomic, clinical, and ecological factors that are critical to microbiome assembly, including microbial transmission, persistence, and competition. Together, these findings lay the groundwork for future developments of precision personalized microbiota modulation therapies.
Characterization of dietary and genetic influences on the gastrointestinal microbiota
(2023) Bubeck, Alena Marie; Fricke, Florian W.
Although the gut microbiota is known to contribute fundamentally to human health, e.g. by promoting the maturation of the immune system and intestinal homeostasis, the factors shaping its composition are only poorly understood. Extrinsic and intrinsic influences can disturb the tightly controlled equilibrium between the microbiome and the host and induce dysbiosis, which has been linked to diverse health conditions such as obesity, atherosclerotic cardiovascular disease (ACVD) and inflammatory bowel disease (IBD). Therefore, understanding events leading to microbial perturbations and the prediction of associated health outcomes could aid in the prevention and treatment of these conditions. In this work, the impact of dietary and genetic factors on gastrointestinal microbiota compositions were determined, with the diet serving as an exemplary extrinsic, modifiable microbiota-relevant factor and with a genetic deficiency in a mouse model for intestinal inflammation serving as an exemplary intrinsic, non-modifiable microbiota-relevant factor. In both studies, microbial communities obtained from either a human or a murine cohort, respectively, were taxonomically characterized by 16S rRNA gene amplicon sequencing and analyzed in the context of metabolic and inflammatory implications for the host. In ACVD, the reduction of excess blood cholesterol, which is a main risk factor, is tackled by clinical interventions aiming to reduce cholesterol uptake from exogenous, dietary sources or by inhibiting endogenous cholesterol biosynthesis. Cholesterol-to-coprostanol conversion by the intestinal microbiota has also been suggested to reduce intestinal and serum cholesterol availability, but the dependencies of cholesterol conversion on specific bacterial taxa and dietary habits, as well as its association with serum lipid levels remain largely unknown. To study microbiota contributions to human cholesterol metabolism under varying conditions, fecal microbiota and lipid profiles, as well as serum lipid biomarkers, were determined in two independent human cohorts, including individuals with (CARBFUNC study) and without obesity (KETO study) on very low-carbohydrate high-fat diets (LCHF) for three to six months and six weeks, respectively. Across these two geographically independent studies, conserved distributions of cholesterol high and low-converter types were measured. Also, cholesterol conversion was most dominantly linked to the relative abundance of the cholesterol-converting bacterial species Eubacterium coprostanoligenes, which was further increased in low-converters by LCHF diets, shifting them towards a high-conversion state. Lean cholesterol high-converters, which were characterized by adverse serum lipid profiles even before the LCHF diet, responded to the intervention with increased LDL-C, independently of fat, cholesterol and saturated fatty acid intake. These findings identify the cholesterol high-converter type as a potential predictive biomarker for an increased LDL-C response to LCHF diet in metabolically healthy lean individuals. Although the etiology of IBD has not been fully resolved, an interplay between the intestinal microbiota, environmental factors and an individual’s genetic susceptibility is thought to trigger chronic inflammation by a dysregulation of the immune response in the gut. To identify colitis-associated microbiota alterations throughout the development of spontaneous colitis, mice with a genetic deficiency of the anti-inflammatory cytokine Interleukin-10 (IL-10) from different litters were co-housed with wild-type mice and monitored for 20 weeks. The scoring of mice based on their phenotype and stool consistency mirrored the state of mucosal inflammation as assessed based on histopathological examinations and cytokine expression profiles. Also, the state of colitis was characterized by global microbiota alterations and susceptibility to colitis was dependent on litter-specific microbiome compositions that mice adopted early on in their lives. Colitis development was further associated with the presence of the bacterial genus Akkermansia in mature mice shortly before symptoms manifested. This genus was also a good predictor of colitis-related mice withdrawal, suggesting the potential of Akkermansia to serve as an early onset, subclinical colitis marker. In summary, fecal microbiota characterizations in response to LCHF diets in humans and throughout the development of intestinal inflammation in a colitis mouse model highlight the potential of personalized microbiome-based patient classifications to predict clinical outcomes and improve treatment approaches.
Identification and quantification of tocomonoenol isomers in plants and microalgae and investigation of their metabolism in liver cells
(2022) Montoya Arroyo, Alexander; Frank, Jan
Tocopherols (T), tocomonoenols (T1), and tocotrienols (T3) are tocochromanols, a group of bioactive compounds composed of a chromanol ring and a 16-carbon sidechain with biological functions, such as the protection of lipid membranes from oxidation and the modulation of cellular signaling. T have saturated sidechains, while T1 and T3 have a single or three double bonds in theirs, respectively. The prefixes alpha-, beta-, gamma-, and delta- are assigned based on the number and positions of methyl groups on the chromanol ring. alpha-, beta-, gamma-, and delta-congeners of T1 have been reported, with alpha-T1 being the predominantly identified congener. Two different alpha-T1 isomers are known, 11-alpha-T1, which has been mainly found in land plants, and 12-alpha-T1, which has been mostly detected in marine organisms. However, little is known regarding the occurrence of T1 in photosynthetic organisms and their metabolism in the liver, a strong determinant of bioavailability and bioactivity. The aim of this thesis was to evaluate underutilized plant-based food sources, cyanobacteria, and microalgae as potential sources of T1 and to characterize the uptake and conversion into metabolites of T1 in cultured liver cells in comparison to T and T3. Acrocomia aculeata fruits were analyzed for alpha-T1 due to its phylogenetic relationship with Elaeis spp, the most common source of this congener. No alpha-T1 was detected in oils from endosperm and mesocarp of wild fruits of Acrocomia aculeata from Costa Rica. Aerial parts of the local underutilized leafy vegetable Urtica leptophylla were evaluated as source of T1 due to its agronomical potential and previous reports of T1 in leaves of plants. LC-MS analyses indicated that leaves and flowers of Costa Rican Urtica leptophylla contain minor amounts of alpha-T1 and gamma-T1. Cyanobacteria and microalgae from different species and origins were analyzed as source of alpha-T1 due to their role as primary producers in aquatic ecosystems and the reported presence of 12-alpha-T1 in marine phytoplankton. alpha-T1 in cyanobacteria and microalgae ranged from traces up to 17% of the total tocochromanol content. alpha-T1 concentrations alone were higher than the sum of all four T3. 11-alpha-T1 was the major alpha-T1 isomer in cyanobacteria and microalgae, as determined by GC-MS. Hence, 11-alpha-T1 is a product of biosynthetic pathways even in aquatic organisms. The effect of nitrogen depletion during the cultivation of microalgae on their alpha-T1 content was investigated. Nitrogen depletion did neither significantly affect the relative or absolute content of alpha-T1, despite an increase in tocochromanol content, nor the proportion of 11-alpha-T1/12-alpha-T1 in microalgae. The uptake and conversion into metabolites of 11-alpha-T1 in HepG2 liver cells was compared to those of alpha-T3 and alpha-T. Cellular uptake of alpha-T1 in liver cells was higher than that of alpha-T. 11-alpha-T1, similar to alpha-T, was converted mostly to alpha-carboxymethylhydroxychroman in a time dependent manner, but to lower extend than alpha-T3. The effect of both ring methylation and sidechain saturation on the uptake and metabolism of the alpha- and gamma-congeners of T1, T and T3 was studied in HepG2 cells. gamma-Congeners were metabolized at higher extent than alpha-congeners and metabolite production increased with increasing number of double bonds in the sidechain independently of chromanol ring methylation. In conclusion, alpha-T1 is present with up to 17% of total tocochromanols in cultured microalgae, which thus are an important new source of this congener. gamma-T1 is only a minor tocochromanol in U. leptophylla flowers. 11-alpha-T1, and not 12-alpha-T1, is the major alpha-T1 isomer in cyanobacteria and microalgae and nitrogen depletion of microalgae does not significantly affect alpha-T1 concentration. The metabolic conversion of alpha-T1 into alpha-carboxymethylhydroxychroman in HepG2 cells is similar to that of alpha-T and significantly lower than that of alpha-T3, suggesting that it may be handled by the organism similar to alpha-T. In conclusion, novel potential food sources of alpha-T1 have been identified and, because of similarities with alpha-T, its pharmacokinetics and biological activities warrant further investigation.
Maternal psychosocial stressors, depression and its implications on maternal and infant nutrition : a longitudinal birth cohort study in South West Ethiopia
(2022) Woldetensay, Yitbarek Kidane; Scherbaum, Veronika
This thesis draws on theoretical background and a conceptual model of how selected psychosocial stressors (household food insecurity and intimate partner violence) and coping strategies (maternal social support) are linked to psychological distress (maternal depression) which can influence the nutritional status of mothers and infants. The scientific contribution of this work is threefold. First, it adds to the existing literature on the links between psychosocial stressors, social support and depression, by showing the degree to which household food insecurity and intimate partner violence during pregnancy are associated with the risk of antenatal depressive symptoms, and whether maternal social support plays a buffering role in this process. Second, it describes the longitudinal relationship of prenatal and postnatal depressive symptoms with infant feeding practices. Finally, this work contributes to the literature on depression by validating one of the most commonly applied depression measurement tools, the patient health questionnaire (PHQ-9), in a new culture and language. This thesis includes three research articles; two were published in peer-reviewed journals and the third manuscript is currently under peer review. The first article is a validation study of the depressive symptoms measurement tool in a new culture and language in a rural setting. The other two papers are based on ENGINE birth cohort data, a prospective community-based birth cohort study conducted by Tufts University in collaboration with Jimma and Hawassa Universities and the Ethiopian Public Health Institute in the southwest part of Ethiopia. The first article validated an Afaan Oromo language version of the Patient Health Questionnaire (PHQ-9). PHQ-9 is one of the most commonly used depressive symptoms measurement scales. Few validation studies have been conducted in sub-Saharan Africa for scales seeking to detect depression in pregnant women and to the author’s knowledge this is the first validation of the PHQ-9 in this language. The main finding of the first paper was that the PHQ-9 scale has acceptable reliability and validity as a screening instrument for depressive symptoms among Afaan Oromo speaking Ethiopian pregnant women. The PHQ-9 score of eight or above was found to be an optimal cut-off point to diagnose prenatal depressive symptoms with a sensitivity and specificity of 80.8% and 79.5% respectively. The second article tested hypotheses derived from Lazarus and Folkman’s stress and coping theory. This theory provides a basis for understanding the impacts of psychosocial stressors on the development and prognosis of depression and the buffering effect of coping mechanisms. The hypotheses are as follows: increased psychosocial stressors, specifically intimate partner violence and household food insecurity during pregnancy, lead to higher prenatal depressive symptoms, and strong social support from friends,families and partners have a buffering effect. The present results supported these hypotheses by demonstrating that both household food insecurity and intimate partner violence were positively associated with prenatal depressive symptoms. Simultaneously, strong social support from friends, families and partners was negatively associated with prenatal depressive symptoms. The second article tested another hypothesis that the risk of prenatal depression is higher in anemic pregnant women. The current study supported this hypothesis by demonstrating that anemic pregnant women experienced a higher risk of prenatal depressive symptoms than their non-anemic counterparts. The second paper also investigated the degree to which socio-demographic factors such as age, marital status, educational status, and family size predicted the risk for prenatal depression among pregnant women. The results suggested that unmarried pregnant women and women living in households with large family size were at higher risk of prenatal depressive symptoms. The third paper tested one hypothesis that infants born to mothers with maternal depressive symptoms (prenatal and postnatal) are more likely to have poor infant feeding practices than their counterparts. The present study findings supported this hypothesis by showing that immediate postnatal depressive symptoms are negatively associated with infant feeding practices. However, there was no association between prenatal depressive symptoms and infant feeding practices. The results also showed that intimate partner violence was negatively associated with infant feeding practices. In this study, strong maternal social support and active social participation were positive predictors of infant feeding practices. Contrary to expectations, the present study also found that household food insecurity and infant morbidity episodes were positively associated with infant feeding practices. Finally, the third article’s findings suggested that maternal educational status was positively associated with infant feeding practices and preterm babies were at higher risk of poor infant feeding practices. Infant gender did not affect infant feeding practices in this study. Overall, this PhD thesis provided support for Lazarus and Folkman’s stress and coping theory by demonstrating that psychosocial stressors were positively associated with prenatal depressive symptoms and perceived maternal social support was negatively associated with prenatal depressive symptoms. The thesis also found that anemia during pregnancy is a predictor of prenatal depressive symptoms. Additionally, early postnatal depression and intimate partner violence negatively affect infant feeding practices, whereas perceived maternal social support and active social participation predict appropriate infant feeding practices. The study has a number of implications for practice and future research including prioritization of early screening for maternal depressive symptoms and intimate partnerviolence, and promotion of social support and active social participation as a means of preventing maternal depression and improving maternal and infant nutritional status.
The influence of phosphate-availability and phytic acid on the profiles of fatty acids, (poly)phenols, carotenoids, and tocochromanols in maize (Zea mays L.) grains – from field experiments to human in vitro digestion studies
(2022) Lux, Peter Erwin; Frank, Jan
Phosphorus (P) is an essential element for living organisms and involved in phosphorylation reactions, including the biosynthesis of several organic micronutrients. Since P is taken up by plants from soil as phosphates, phosphate fertilizers are applied on fields to support the P-supply for crops. Today, shrinking global P-resources demand a reduction in the application of P-containing fertilizers, but knowledge about possible effects of a reduced phosphate-availability in soils on the quality of maize grains is lacking. Thus, it was hypothesized that a reduced phosphate-availability in soil influences the concentrations of dietary organic compounds (phenolics, fatty acids, carotenoids, and tocochromanols) in grains of maize during cultivation. Moreover, concentration differences in the P-storage form phytic acid in maize grains may impact the oxidative stability of these organic compounds during processing and digestion. Fertilizer experiments with maize hybrids were conducted at study sites with low to high phosphate concentrations in soil (1.6 to 20.6 mg CAL-P/100 g soil) in Germany. GC-MS or HPLC-(MS) analyses of the ground maize grains revealed the identity of fatty acids, insoluble (mostly diferulic and triferulic acids) and soluble (poly)phenols, carotenoids, and tocochromanols. The concentrations of these (poly)phenols, carotenoids, and tocochromanols as well as the fatty acid composition in the grains of the maize plants grown with or without phosphate fertilizer were not significantly (p < 0.05) different. Interaction effects between phosphate application and the locations on the fatty acid composition as well as on carotenoids and tocochromanols were considered as insignificant, concluding that a reduction in phosphate fertilization could be implemented on most fields in Germany when only considering these dietary compounds. Lastly, the influence of phytic acid on oxidation processes in maize during processing of porridge and in vitro digestion was examined. Porridges were prepared from maize flour containing either high phytic acid concentration or low phytic acid concentration supplemented with or without phytate. The porridges were digested using a human in vitro digestion model, resulting in a decrease in tocochromanols, carotenoids and unsaturated fatty acids. Oxidation products (alpha-tocopherylquinone, malondialdehyde) were formed in all samples, implying that phytic acid addition did not show the expected protective effect. The addition of phytate evoked a significant reduction in the micellarization efficiency of most carotenoids. Thus, the knowledge about phytic acid as antinutrient was extended.
Relative bioavailability of curcumin as affected by formulation strategies : from in vitro digestion and cell culture experiments to human clinical trials
(2022) Flory, Sandra; Frank, Jan
Curcumin, the bioactive component of turmeric (Curcuma longa L.), has been used for thousands of years in traditional medicine for the prevention or treatment of several diseases and symptoms. Nowadays, curcumin is investigated worldwide as a nutritional supplement. To overcome the central limitation of its naturally low oral bioavailability, several formulation strategies have been developed, such as its co-administration with turmeric oils or piperine to inhibit its metabolism and efflux or its incorporation into micelles, cyclodextrin complexes or phospholipid bilayers to improve its stability and solubility. So far, the different formulations have not been compared directly, in one cohort of participants and at equal doses. The present doctoral thesis aimed, for the first time, at a direct comparison of the bioavailability of curcumin in form of a native curcuma extract or seven formulations, namely polysorbate 80 micelles, g-cyclodextrin complexes, liposomes, phytosomes, submicron-particle curcumin or curcumin administered with turmeric oils or piperine, in healthy adults. The project further aimed to investigate several critical factors for curcumin bioavailability in vitro and to explain thereby the observations made in vivo. In a randomized, double-blind crossover trial with 12 healthy participants (6 females, 6 males), curcumin pharmacokinetics, namely AUC (area under the plasma concentration-time curve), Cmax (maximum plasma concentration) and tmax (time to reach Cmax) were compared after administration of a single oral dose of 207 mg curcumin in form of a native curcuma extract or one of the seven formulations. Curcumin incorporated into polysorbate 80 micelles or g-cyclodextrin complexes showed 57-fold and 30-fold improved bioavailability compared to the native extract, whereas all other formulations showed no or minor effects. tmax of the better bioavailable formulations was smaller (1 to 2 hours) compared to all others (up to 7 hours). To compare the formulations regarding their digestion characteristics and transepithelial transport, in vitro digestion experiments followed by Caco-2 cell transport assays were conducted with the formulations normalized to their curcumin content. In parallel to the effects in vivo, curcumin showed higher stability, solubility and micellization efficiency when it was incorporated into polysorbate 80 micelles (100%, 80%, 55%) or g-cyclodextrin complexes (73%, 33%, 23%), whereas curcumin permeability through Caco-2 cell monolayers was not affected by its formulation. In the next study, curcumin efflux, partially mediated by P-glycoprotein (P-gp), was investigated, because the inhibition of curcumin efflux from the intestinal cells back to the intestinal lumen is targeted by the co-administration of curcumin with turmeric oils or piperine. In LS180 (colon adenocarcinoma) cells, native curcuma extract and the seven formulations were studied regarding cellular curcumin uptake within 1 hour and efflux within further 8 hours, as well as their effects on P-gp activity. Independently from its formulation, curcumin inhibited the activity of P-gp. Cellular curcumin uptake and efflux showed significant variability between formulations but no consistent effects. Cellular uptake and efflux may thus not be important for curcumin bioavailability in vivo. Another potential factor influencing bioavailability, that was investigated for native and micellar curcumin, was the time-dependent intracellular distribution in intestinal cells. Uptake and intracellular distribution in Caco-2 cells mainly did not differ between native and micellar curcumin. After 30 minutes, both were localized in lysosomes and mitochondria, after 180 minutes in peroxisomes and native curcumin also in mitochondria. The temporary localization in lysosomes is in line with the involvement of endocytosis in cellular uptake of curcumin. Nevertheless, the intracellular localization of curcumin was not affected by its incorporation into polysorbate 80 micelles. The data generated in this doctoral project thus demonstrate that the incorporation of curcumin into polysorbate 80 micelles or g-cyclodextrin complexes successfully improve its bioavailability. The improved bioavailability of both formulations can be explained by enhanced digestive stability, solubility and micellization efficiency and appears to be independent from post-digestive processes, such as intestinal permeability, cellular uptake, cellular efflux or intracellular distribution. Consequently, the present doctoral thesis delivers relevant information for the therapeutical application of curcumin, for the development of highly bioavailable formulations, as well as the basis for further clinical research on the health beneficial effects of curcumin.
Nutrition during pregnancy and early infancy in urban and rural areas of Deyang region, Southwestern China
(2022) Gao, Haoyue; Birner, Regina
In the past years, China has made remarkable progress in achieving the Millennium Development Goals. However, there are still significant urban-rural disparities and regional differences in the main indicators of child nutrition, maternal and child health. Inadequate dietary intake with regard to certain macro- and micronutrients occurs among women during the reproductive period. Despite the efforts that have been made to promote breastfeeding in China since the 1990s, there is still a very low prevalence of exclusive breastfeeding during the first six months of life. Therefore, the purpose of this observational study was to assess dietary intake and identify risk factors for nutritional inadequacy in pregnant women and to assess the current situation of infant feeding practices during the postpartum hospital stay in urban and rural settings of the study area in China. Between 2010 and 2012, cross-sectional sampling was used in two urban hospitals and five rural clinics randomly selected in the Deyang region. A total of 203 pregnant women in the third trimester and 204 pairs of mothers and newborns were recruited on the basis of informed consent during antenatal clinic sessions and in postnatal wards respectively. Semi-structured interviews on anthropometric and socio-demographic information were conducted for both the pregnant women and postpartum mothers. In addition, data on 24-h dietary recalls of pregnant women, birth outcomes and infant feeding practices of postpartum mothers were collected. Prior to pregnancy, 26.3% of the pregnant women were found to be underweight, while only 5.1% were overweight, based on self-reported pre-pregnancy weight. The women’s overall dietary energy intake during the third trimester originated excessively from fat, was low in carbohydrates, and reached the lower limits for protein. Compared to rural areas, women living in urban areas had significantly higher reference nutrient intake fulfilment levels for energy, fat, protein, vitamin A, Zn, Fe, Ca and riboflavin. The likelihood of pregnant women following certain traditional food recommendations such as avoiding beef, mutton, fish etc. was higher in rural than in urban areas. Both the short-term (e.g. gestational weight gain) and long-term nutritional status (e.g. height) of the mothers affected the birth outcomes (e.g. type of delivery). The decision for a Caesarean Section (CS) was not only related to medical/obstetrical issues but also nutritional and social demographic factors. The short stature of mothers, excessive gestational weight gain, larger-sized newborns, higher educational level or older ages of mothers were significantly positively related to the probability of CS. Although almost all of the mothers started breastfeeding after childbirth, the high prevalence of CS delayed initiation of breastfeeding. Due to the widespread use of infant formula, the prevalence of exclusive breastfeeding (EBF) was only 8.0 % one week after delivery. Regardless of prelacteal feedings, almost exclusive breastfeeding (AEBF) was 34.5%. Breastfeeding initiation within two days after birth was positively associated with AEBF, whereas smaller newborns, mothers with higher education level, mothers lack of knowledge about the importance of colostrum were negatively associated with AEBF. As most mothers lacked knowledge of the lactation physiology, higher formal education was often not related to a better knowledge of breastfeeding. In conclusion, culturally sensitive nutrition education sessions are necessary for both urban and rural women. Special attention must be given to the nutritional status, especially of rural women, before or, at the latest, during pregnancy. Personalized nutritional guidance for expectant mothers should be based on comprehensive consideration of all aspects, such as dietary intake, place of origin (rural vs. urban), body height, and gestational weight gain etc. Better guidance for the gestational weight gain of Chinese mothers needs to be established. Although disparities between urban and rural areas exist, the situation of infant feeding is inadequate in both settings. The high prevalence of CS, the mothers’ poor knowledge of the physiology of breast milk production, the mothers’ lack of breastfeeding confidence, the widespread advertising of breast milk substitutes, and the changing perception of the function of breasts, may influence the unfavorable breastfeeding behavior observed in the study area. In order to prevent CS and to promote breastfeeding, education about the complications and disadvantages of CS especially among mothers with higher education, older childbearing age, and primiparas needs to be strengthened. Supporting adequate maternal nutrition during the reproductive period and creating a good breastfeeding environment requires the efforts of the whole society.
Ready-to-Use Food (RUF) from composite flour of local commodities in Banten Province, Indonesia for prevention and rehabilitation of malnutrition in children under five
(2021) Fetriyuna, Fetriyuna; Biesalski, Hans-Konrad
Indonesia faces a serious problem of malnutrition especially stunting and underweight caused by micronutrient deficiencies. The high prevalence of undernutrition was also found in Banten Province, Java Island, the most populated island in Indonesia. The Indonesian Demographic and Health Survey (DHS) in 2012 reported that of about 24.5 million children under five years of old, approximately 37% and 12% of them were stunted and wasted. Furthermore, based on the Basic Health Survey (Riskesdas) in 2013 and 2018, the prevalence of stunting in children under five years old was 37.2 % and 30.8%, while the proportion of underweight children was 19.6% and 17.7%. The undernourished children have an increased mortality rate, risk of illness and infections, cognitive shortages, delayed development, and poor school performance. Ready-to-use food (RUF) is a kind of food easily consumed without much preparation and can be made using various techniques for targeted consumers. RUF can be produced using locally available food resources, which may be affordable for most people suffering from undernutrition. To improve and accomplish the nutrient content of RUF to meet the standard for rehabilitation food for malnutrition, the combination of various food resources might enhance the nutritional composition. This nutritious formulation must have the following attributes: good nutritional quality in macro and micronutrient content, highly palatable taste, consistency, and texture suitable for feeding to children, no additional processing required prior to feeding, product stability, long shelf life, and readily available ingredients. This research was divided into three main objectives. The first objective was a baseline assessment of the nutritional condition of the target group of children below five years old. The second section was RUF biscuit product development. The third objective was to investigate the nutritional outcomes of the field study in Banten Indonesia, particularly in WHZ and weight after the RUF provision and weight gain of the children and acceptability. The RUF biscuit was made from the composite flour of local food resources available in Banten province Indonesia which are still underutilized. The result of nutrition content analysis in local food resources showed that the foods have a high nutritional composition as a source of macronutrients and micronutrients (vitamins and minerals). Due to poor handling of post-harvest and processing into the intermediate product (flour), some vitamins were lost during the process. Based on the baseline assessment in 2017, of the 105 children, 4.8% and 11.4% of them were wasted and overweight-obese, while 40% and 23.8% of the children were stunted and underweight. Based on the mid-upper-arm circumference, 16.1% of children were categorized as the risk of chronic energy deficiency (CED). In addition, it was found that most of the children have insufficient intake of protein, Vitamin A, Iron, and Zinc. Twelve recipes developed using the combination of local food resources showed the macronutrient composition that met the requirement for the treatment of Moderate Acute Malnutrition (MAM) children, but not for micronutrient (vitamin and minerals). Five recipes (recipes 3, 4, 7, 11, and 12) were selected based on nutritional composition and sensory evaluation. The selected recipes were continued to further research of acceptability study in Banten, Indonesia. Before the acceptability study in the field, the micronutrient composition of the recipes chosen was improved according to the MAM treatment recommendation. It fortified the RUF with micronutrient premix. The micronutrient stability test was performed to analyze the loss due to baking. Around 15-28% of the vitamins (A, B1, B2, and E) were lost after baking, which was slightly lower than the other published articles, perhaps due to the short process of baking (15 minutes). Furthermore, the micronutrient sources were from the vitamin-mineral premix with higher stability than that from foods. Minerals content was stable during the baking process. Before the acceptability study started, five fortified cookie recipes were tested to select two for the acceptance and intervention pilot study. Most of the children in the acceptance study reported that they accepted and liked the RUF cookies. The consumption of RUF biscuits for two weeks led to an improvement in the nutritional status: Reduction of severe wasting (WHZ) from 5.7% to 4.3% and of severe underweight (WAZ) from 8.6% to 4.3%. There was no difference in terms of stunting (HAZ). Thus, it has been shown that the developed RUF biscuits have the potential to reduce malnutrition in the under 5-year-olds in the target area.
Baseline assessment and effect of a supplementary community-based nutrition intervention study on the prevention/treatment of anemia among young Adivasi children in West Bengal, India
(2020) Stiller, Caroline K.; Biesalski, Hans-Konrad
Background: India´s Adivasi scheduled tribe population is disproportionately affected by anemia and undernutrition. On the avenue to sustainably promote child health locally available resources have to be maxed out. Subsequently, designed recipes may find entrance and modify dietary routines on household level, through interactive home-based cooking trainings, community awareness and homestead food production. Study design: From February 2015 onwards, the cluster-randomized controlled feeding trial was conducted in 21 tribal villages Birbhum District, West Bengal. The intervention lasted for 18 months and embraced four assessment points (t0, t6, t12, t18) including medical checkup, measurement of Hb concentrations (HemoCue Hb201+) as well as anthropometric indices. A semi-structured household (HH) survey was part of the baseline assessment. The research comprises one control group (CG) and three intervention groups (diversified meals only (IG1), with the addition of locally producible Amaranthus tricolor/Moringa oleifera leaf powders (ALP/MLP) in the ratio 2:1 (IG2), or with an adjusted amount of commercially produced micronutrient powder (MNP) TopNutri (IG3)). Supplementary meals were provided three times a week during an on-the-spot community feeding program. 293 children (6-39 months) were valid for the pre-/post intervention effect analysis. Objectives: To assess the overall burden of anemia and undernutrition and to investigate nutrition-sensitive and nutrition-specific factors determining the maternal and child nutritional status at baseline, moreover to identify independent drivers of anemia in Adivasi children (Article I and II). To design improved recipes and to evaluate their effect in a supplementary feeding intervention on the primary outcome variable hemoglobin (Hb), and the secondary outcome variables stunting (HAZ), underweight (WAZ), and wasting (WHZ) (Article III). The first article (Chapter 4) provides data on the maternal nutritional status and child feeding practices, and describes socio-demographic characteristics, family planning methods, use of antenatal care services and birth, childcare, mother-child dyad analysis. Moderate/severe forms of anemia and underweight were prevalent in every second mother. Child feeding practices and child caring were found to be suboptimal. Infants (6 to 11 months) were particularly vulnerable with merely every fourth child fulfilling the minimum acceptable diet (2 to 3 meals per day and  4 food groups (FG) per day). On HH level the serving of animal-sourced foods, legumes, vegetables and fruits has to be scaled up. The second article (Chapter 5) assessed the prevalence of anemia and undernutrition among Santal Adivasi children, determined independent predictors of moderate/severe anemia, and depicted agricultural assets, livelihood, aspects of food security and hygiene, morbidity rates, health seeking behavior. Binary Logistic regression assessed five independent predictors of moderate/severe anemia in children (Hb<10g/dl): child´s age<24 months, low WAZ scores, any morbidity (fever, diarrhea, or respiratory infection), low maternal Hb concentrations, lack of dietary diversification (low count of FG consumed during the previous 24 hours). The third arcticle (Chapter 6) assessed the intervention effects of the three study meals on the prevention/treatment of anemia (and undernutrition) in Adivasi children. IG1 (diversified meals only) showed significant higher Hb concentrations as compared to CG during the intervention (adjusted for age and Hb concentrations at baseline, time between assessment points, and gender). The mean Hb of IG2 or IG3, remained comparable to CG. Concluding remarks: Distressing rates of anemia and undernutrition were found among Adivasi mothers and children. To address identified drivers of anemia in Adivasi children and lower the burden of undernutrition multi-sectoral programmatic actions comprising the key pillars nutrition, agriculture and health care are recommended for timely intervention before the child reaches two years of age; accompanied by hygienic and sanitary activities, with interactive awareness trainings being in the center of actions. Lack of dietary diversification was not only found as predictor for anemia at baseline, moreover the low-dose intervention trial confirmed diversified meals only (IG1) being a successful food-based approach in significantly increasing the Hb concentration as opposed to the CG, thus is suggested as useful preventive strategy to overcome nutrition-related anemia amongst Santal Adivasi children (aside iron therapy). Study findings indicate the adding of MNP (IG3) as being beneficial for decreasing the burden of undernutrition and infectious diseases.
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.
Dietary intake, nutritional status of lactating women and their 6-23-months-old children in Genta Afeshum District, Rural Ethiopia : adaptation and validation of calculator for inadequate micronutrient intake (CIMI)
(2020) Desalegn, Beruk; Frank, Jan
Religious fasting is one of the categories of dietary or food taboos, which may affect the dietary intake and nutritional status of individuals. In Ethiopia, about half of the population are followers of Ethiopian Orthodox Tewahedo religion, and approximately 250 days per annum are fasting days. In these fasting days, lactating and pregnant women and children are exempted from fasting. However, lactating and pregnant women fast and are not also happy to prepare non-fasting foods for their children during the fasting days due to fear of contamination of family food. Early identification of micronutrient deficiencies in Ethiopia are flouted, as most often the quantitative dietary data are not available. As a result, the hidden hunger might have not been addressed properly, where it remains high and persistent. Therefore, easy to use, less costly and applicable assessment tool which can estimate the quantitative dietary intake of an individual or a community is urgently needed to achieve the national and international goals set for eradicating malnutrition. The Calculator for Inadequate Micronutrient Intake (CIM) is a simple, easy-to-use, informative, web-based application of quantitative dietary assessment method, which was first developed in Indonesia for Indonesian population. It estimates energy and nutrient intake correctly, and identifies nutrient inadequacy according to FAO/WHO recommended nutrient intake (RNI) regarding age, sex and physiological stage. Thus, the present study was conducted with the aim of assessing and comparing the nutritional status and dietary intake of lactating women and their 6-23-months-old children in fasting and non-fasting periods, and to adapt and validate the CIMI program for Ethiopian population. This study was conducted in rural Genta Afeshum district, in Tigray, Ethiopia, where almost all people in the woreda are followers of Ethiopian Orthodox Christianity. A longitudinal study was conducted using575 and 522 lactating women and their 6-23-months-old children in the lent fasting and non-fasting. In the present study it was found out that the prevalence of underweight (BMI < 18.5 kg/m2) in fasting lactating women was high (50.6%) which is associated with maternal age, maternal illness within four weeks preceding the fasting survey, fasting status during their pregnancy and lactation period of their children included in this study. Additional predictor variables for maternal underweight were grandfathers‘ as household decision maker, use of non-improved water source, household aid experience and the absence of chicken in the household. The average number of meals, diet diversity, and animal source foods consumption scores were significantly higher in non-fasting compared to fasting periods, regardless of the fasting status (p < 0.001, p < 0.05 and p < 0.001, respectively). Whereas, 31.6–33.7%, 11.7–15.7% and 4.4–4.8% of the 6-23-months-old children in the study population were stunted, underweight and wasted, respectively. In the fasting period, the weight-for-length (WLZ) and length-for-age (LAZ) values for the 6-23-months-old children of non-fasting mothers were significantly higher (p < 0.05) than the children of the fasting mothers‘ group. Similarly, the median weight-for-age (WAZ) and diet diversity score (DDS) of children of fasting mothers were also significantly lower in fasting compared to non-fasting period. The proportion of the 6-23-months-old children who met the minimum acceptable diet (MAD) was small (2.3-6.7%) in the study population; however, this proportion was significantly (p < 0.001) higher in the non-fasting than fasting period in the children of fasting mothers. Age of the child, maternal fasting status during pregnancy and lactation periods, maternal education and occupation were associated with child underweight. Likewise, age of the child, colostrum intake status, maternal fasting status during pregnancy and lactation period and toilet presence in the household were associated with child stunting. But, maternal fasting status during lactation period and maternal education predicted wasting in the children precisely. The average energy, protein and almost all micronutrients intakes of children and women were lower in fasting compared to non-fasting period. At the same time, the prevalence of inadequate intakes of energy, protein and most micronutrients were higher in both the children and lactating women during fasting than non-fasting period. The result of this study revealed that the correlation coefficients for the average dietary nutrient intake calculated by CIMI and the reference software NutriSurvey (NS) were between 0.741-0.956 for the children and between 0.779-0.920 for the lactating women groups. As a conclusion, the dietary pattern and nutritional status of lactating women and their breastfed children are affected during the fasting period. Therefore, the existing multi-sectoral nutrition intervention strategies in Ethiopia should include religious institutions in a sustainable manner. CIMI adapted for the rural Ethiopian setting estimates the average nutrient intake accurately; and identifies inadequate micronutrient intake of individuals enabling enumerators to provide feedback and suggest improvements. Thus, CIMI can be used in Ethiopia, as a simple dietary assessment tool by nutrition and related researchers, policy makers, implementers and evaluators.
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.
Tocotrienols, tocopherols and tocomonoenols : characterization in Costa Rican palm oils, and intracellular and tissue distribution as a function of the hepatic alpha-tocopherol transfer protein
(2018) Irías-Mata, Andrea Paola; Frank, Jan
Vitamin E is a generic term for a group of micronutrients exhibiting the biological activity of alpha-tocopherol. Initially, four tocopherols (T) and four tocotrienols (T3) were recognized as the naturally occurring vitamin E compounds. The main difference among T and T3 is the 3-fold unsaturated 16-carbon side chain of the T3 compared to the saturated 16-carbon side chain of the T. Recently, a group of four vitamin E compounds with a single double bond at carbon 11 were discovered, namely tocomonoenols (T1). Edible oils are the major source of T, T3, and of T1. As a fat-soluble vitamin, the vitamin E is absorbed after oral intake and transported in the circulation to the liver, where vitamin E undergoes sorting by the action of the alpha-hepatic-tocopherol transfer protein (TTP) and the cytochrome P450 (CYP) enzymes. alpha-T is preferentially secreted into the bloodstream, while the non-alpha-T congeners are metabolized by CYP to the carboxyethylhydroxychromanols (CEHC), which are excreted via urine and feces. The TTP has been recognized as necessary for the maintenance of normal alpha-T concentrations in plasma and extrahepatic tissues. Interestingly, TTP might also protect the non-alpha-T congeners from side-chain degradation, and therefore prevent their metabolic degradation. The present thesis aimed at increasing our knowledge of the non-αT congeners of vitamin E with respect to their occurrence in food, their intracellular localization upon uptake into liver cells, and their tissue distribution in mammals. A potential role of the TTP in the intracellular and intra-organismic trafficking of the non-alpha-T congeners was a second focus of the current investigations. To this purpose, the vitamin E profiles and contents in oils of three Elaeis Guineensis, two Elaeis Oleifera, and one hybrid OxG palm fruit genotypes from Costa Rica were determined after mechanical extraction with a screw press and chemical extraction with hexane. Vitamin E profiles in the palm oils were similar, irrespective of the genotype and extraction procedure, and alpha- and gamma-T3 were the most abundant congeners. alpha-T1 was found in oils from five of the six varieties. Hexane extraction yielded up to 2.5-fold higher total vitamin E compared to screw press extraction. The two most abundant tocotrienols in the oils were selected for further studies with respect to their cellular uptake and intracellular distribution in cultured liver cells with and without stable expression of TTP and compared to their respective tocopherol counterparts. After uptake, all four congeners were primarily associated with the lysosomes, endoplasmic reticulum and plasma membrane. Overall, the results conclude that neither the structural differences between the four congeners, nor the TTP-expression are important factors behind the intracellular trafficking (uptake and distribution) of the congeners in cultured liver cells. Finally, an animal study was performed to examine the tissue distribution of alpha-T1 in mice in comparison to alpha-T. Besides was investigated the influence of TTP. Wild-type and TTP knockout mice were fed a standard diet with either alpha-T or alpha-T1 for 2 weeks. Concentrations were measured in blood and several tissues. alpha-T1 was only found in blood, not in tissues. Loss of TTP function in knockout mice resulted in almost complete depletion of alpha-T in all tissues. Interestingly, alpha-T1 was still present in blood. In conclusion, alpha-T1 reached the blood in mice with and without TTP function, suggesting that TTP may not, or only to a limited extent, be required for the secretion of alpha-T1 into the systemic circulation. Since more is known about alpha-T than the non-alpha-T congeners, new opportunities for further research on the biological activities and consequent health benefits of the non-alpha-T congeners have arisen based on the contributions of the present thesis.
Establishment of defined culture conditions for the differentiation, long-term maintenance and co-culture of adipose-derived stem cells for the setup of human vascularized adipose tissue
(2018) Volz, Ann-Cathrin; Kluger, Petra Juliane
Most current attempts in engineering adipose tissue are based on the supplementation with human or animal-derived sera. However, especially the use of animal-derived serum is linked to many disadvantages, like potential contaminations, ethical issues and in general the missing identification of many ingredients. Therefore, serum supplementation impedes the actual application of engineered adipose tissue constructs as implants, to substitute lost tissue after tumor resection, severe burnings or trauma. Equally, due to a potential cover up of the cellular response by unidentified components, it impairs the in vitro use of such models as test systems to elucidate mechanisms of disease development, screen for new drugs or generally assess pharmaceutical safety levels. To be capable for functional anastomosis with the host tissue after implantation and for the use in time- and maturation-dependent in vitro purposes, engineered constructs have to exhibit a minimum sustainability. So far, only few authors addressed the serum-free, defined differentiation of adipocytes. And there are hardly any trials available on the defined maintenance of adipocytes. In this study, the development of a defined culture medium for the adipogenic differentiation of primary human adipose-derived stem cells (ASCs) was aimed. Based on the addition of specific factors for the replacement of serum, ASCs were differentiated to viable and characteristic adipocytes for 14 days, which was proven through the accumulation of lipids, the expression of perilipin A and by the release of leptin and glycerol. Furthermore, a defined maintenance medium was developed, which supported the maturation and stability of cells for a long-term period of additional 42 days until day 56. In order to pursue the goal of a physiological tissue substitute of relevant size, the integration of a vascular component is of fundamental importance to allow sufficient nutrient supply of all peripheral tissue areas. For this purpose, a natural vascular system based on a cellular component would be ideal. Due to the lack of an adequate co-culture medium, a major challenge in adipose tissue vascularization is represented by the setup of an adipocyte/endothelial cell (EC) co-culture. In this study, the development of a tissue-tailored co-culture medium based on adipocyte- and EC-factors was developed. Thereby the critical role of epidermal growth factor (EGF) and hydrocortisone (HC) in adipocyte/microvascular (mv)EC co-culture was determined. Through the adjustment of their supplementation, a functional co-culture of adipocytes and mvECs was achieved. In there, mvECs maintained the cell-specific expression of von Willebrand factor (vWF) and cluster of differentiation 31 (CD31). Additionally, cells kept their ability to incorporate acetylated low density lipoprotein (acLDL). By combining the experiences from both mentioned attempts, a defined adipocyte/EC co-culture medium was developed. Next to the maintenance of functional and characteristic adipocytes, the medium facilitated the formation of vascular-like structures in the direct co-culture. To be able to establish tissue constructs of relevant size, current in vitro attempts have to be transferred to a three-dimensional (3D) environment. In this trial, a 3D adipose tissue model was set up based on the differentiation of ASCs in a collagen type I hydrogel in co-culture with mvECs for 21 days in total. The comparison of these models with native adipose tissue demonstrated high accordance in the gene expression levels related to differentiation and fatty acid metabolism. Some deviations were found mostly in maturation-dependent genes linked to tissue functionality and angiogenic mediation. Differentiation and the maintenance of a homeostatic tissue state highly rely on the physical and chemical characteristics of the applied scaffold. As another part, the influence of a novel cellulose-based material (CBM) on defined adipogenic differentiation of ASCs and the defined maintenance of mvECs was investigated in this thesis. An accelerating effect of CBM on the defined differentiation of ASCs was proven by enhanced release of leptin and the increased expression of perilipin A. CBM was further shown to facilitate the formation of vascular-like structures by mvECs under defined conditions in the absence of another supporting cell type. Additionally, the successful co-culture of adipocytes and mvECs was demonstrated on CBM under defined conditions. Summarized, defined culture media for the differentiation, maintenance and co-culture of primary ASC and mvECs were developed. The supporting effect of CBM on the defined establishment of cultures was proven. Further the successful setup of a 3D adipocyte/mvEC co-culture model with a high predictive power was shown. Combined these achievements can be used for the in vitro setup of a 3D vascularized adipose tissue under defined conditions.
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.

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