Institut für Ernährungsmedizin

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The role of NLRC5 in obesity
(2024) Bauer, Sarah Katharina; Kufer, Thomas
Obesity and its associated morbidities are major global health problems. It has become evident in the last decades that the state of obesity is intimately linked with our immune system. Pattern recognition receptors (PRRs), the main sensor molecules of the innate immune system, were shown to play an essential role in the pathology of obesity and its associated morbidities. Among others, members of the nucleotide-binding and oligomerization domain (NOD) -like receptors (NLRs), a family of cytosolic PRRs, were associated with the obesity-accompanying low-grade inflammatory response contributing to obesity-associated morbidities. NLRC5 is a NLR protein functioning as key transcriptional regulator of major histocompatibility complex (MHC) class I genes responsible for antigen presentation. Recent observations now suggest novel roles of NLRC5 in metabolic trades, but so far, no confirmation of these singular observations is available, and the underlying mechanisms remain elusive. The aim of this thesis was to characterize the role of the NLR protein NLRC5 in obesity. To this end, two Nlrc5 deficient mouse lines (Nlrc5 deltaExon4-7 and Nlrc5 deltaExon4) were subjected to high-fat diet (HFD) feeding and phenotypic, morphological, and biochemical analyses were performed. Female Nlrc5 deltaExon4-7 mice presented with higher body and adipose tissue (AT) weight gain and larger adipocytes compared to wildtype (WT) animals. This phenotype, however, could not be recapitulated in the Nlrc5 deltaExon4 mouse line. Microbiome analysis revealed subtle alterations of the faecal microbiome by diet:genotype interactions. To further characterize the effect of NLRC5 deficiency on adipocyte differentiation, the CRISPR/Cas9 gene editing system was used to modify Nlrc5 expression in the 3T3-L1 preadipocyte cell line. Using inducible HeLa cell lines with stable GFP-NLRC5 expression we showed NLRC5 to interact with the master regulator of adipogenesis peroxisome proliferator-activated receptor y (PPARy) and to enhance the expression of PPARy target genes. In addition, a contribution of NLRC5 to PPARy’s anti-inflammatory actions was revealed using NLRC5 deficient THP-1 macrophage-like cells and bone marrow-derived macrophages from Nlrc5 deltaExon4-7 mice. To elucidate the mechanism behind the synergy between NLRC5 and PPARy, reporter gene and chromatin immunoprecipitation (ChIP) assays were performed. Lastly, the expression of multiple NLR family members was correlated with body mass index (BMI) in obese human patients and investigated in the adipose tissue and liver of HFD-fed mice, the latter revealing Nlrp10 to be highly upregulated by HFD feeding. Taken together, this thesis provides a comprehensive characterization of Nlrc5 deficient mice on HFD and reveals a function of NLRC5 as transcriptional co-regulator of PPARy targets and its anti-inflammatory properties. In addition, this work provides first insights into the potential mechanism behind the synergistic transcriptional regulation by NLRC5 and PPARy and extends the knowledge on the regulation of NLR expression by HFD feeding.
Die Mikroalge Phaeodactylum tricornutum : Bioverfügbarkeit, Sicherheit und potenzieller gesundheitlicher Nutzen für die humane Ernährung
(2023) Kopp, Lena Janine; Bischoff, Stephan C.
The dissertation by Lena Kopp investigated the suitability of the microalga Phaeodactylum tricornutum (PT) for human nutrition. PT contains essential nutrients such as the long-chain omega-3 fatty acid eicosapentaenoic acid (EPA), which is otherwise found mainly in fish. In addition, PT contains a high content of other nutrients such as proteins, carotenoids (in particular fucoxanthin), vitamins and β-glucans, which have nutritive and therapeutic potential. Clinical and animal studies have shown that the PT biomass ingestion is safe and has potential health effects, such as anti-inflammatory and prebiotic effects. The results suggest that PT can be used as a food for human nutrition with possible health-promoting effects.
Immunomodulatory effects of resveratrol on human intestinal mast cell signaling in vitro and mast cell associated enteritis and colitis in mice
(2023) Bilotta, Sabrina; Lorentz, Axel
By releasing their pre-stored or de novo synthesized mediators, mast cells (MC) are important immunoregulatory cells responsible for a variety of inflammatory reactions. Although known to be major effector cells in immunoglobuline (Ig) E dependent allergic reactions, MC have been widely shown to play a role in various inflammations of the gut. Diseases of the gastrointestinal tract (GIT) are widespread and multicausal. Those affected suffer from the sometimes severe symptoms and may experience restrictions on their daily life. Even if conventional medication is applied routinely, aim of the past and current research is to establish supportive and/or alternative medication that is based on natural substances. These may be on the basis of small natural components like resveratrol, a stilbene mostly found in grapes. Numerous positive properties are attributed to resveratrol. These are anti-inflammatory, anti-cancerogenic, anti-oxidative, as well as neuroprotective effects. The use of substances of natural origin as so-called nutraceuticals can help to increase the acceptance of medication by those affected, but also to reduce and overcome the side effects associated with conventional treatment. Effects of resveratrol were examined on the reactivity of MC isolated from patients’ tissue undergoing bowel resection. The results of this work show that resveratrol exhibited potent inhibitory effects on high affinity IgE receptor mediated activation of MC, strongly inhibiting not only MC degranulation, but also gene expression of the pro-inflammatory cytokines C-X-C motif chemokine ligand (CXCL) 8, C-C motif chemokine ligand (CCL) 2, CCL3, CCL4 and tumor necrosis factor (TNF-) α. Ultimately, the intracellular signaling cascade triggered during MC activation via IgE receptor leads to mediator release. Following IgE receptor mediated activation, phosphorylation of signaling molecules like extracellular signal-regulated kinase (ERK) 1/2 and signal transducer and activator of transcription (STAT) 3, occurs. ERK1/2 was found to be responsible for phosphorylation of mitochondrial STAT3, which contributes significantly to MC degranulation. Treatment with resveratrol was able to inhibit the phosphorylation of STAT3 by more than 50 % and that of ERK1/2 by almost 100 %. Furthermore, the experiments performed succeeded in isolating the mitochondrial fraction from relatively low human intestinal MC (hiMC) numbers. Also, in this fraction we could detect phosphorylation of STAT3 and ERK1/2 after MC activation, which was reduced after treatment with resveratrol. Having shown the strong inhibitory effects in vitro, we set out to examine immunomodulatory effects of resveratrol in vivo. Presence and activity of MC are closely related to intestinal inflammations in consequence of food allergy (FA) and inflammatory bowel disease (IBD). In mice, FA can be studied using the ovalbumin (OVA)-induced allergic enteritis model and colitis can be studied using the IL-10 knockout (-/-) mice, which develop a spontaneous form of chronic colitis. We could show that the oral application of resveratrol inhibited the increase of MC numbers in the colon and duodenum of affected animals in both experimental settings. Less pronounced but still visible effects of resveratrol administration were observed in the colon with regard to epithelial damage, cell infiltration and reduction of goblet cell numbers. In all cases, based on a scoring system, the damage decreased to the level of the corresponding controls receiving no additive and in which no allergic enteritis was induced or nor colitis developed. Overall, allergic enteritis resulted in a weaker symptomatology, and IL-10-/- animals showed a delayed appearance of the typical symptoms. The results of this thesis show a strong inhibitory effect of resveratrol on hiMC. This could be detected for mediator release as well as on gene expression levels and in the phosphorylation of the signaling molecules ERK1/2 and STAT3, which we could also identify in the mitochondria of hiMC. We observed positive influences on MC-associated parameters in the OVA enteritis and IL-10-/- colitis mouse models. With regard to its use as nutraceutical, resveratrol could therefore come more of a focus in the future.
Einfluss der mediterranen Ernährung auf das Fettsäuremuster von Erythrozytenmembranen sowie auf Darmmikrobiota- und Darmbarriere-assoziierte Biomarker : Mechanismen und klinische Anwendungen
(2022) Seethaler, Benjamin; Bischoff, Stephan C.
Dissertation from Benjamin Seethaler: "Effect of the Mediterranean diet on the fatty acid pattern of erythrocyte membranes and on gut microbiota- and gut barrier-associated biomarkers - mechanisms and clinical applications". In summary, the results of the PhD project offer new insights into the biomedical mechanisms of action and health effects of the Mediterranean diet. Of particular importance is the relationship we have shown between dietary fiber from the Mediterranean diet, its fermentation to short-chain fatty acids, and its beneficial influence on impaired intestinal barrier function. In the future, our studies may provide the basis for personalized nutritional therapy to improve impaired gut barrier function in high-risk breast cancer patients. Furthermore, we were able to establish LBP and zonulin as biomarkers to detect gut barrier function and to determine and assess gut barrier disorders. This method validation simplifies or enables the assessment of intestinal barrier function in clinical practice or clinical trials.
Einfluss verschiedener Getreidearten und Herstellungsverfahren auf den Gehalt immunogener Substanzen in Brot sowie in vivo auf die Verträglichkeit an der Maus und im Menschen
(2022) Zimmermann, Julia; Bischoff, Stephan C.
There are three medical conditions that are triggered by consumption of cereals. Celiac disease, wheat allergy and non-celiac wheat sensitivity (NCWS). While the underlying triggers and mechanisms of the first two entities have been extensively studied, there is still uncertainty in this regard for NCWS. Symptoms are nonspecific and diagnostic markers are lacking. Besides bacterial fermentable carbohydrates (FODMAPs), selected cereal proteins such as gluten or α-amylase trypsin inhibitors (ATIs) are in the focus of research as triggers. The aim of the present work was firstly to investigate the influence of the choice of cereal (common wheat, spelt, rye) and the production of bread (degree of milling and choice between yeast and sourdough) on the presence of potentially immunogenic proteins based on proteomic analysis. In a second step, the tolerability of selected breads should be investigated in a transgenic mouse model with intestinal inflammation and in a human study in patients with NCWS and subjective spelt tolerance. This was to narrow down possible triggers of NCWS and to investigate underlying mechanisms. Within the project, protein composition of bread and flour samples was analyzed based on a quantitative proteomics method (nano-UHPLC-ESI-MS based). In addition, a list of known and potentially immunogenic cereal proteins was generated based on Pfam annotation, which was used for the analysis of allergens in flour and bread. This showed that neither the absolute number nor the abundance of these allergenic proteins were dependent on the degree of milling of the flour or the fermentation process of the dough, which means that they are not selectively degraded during bread production. However, such proteins were identified in higher numbers and higher relative amounts in spelt and wheat samples compared to rye samples. Furthermore, different bread types from the proteome analysis were investigated in a mouse model with intestinal inflammation. This did not demonstrate better tolerability of rye bread compared to spelt and wheat bread. Instead, there was a trend for sourdough bread to have less negative effects on intestinal inflammation compared to yeast dough bread. It also turned out that inflammation was increased independently of gluten. No differences were found between wheat and spelt in either the proteomic analysis or the animal studies in this project. However, in a subgroup of NCWS patients, spelt bread is subjectively better tolerated than wheat bread, which could be due to both genetics and the different production of wheat and spelt bread. In order to verify the phenomenon and identify underlying mechanisms, if any, a clinical study was conducted in patients of this subgroup. The aim of the blinded study was to investigate whether spelt bread is actually better tolerated than wheat bread and whether the production process (16h dough or 1h dough + baking agent) has an influence. After each bread (4 days each + 3 days washout), gastrointestinal symptoms were assessed using the Irritable Bowel Syndrome Severity Scoring System (IBS-SSS) questionnaire. Extraintestinal symptoms and various blood and stool parameters were also analyzed. It was found that spelt bread was not better tolerated compared to wheat bread after blinded consumption and that FODMAP-rich bread was not worse tolerated compared to FODMAP-poor bread.
Characterization of the role of the NLR proteins NLRC5 and NLRP11 in the immune response
(2021) Kienes, Ioannis; Kufer, Thomas
Recognition of conserved molecular patterns by pattern recognition receptors (PRRs) is crucial for the initiation of an innate immune response. Within PRRs, the NOD-like receptor (NLR) family is, in humans, a group of 22 cytosolic proteins, which function as PRRs of the innate immune system and as regulators of adaptive immune responses. However, it has become evident, that several NLR proteins also function as regulators of innate immune responses. In this thesis the function of human NLR proteins NLRC5 and NLRP11 in immune responses was further characterized. The first part of this thesis was focused on the NOD-like receptor NLRC5. NLRC5 and major histocompatibility complex (MHC) class II transcriptional activator (CIITA) are the master regulators of MHC class I and II transcription, respectively. Both proteins can translocate into the nucleus, where they induce transcription of MHC class I and class II, respectively. As NLRC5 and CIITA do not possess intrinsic DNA binding capacities, they exert their function by binding to a common multiprotein complex, termed MHC enhanceosome and through recruitment of further transcriptional regulators. Although MHC enhanceosome components are, as known thus far, identical, NLRC5 and CIITA are specific for their respective transcriptional targets. In this work we employed several techniques to identify novel interaction partners of NLRC5 to understand the mechanisms behind this specificity. As the N-terminal domain death-domain like fold (DD) of NLRC5 has previously been shown to be involved in conferring specificity, we adapted a protocol for proximal ligation by fusion of the NLRC5 DD to biotin ligase from Aquifex aeolicus (BioID2) to unravel the interactome of this NLRC5 domain. By enrichment of biotinylated proteins through streptavidin-biotin precipitation and analysis of the proteins by LC-MS/MS, we aimed to identify novel putative interactors with functions in transcriptional regulation. Additionally, we used yeast 2 hybrid screening of the NLRC5 DD against a library of human CD4+ and CD8+ T cells for the identification of novel interaction partners. This led to the identification of the paired amphipathic helix protein Sin3A (Sin3A) and the negative elongation factor B (NELFB) as interactors of NLRC5 DD. Characterization of their role in transcriptional regulation of MHC class I revealed an inhibitory role of both proteins. However, as we also observed repression of CIITA-mediated MHC class II transcription, both proteins are likely not involved in determination of target specificity of NLRC5. Translocation of NLRC5 into the nucleus is essential for the induction of MHC class I transcription. It has however previously been shown, that forced nuclear localization of NLRC5 strongly diminishes its transcriptional activity. We therefore employed co-immunoprecipitation of differentially localized NLRC5 constructs to identify interaction partners which might be involved in post translational regulation of NLRC5. Further, to advance our understanding of the NLRC5 DD, we aimed to elucidate its crystal structure. For this, we established a protocol for large-scale recombinant expression and purification of the NLRC5 DD for subsequent crystallization of the recombinant protein. The second part of this thesis was focused on NLRP11. Tight regulation of inflammatory cytokine and interferon (IFN) production in innate immunity is pivotal for optimal control of pathogens and avoidance of immunopathology. NLRP11 has previously been shown to regulate type I IFN and other pro-inflammatory responses. To gain a deeper understanding of the underlying mechanism, we aimed to identify novel NLRP11 interactors, through which the inhibition is conferred. Here we generated cell lines stably expressing NLRP11-eGFP as novel tools to elucidate the functions of NLRP11. We identified the ATP-dependent RNA helicase DDX3X as a novel binding partner of NLRP11 by co immunoprecipitation and LC-MS/MS. DDX3X is known to enhance type I IFN responses and NLRP3 inflammasome activation. We demonstrate that NLRP11 can abolish IKKe-mediated phosphorylation of DDX3X, resulting in lower type I IFN induction upon viral infection. These effects were dependent on the leucine-rich repeat (LRR) domain of NLRP11 that we mapped as the interaction domain for DDX3X. In addition, NLRP11 also suppressed NLRP3-mediated caspase-1 activation in an LRR domain-dependent manner, suggesting, that NLRP11 might sequester DDX3X and prevent it from promoting NLRP3-induced inflammasome activation. Taken together, our data revealed DDX3X as a central target of NLRP11, which can mediate the effects of NLRP11 on type I IFN induction, as well as NLRP3 inflammasome activation. This expands our knowledge of the molecular mechanisms underlying NLRP11 function in innate immunity and suggests that both NLRP11 and DDX3X might be promising targets for modulation of innate immune responses.
Effekte der Proteinzufuhr während einer Gewichtsabnahme auf fettfreie Masse, Ruheenergieumsatz und physische Funktion bei übergewichtigen postmenopausalen Frauen - eine randomisierte, kontrollierte Studie
(2020) Englert, Isabell; Kohlenberg-Müller, Kathrin
Aim Weight loss in old age increases the risk of sarcopenia caused by the age-related reduction of fat-free mass (FFM). Due to the strong correlation between FFM and resting energy expenditure (REE), the maintenance of this must also be considered. In addition, the physical function (PF) must be maintained. The objective was to investigate the impact of protein intake on changes in fat-free mass (FFM), resting energy expenditure (REE), and physical function (PF) during weight loss. Methods 54 postmenopausal women (BMI 30.9 ± 3.4 kg/m²; 59 ± 7 years of age) were randomized into two groups with 0.8 g (K) or 1.5 g protein/kg body weight/d (P) energy-restricted diets (- 750 kcal of individual energy requirements) for 12 weeks, followed by six months weight maintenance with ad libitum food intake. The protein dose was evenly distributed to two liquid and one solid meal. The shakes of the P group were additionally enriched with pure whey protein. Four seminars were held to provide information on the course of the study and in particular on healthy nutrition. At the beginning and at the end of the study, the subjects kept a 7-day nutrition protocol. FFM (by bioelectrical impedance analysis), REE (by indirect calorimetry), PF (strength, endurance, and balance by short physical performance battery test (SPPB), 400 m walking speed and handgrip strength by hand dynamometer), blood parameters (lipid and carbohydrate profile, urea, vitamin D, calcium, magnesium, liver and kidney values from serum) and blood pressure were measured at baseline, after weight loss, and after follow up. The evaluation was primarily based on an intention to treat analysis with correlation and regression analysis, paired and unpaired t-tests, whereby the significance level was set ≤ at 0.05. The values given are continuous mean values ± standard deviation. Results 46 women completed the weight loss intervention and 29 were followed up after weight maintenance. Weight loss was -4.6 ± 3.6 kg (P) and -5.2 ± 3.4 kg (K) (both p < 0.001) and weight regain during follow up was 1.3 ± 2.8 kg (P, p = 0.028) and 0.4 ± 2.5 kg (K) (p = 0.392) with no differences between protein groups. Similar losses in FFM (-0.9 ± 1.1 kg (P) vs. -1.0 ± 1.3 kg (K)) and REE (-206 ± 136 kcal/d (P) vs. -239 ± 134 kcal/d (K), both p < 0.001) were observed in both groups. At follow-up, no changes in FFM were detected in both groups whereas in the NP group the REE increased again (138 ± 296, p = 0.02). The main determinants of the FFM loss were the energy deficit and the speed of weight loss. In the NP group, SPPB score improved with weight loss (0.6 ± 0.8, p < 0.001) and handgrip strength decreased (-1.7 ± 3.4 kg, p < 0.001) whereas no changes were observed in the HP group. The blood profile improved, especially regarding the carbohydrate profile, due to weight loss, and blood pressure. Conclusion A high protein weight loss diet without exercise had no impact on preserving FFM and REE but may help maintain muscle strength in postmenopausal women. As the handgrip strength can be a sensitive parameter for incipient sarcopenia even before the muscle mass decreases noticeably, it can be concluded that an increased protein intake during weight loss can counteract the risk of sarcopenia. Energy deficit and speed of weight loss should be considered as confounders in future studies. In addition, further strategies must be pursued to maintain FFM in weight loss in old age.
The dietary quality of food pantry users from a socio-ecological perspective
(2019) Simmet, Anja; Ströbele-Benschop, Nanette
In Germany, around 1.5 Mio people with a low income receive food for a small fee from one of around 940 so-called “Tafel”. In high-income countries like Germany, users of food pantries are a particularly vulnerable population group, as they are characterized by cumulative health risks. They often suffer from food insecurity and from chronic diseases such as hypertension, diabetes mellitus type 2 or obesity. Given that these diseases as well as food insecurity both relate to diet, the dietary quality plays a critical role in the health status of food pantry users. To illustrate the dietary quality and the different levels of factors influencing the dietary quality among food pantry users, this thesis has the following aims: 1. to provide a summary of the scientific evidence about the dietary quality of food pantry users in high-income countries; 2. to provide a summary of the scientific evidence about the nutritional quality of food provided by food pantries in high-income countries; 3. to examine the distribution of Tafel food pantries and food banks and to provide a representative picture of their resources (e.g. food, volunteers etc.), activities (e.g. provided programs) and users in Germany; 4. to examine the distribution of Tafel food pantries and to identify compositional and physical environmental correlates of food pantry use in Berlin. To reach these aims, two systematic reviews were conducted (first and second publication). In addition, an explorative cross-sectional study consisting of an analysis of secondary data and a comprehensive survey among all Tafel belonging to the federal association “Tafel Deutschland” was performed (third publication). Finally, an ecological study was conducted by analyzing and mapping food pantry use and compositional and physical characteristics of areas in Berlin (fourth publication). The first review revealed that the dietary quality among the reviewed food pantry users tended to be low as reflected by an inadequate intake of energy, fruit and vegetables, dairy products and calcium compared to national recommendations. The reviewed food pantry users had, in particular, a lower consumption of dairy products compared to the general populations. The second review demonstrated that the nutritional quality of reviewed pre-packed food bags provided by food pantries was highly variable within and between included studies. It also showed that the nutritional quality of most of the food bags was low, reflected, in particular, by a low provision of dairy products, vitamins A and C and calcium compared to national recommendations. None of the studies included in the reviews were nationally representative. The third publication showed that the German food bank system Tafel Deutschland provided a comprehensive net of food pantries, social supermarkets, food banks and other services. However, the number of Tafel per 10,000 welfare recipients was lower in eastern Germany (M = 1.37) compared to western Germany ((M = 2.12), t(162.54) = 4.2424, p < 0.0001). In contrast to the results of the studies included in the second review, the Tafel mainly provided perishable food such as fruits and vegetables (41.42% of the amount of food distributed), bakery products (19.85%) and dairy products (13.39%). However, due to the dependence on donors such as retailers, the amount of food distributed varied widely. The assistance of most local Tafel is based on volunteer labor as 89.97% of Tafel’s staff were volunteers. In 79 districts, in which all Tafel participated in the survey, there was an average of 179 Tafel beneficiaries per 1000 welfare recipients and 17 Tafel beneficiaries per 1000 residents overall. An even lower usage was found by the fourth publication which revealed that only two out of 1000 adult inhabitants and six out of 1000 children received assistance from one of the 44 investigated Tafel food pantries in Berlin. Tafel use by adults (A) or by children (B) was related to the percentage of welfare recipients (A, β = 0.17, p < 0.001 und B, β = 0.16, p = 0.002), the percentage of inhabitants with migration background (A, β = - 0.08, p = 0.002), the number of discount grocery percentage stores per 1000 children (B, β = 5.65, p = 0.010), and the number of stops of the public transport within a radius of 500 meters (A, β = - 0.24, p = 0.020). The most important limitation of both studies was the unknown reliability of the data collected from the Tafel. The dietary quality of food pantry users in high-income countries was seen to be low. Although food pantries did not provide a full meal plan for a healthy diet, they may have a positive impact on the dietary quality of its users. However, to better understand the role of food pantries in the complex interplay of individual, social and environmental influences on the diet of food pantry users, multi-level approaches should be used in the future. Moreover, researchers are strongly recommended to investigate the mechanisms by which using a food pantry might impact users’ dietary quality. In addition to these implications for researchers, this thesis makes several recommendations to practitioners at food pantries in Germany and other high-income countries. Following these recommendations could make the position of food pantries in civil society into an important entry point for health promotion among a part of the food insecure population in the future.
Metabolic chamber studies on energy- and macronutrient metabolism : impact of meal skipping and energy flux
(2018) Nas, Alessa; Bosy-Westphal, Anja
The classical concept of body weight regulation attributes the development of obesity to a chronically positive energy balance. There is, however, evidence indicating that beyond this basic concept, the effectiveness of body weight regulation is affected by the circadian regulation of metabolism and the level of energy flux (EF, level of energy balance). Meal skipping affects circadian regulation and might therefore also affect the regulation of body weight. In addition, an asymmetric regulation of body weight is hypothesized with improved effectiveness when EF is high (active lifestyle) and less effectiveness at a low EF (sedentary lifestyle). Metabolic chambers offer the opportunity to acquire short-term parameters of energy and macronutrient balance that precede long-term weight gain and therefore, can help to understand the impact of nutrition and physical activity interventions on body weight regulation. This thesis presents the implementation of a metabolic chamber system (Chapter II) and investigates the acute impact of meal skipping (Chapter III) and energy flux (Chapter IV) on energy and macronutrient metabolism by performing two well-controlled, cross-over intervention studies using metabolic chambers. The implementation of the metabolic chambers revealed, that thorough considerations must be made in terms of the metabolic chamber environment (room ventilation and position of analyzer unit), the additional devices (e.g. air conditioner) used as well as the study protocol, in order to obtain good data quality. The study on meal skipping includes 17 healthy participants who underwent 3 isocaloric 24-h interventions (55%, 30%, and 15% carbohydrate, fat and protein, respectively): a breakfast skipping day (BSD) and a dinner skipping day (DSD) separated by a conventional 3-meal-structure day (control). Energy and macronutrient balance were measured and postprandial glucose and insulin concentrations, as well as 24-h glycemia and 24-h insulin secretion (C-peptide), were analyzed. When compared with the 3-meal control, 24-h energy expenditure was higher on DSD (DSD: +69 kcal/d; p < 0.05), but not on BSD. Whereas, fat oxidation increased on the BSD only (+13 g/d; p < 0.01). Spontaneous physical activity, 24-h glycemia, and 24-h insulin secretion did not differ between intervention days. The postprandial homeostasis model assessment index (+54%) and glucose concentrations after lunch (+46%) were, however, higher on the BSD than on the DSD (both p < 0.05). When compared with 3 meals/d, dinner skipping increased energy expenditure. In contrast, higher postprandial insulin concentrations and increased fat oxidation with breakfast skipping show the development of metabolic inflexibility in response to prolonged fasting that may in the long-term lead to impaired glucose homeostasis. The study on energy flux includes 16 healthy participants who underwent three 24-h interventions with different levels of EF: (i) low EF, physical activity level (PAL) = 1.3 – 1.4 (ii) medium EF, PAL = 1.5 – 1.6 and (iii) high EF, PAL = 1.7 – 1.8 each at energy balance (EB), caloric restriction (CR), and overfeeding (OF) (100%, 75% and 125% of individual energy requirement with 50% carbohydrate, 35% fat, 15% protein). Different levels of EF were accomplished by walking (4 km/h) on a treadmill (0, 165 and 330 min). Sleeping energy expenditure (SEE), 24-h macronutrient oxidation and relative macronutrient balance (oxidation relative to intake) were determined. During EB and OF, 24-h fat oxidation increased with higher EF. This resulted in a higher relative fat balance at medium EF (EB: +17%, OF: +14%) and high EF (EB: +23%, OF: +17%) compared to low EF (all p < 0.05). SEE during EB and OF was higher at medium (EB: +5 kcal/3h and OF: +12 kcal/3h) and high (EB: +7 kcal/3h and OF: +18 kcal/3h) EF compared to low EF (all, p < 0.05). In contrast, during CR 24-h fat oxidation was only higher at high EF compared to low EF and neither relative fat balance nor SEE differed between the EF levels. A higher EF might have beneficial effects on body weight regulation during short-term overfeeding and energy balance because it increased SEE and improved relative fat balance. However, during short-term caloric restriction, a higher EF had no impact on the regulation of energy or fat balance. Therefore, a high EF especially can attenuate the adverse effects of short-term overfeeding. Altogether, this thesis emphasizes the importance of physical activity in daily life and suggests that the adverse metabolic outcome of breakfast skipping (caused by a positive energy balance after lunch with a preceding prolonged fasting period) might be attenuated by a high EF.
Ernährung von sozial benachteiligten Menschen am Beispiel von Tafelkunden : Betrachtung des Ernährungs- und Gesundheitsverhaltens, der Verbreitung von Ernährungsarmut und des Obst- und Gemüsekonsums
(2018) Depa, Julia; Ströbele-Benschop, Nanette
In industrialized countries the distribution of mortality of morbidity follows a social gradient. Hence, among people with a lower socioeconomic status (SES) the nutritional quality is poor and low fruit and vegetable consumption occur more frequently compared to people with a higher SES. A particularly vulnerable group are socially disadvantaged people such as food bank users. Food insecurity (FI) is also more common in this population group. Food banks exist worldwide and distribute donated groceries (e.g. from food retailers) to socially disadvantaged people. In Germany, 1.5 million users are supported by over 900 food banks providing mainly fresh fruits and vegetables (FV). In Germany, little is known about the diet of food bank users. The following research questions were developed for this thesis: 1. Are there differences in health and nutrition status among people using food banks in different types of cities and can differences in these variables be found when comparing food bank users with the as low SES defined German population (chapter 2, first publication)? 2. How widespread is FI among food bank users and which socio-demographic, food bank-related and health variables are associated with FI (chapter 3, second publication)? 3. Are there differences in FV intake between male food bank users and male eligible non-food bank users and can FV intake among this study population be increased by an intervention providing weekly free and personally selected FV (14 portions/ per week) for four weeks (chapter 4, third publication)? For the publications of this thesis data of food bank users regarding socio-demographic, health- and nutrition-related variables were collected in different cities. The questions were taken from the questionnaire of the national study DEGS (German Health Interview and Examination Survey for Adults) and GEDA (German Health Update) and from the FIES (Food Insecurity and Experience Scale). Additionally, questionnaires were adapted to the study population. In all publications cross-sectional study designs were used. Except in the third publication for the second part of the research questions an intervention study using a longitudinal design was conducted. The first publication shows that food bank users from the three examined cities (Berlin n=94, Ludwigsburg n=64, Fulda n=114) are not a homogenous group. Food bank users assess their self-rated health mostly worse than people from the low SES German population (proportion self-rated health as moderate, bad or very bad: men 67.4% vs. 43.5%, women 68.8% vs. 36.7%). Additionally, they consume less fruit daily (proportion of daily fruit consumption: men 39.8% vs. 43.5%, women 56.2% vs. 62.4%). The second publication reveals with 70.2% a high rate of FI among food bank users (Stuttgart n=510, Karlsruhe n=186, Berlin n=337). Especially age (r τ = -0.224, p<0.000) and smoking (V=0.219, p<0.000) are strongly associated with FI. The third publication clarifies that male food banks users (n=24) from Stuttgart did not differ in consumed FV amount (2.2 portions/day vs. 1.8 portions/day) and variety (17 types/month vs. 14.4 types/month) compared to non-food bank users (n=28). Besides, the weekly provision of free fruit and vegetables for four weeks (14 portions/ month) increases the consumed fruit and vegetable amount (difference-IG 1.1 portions/day vs. difference-CG -0.2 portions/day) and variety (difference-IG 2.6 types/month vs. difference-CG -1.2 types/month) among the intervention group (n=25) compared to the control group (n=27). It is important to note the high amount of smokers among food bank users both in the first (46.9%) and the third publication (66.7%). The reported results correspond to research from abroad. Results of the publications are limited by unbalanced standardization and representation procedures (first and second publication) and the cross-sectional design (first and second publication and first research question of the third publication). It can be concluded that food banks are a suitable option to target socially disadvantaged people and to explore their nutrition and health behavior as well as a suitable option to provide intervention opportunities. The extent of health inequality is probably underestimated. FI is widespread among food bank users and smoking as poverty factor among food bank users should be further examined. The free provision of fruit and vegetables seems to be an appropriate possibility to increase the consumption and variety of fruit and vegetables among socially disadvantaged people.
Strategien zur Optimierung der Ernährung in schulischen Einrichtungen mit besonderem Fokus auf die Nährstoffversorgung
(2014) Böhringer, Stefanie; Grimm, Peter
Changes in society, such as growing number of employed women lead to a larger amount of schools in Baden-Württemberg that offer full time school programs. Due to this, the number of all day school pupils has more than tripled over the last 10 years (Landesinstitut für Schulentwicklung et al. 2011). As a result, in 2008, the Federal Ministry of Food, Agriculture and Consumer Protection set up the Vernetzungsstelle Schulverpflegung in Baden-Württemberg in joint financing with the Ministry of Rural Affairs and Consumer Protection. The Vernetzungsstelle Schulverpflegung Baden-Württemberg supports the schools in this new additional task of lunch catering and improves the quality and acceptance of the school meals in Baden-Württemberg. This study aims to evaluate and enhance the methods of the Vernetzungsstelle Schulverpflegung Baden-Württemberg to optimize the nutrition in educational institutions. For this purpose, ten schools with the corresponding seven canteens and 6000 pupils had been reviewed from June 2010 until October 2011 by the Vernetzungsstelle Schulverpflegung Baden-Württemberg. This supervision targets to improve the nutrition consumption of the pupils and thereby increase the amount of pupils with normal body-mass-index In order to reach this aim, both, the quality as well as the general requirements (e.g. menu, reservation) of the school lunch needs to be improved. Furthermore, the satisfaction of the customers as well as the resulting weekly participation in lunch at school canteens should be increased. Additionally, the pupils shall be sensitized for an appropriate diet. The “round table” is the fundament of the supervision of schools by the Vernetzungsstelle Schulverpflegung Baden-Württemberg. Several target groups come together to organize the improvement of the school canteen. For each school canteen, individual important optimizing and advertising strategies take place. Optimizing strategies include the training of caterers and canteen personal. Advertising strategies comprise several events, like pasta day or Santa Clause day, which help to raise the acceptance of the school canteen. To be able to measure the changes caused by intervention, an analyses takes place at the beginning and the end of the the study. For that, the surrounding conditions are evaluated by an expert interview with the responsible person of the school canteen. The satisfaction of the customers, such as pupils, teachers and parents is evaluated by written questionnaire. The quality of the lunch is analyzed by the criteria of the DGE quality standards of school feeding. Additionally, anthropometric data, like body height, body weight and abdominal girth, of six and seventh class pupils are measured. A bioelectric impedance analysis determines the body composition. Furthermore, a food frequency questionnaire and a 24h recall are conducted. Results of the study show, that during the time of intervention, on average 8 optimizing strategies and 15 advertising strategies are executed. 2991 (2966) pupils, 1939 (1785) parents and 143 (114) teachers take part at the first (second) survey. The cooperation of the schools with the Vernetzungsstelle Schulverpflegung Baden-Württemberg leads to an increased acceptance of the scool canteen by pupils, teachers and parents. Pupils and parents upgrade the school canteen judgment (scale 1-5) from 2,8 to 2,4 and teachers upgrade from 2,5 to 2,2. The cooperation doesn´t lead to an improvement of the nutrition composition of the canteen food. The pupils declare that they eat in the city more rarely, however rather bring food from home or eat at home. These changes in lunch habbits, though, do not have any impact on the total nutrition intake of the pupils from the sixth and seven grades (N first evaluation=436, N second evaluation=413). Therefore, neither the antropometroc parameters nor the body composition of the pupils are influenced. The methods of the Vernetzungsstelle Schulverpflegung Baden-Württemberg can be used successfully to raise the acceptance of the school canteen. Reasons, why there have been no changes in the nutrition intake of the pupils, can be discussed versatile. One explanation is that the intervention phase is too short in order to take measurable influence on the nutrition intake. As a result of the cooperation one can say that the school canteen initially needs to be integrated in every day school life, in order to improve the lunch catering. Moreover, the schools should make sure that one person takes responsibility for the catering. In addition to that, all involved parties have to work together on the optimization. Not until these points are settled, optimizing strategies, such as quality improvement of the canteen food, can´t be possible and successful. Finally, the food offer needs to be acceptable for the pupils, in order to be advertising in a good way.
Role of plasminogen activator inhibitor (PAI-1) in the pathogenesis of fructose-induced non-alcoholic fatty liver disease (NAFLD)
(2012) Kanuri, Giridhar; Bischoff, Stephan C.
Non-alcoholic fatty liver disease (NAFLD), a liver disease frequently associated with obesity, type 2 diabetes and dyslipidemia has become a worldwide health problem during the last decades. Results of recent studies suggest that a diet rich in fructose may also be a risk factor for the development of NAFLD. Results of our own group but also other group suggest that TNFα and PAI-1 may be involved in the development of NAFLD in rodents but also humans. Therefore, the aim of the present study was to investigate the role TNFα and PAI-1 in the onset of fructose-induced NAFLD in a mouse model as well as in human NAFLD patients. The specific aims were 1) Are TNFR1-/- mice protected from fructose-induced NAFLD? If yes, what are the molecular mechanisms involved? TNFR1 -/- and wild-type mice were either fed 30% fructose solution or tap water. Chronic fructose feeding caused a significant ~5-fold increase in triglyceride accumulation and neutrophil infiltration in livers of wild-type mice and an ~8-fold increase in plasma alanine aminotransferase (ALT) levels in comparison to control mice. Similar effects of fructose feeding were not found in TNFR 1-/- mice. Indeed, the protective effect of the tumor necrosis factor receptor 1 (TNFR1) deletion against the onset of fructose-induced steatosis was associated with decreased sterol regulatory element-binding protein 1 (SREBP-1), fatty acid synthase (FAS) and plasminogen activator inhibitor 1 (PAI-1) expression in the liver. Furthermore, the protective effect was also associated with protection against alterations markers of insulin signaling cascade (e.g. adenosine monophosphate-activated protein kinase (AMPK), protein kinase B (Akt) levels). However, markers of hepatic lipid peroxidation, inducible nitric oxide synthase (iNOS) protein and adenosine triphosphate (ATP) levels were similar between wild-type and TNFR1 -/- mice fed fructose. Taken together, these data suggest that TNFα plays a casual role in the onset of fructose-induced liver damage as well as insulin resistance in mice through signaling cascades downstream of TNFR1. 2) Are PAI-1-/- mice protected from fructose-induced NAFLD? And if so, what are the molecular mechanisms involved? To address if PAI-1 is also a critical factor in the onset of fructose-induced NAFLD, PAI-1-/- and wild-type mice were either fed a fructose solution or tap water. Chronic fructose feeding in wild-type mice caused a marked increase in hepatic triglycerides, PAI-1 expression and plasma ALT levels in comparison to water controls. A similar effect of fructose feeding was not found in PAI-1-/- mice. PAI-1-/- mice fed fructose were protected from hepatic steatosis despite similar portal endotoxin levels, alterations of markers of insulin resistance and hepatic TNFα protein levels between fructose fed groups. The protective effect of the loss of PAI-1 against the onset of fructose-induced steatosis was associated with a significant increase in phospho-cMet, phospho Akt, expression of apolipoprotein B (ApoB) and activity of microsomal triglyceride transfer protein (MTTP) in livers of PAI-1-/- mice in comparison to fructose fed wild-type mice. Moreover, in PAI-1-/- mice expression of CD1d and markers of CD1d-reactive iNKT cells were markedly higher than in wild-type mice; however, expression of markers of activation of CD1d-reactive iNKT cells (e.g. interleukin 15 (IL-15) and interferon γ (INFγ)) were only found to be increased in livers of fructose fed PAI-1-/- mice. Taken together, these data suggest that PAI-1 plays a causal role in mediating the early phase of fructose-induced liver damage in mice through signalling cascades down-stream of Kupffer cells and TNFα. 3) Are molecular mechanisms identified in mouse studies also relevant to human situation? To determine if the alterations found in livers of animals with NAFLD are also relevant in humans with NAFLD, markers of lipid peroxidation, insulin signaling and number of iNKT cells were determined in 6 controls and 11 NAFLD patients.4-hydroxynonenal (4-HNE) protein adducts levels were significantly higher in livers of NAFLD patients whereas expression of insulin receptor substrate (IRS-1) was reduced by ~80 % in comparison to controls. PAI-1 protein levels primarily found in hepatocytes was significantly higher in NAFLD patients; however, hepatic CD1d and MTTP mRNA expression did not differ between groups. Hepatic c-Met and BCL-2l mRNA expressions were significantly lower in NAFLD patients in comparison to controls and number of CD3ζ positive cells was higher. In contrast, expression of iNKT cell markers (e.g. IL-4 and IL-15) was significantly lower in livers of patients with NAFLD when compared with controls.Taken together, the present study suggests that the molecular mechanism involved in the progression of NAFLD is similar in both rodents and humans. Furthermore, TNFα and PAI-1 may be considered as therapeutic targets for NAFLD.
Einfluss der IgG-Subklassen sowie der immunmodulatorischen Aminosäuren Arginin und Glutamin auf die Aktivierung menschlicher Darmmastzellen
(2011) Lechowski, Sandra; Lorentz, Axel
Mast cells are key effector cells in allergic diseases, like food allergy. Activation of mast cells occurs mainly by crosslinking of immunoglobulin E (IgE) receptor FcεRI. The role of allergen specific immunoglobulin G (IgG) subclasses IgG1, IgG2, IgG3, and IgG4 in activation of intestinal mast cells is not yet fully clarified. On the one hand, mast cells can be activated via IgG1 and by the mechanisms of ?IgG-supercrosslinking?. On the other hand, IgG1 and IgG4 are up regulated during immunotherapy and might be able to attenuate allergic reactions. In this work, we investigated the effect of IgG subclasses on IgE dependent and IgE-independent mediator release of human intestinal mast cells. Mast cells, isolated and purified from human intestinal tissue, were cultured with their growth factor stem cell factor (SCF) in combination with interferon γ (IFN-γ) or with interleukin 4 (IL-4). IFN-γ is known to up regulate expression of the activating IgG receptor FcγRI on mast cells, derived from peripheral blood. IL-4 is known to enhance IgE triggered mediator release from human intestinal mast cells. Expression of FcγRI, FcγRII, FcγRIII and FcεRI was analysed by flow cytometry. IgG subclasses should be isolated from human sera by affinity chromatography. However, the required purity of at least 95 % for each subclass could not be achieved. Thus, intestinal mast cells were stimulated with human myeloma IgG1-4 and their specific anti-IgG1-4 antibodies in combination with IgE/anti-IgE and the release of inflammatory mediators was measured. We could show that human intestinal mast cells cultured with IFN-γ express FcγRI, while FcγRII and FcγRIII were only weakly expressed. Mast cells cultured with IL-4 do not express FcγR. IgG subclasses themselves did not activate intestinal mast cells neither in culture with IFN-γ, nor cultured with IL-4. In combination with IgE/anti-IgE, the IgG subclasses IgG1/anti IgG1, IgG2/anti IgG2 and IgG4/anti IgG4 tended to decrease the release of pre-stored β-hexosaminidase and de novo synthesized leukotriene C4 (LTC4) but the reduction was not significant. To conclude, IFN-γ induced in human intestinal mast cells the expression of FcγRI, but this did not result in stimulation of the cells by IgG. Thus, human intestinal mast cells do not respond or only to a small extend to IgG subclasses and differ from mast cells derived from peripheral blood which could be activated via IgG1. The second part of the work examines the effect of the immunomodulatory amino acids arginine and glutamine on IgE-dependent activation of human intestinal mast cells. Background was the measured anti-inflammatory effect of combined pharmacological doses of both amino acids on intestinal biopsies from patients with Crohn?s disease. Beside allergic diseases, mast cells play a role in chronic intestinal inflammation. Therefore, they could be involved in the described effect. Human intestinal mast cells were incubated over night with combined physiological doses or pharmacological doses of arginine (0.1 mmol/L or 2 mmol/l) and glutamine (0.6 mmol/l or 10 mmol/l). Mast cells were stimulated with IgE/anti-IgE and release of β-hexosaminidase and LTC4, induction of cytokines and activation of signaling molecules was analysed. In human intestinal mast cells, cultured in SCF and IL-4, combined pharmacological concentrations of arginine and glutamine led to decreased release of LTC4 about 40 ± 22 % and to reduced induction of cytokines like CCL2 (monocyte chemotactic protein 1), CCL4 (macrophage inflammatory protein 1 beta), CXCL8 (IL-8), and TNF (tumor growth factor alpha) about an average of 58 ± 35 % compared to physiological doses of arginine and glutamine. The anti-inflammatory effects of arginine and glutamine were associated with decreased activation levels of MAP kinases like ERK1/2, p38, JNK pan und MEK1/2 about 38 ± 17 % as well as serine/threonine kinase Akt about 23 ± 20 %. Therewith, arginine and glutamine reduce activation levels of signaling molecules known to be involved in IgE dependent mast cell cytokine expression. Here, the results show that arginine and glutamine exert anti-inflammatory effects on human intestinal mast cells in vitro. Further investigations are necessary to monitor these findings in vivo prior to use arginine and glutamine as immunomodulatory agents in inflammatory and allergic diseases.
Untersuchungen zur Rolle des intestinalen serotonergen Systems in der Entstehung von Adipositas und der nahrungsinduzierten nicht-alkoholbedingten Fettlebererkrankung im Mausmodell
(2011) Haub, Synia; Bischoff, Stephan C.
The worldwide prevalence of overweight and obesity has increased dramatically throughout the last decades. The causes of obesity are numerous and complex and the understanding of the mechanism(s) involved in the disease process are still limited. The same is true for the development of NAFLD, a liver disease for which a strong association with overweight and obesity has repeatedly been shown. High dietary fat intake has long been thought to be the major cause of obesity and NAFLD. However, other factors like carbohydrate intake may also contribute. Alterations at the level of the intestinal tract have also been discussed to be associated with the development of NAFLD and obesity. The enteric nervous system (ENS) might be of particular importance in this context, regulating the fundamental intestinal functions like peristalsis, secretion and permeability through its main neurotransmitter serotonin (5-HT) and different 5-HT receptors. The termination of the 5-HT action is mediated by the 5-HT selective reuptake transporter (SERT), located on epithelial cells. Based on this background the aim of the present work was to investigate the role of the intestinal serotonergic system and herein particularly of SERT and 5-HT3R in the onset of sugar-induced overweight and NAFLD as well as in the progression of NAFLD in mouse models.
Rolle von SNARE-Proteinen bei der Mediatorfreisetzung humaner Mastzellen
(2010) Frank, Simon P. C.; Lorentz, Axel
Mast cells are involved in a variety of physiological and pathophysiological processes in the body. They posses a wide range of stored and de novo synthesized inflammatory mediators. Therefore they play an important role not only in allergic diseases - as it was originally assumed. The aim of the present work is to acquire a more detailed insight into the processes of mediator release of human intestinal tissue mast cells. To enable the release of histamine, proteases, cytokines, chemokines and other substances, it is necessary that secretory vesicles fuse with the plasma membrane. In this process so-called SNARE proteins are essentially involved. They are generally divided into vesicle membrane (v) - and target membrane (t) -SNAREs. The present work could show that a large number of SNAREs are expressed in human intestinal mast cells. The t-SNAREs SNAP-23, syntaxin-2, -3, -4, -6, Vti1b, and the v SNAREs VAMP-2, -3, -5, -7, -8 could be clearly detected, while SNAP-25, Syntaxin-1b and VAMP-4 were expressed only slightly. A subsequent visualization of these proteins by coupling with fluorescence-marked antibodies showed their localization in the cell. There is a clear arrangement of the t-SNAREs SNAP-23, syntaxin-3, -4 and -6 at the plasma membrane, whereas syntaxin-2, Vti1b, VAMP-3, -7 and -8 are distributed in the cytoplasm in resting cells. However, after activation of the cells for 15 minutes, this arrangement changed for VAMP-7 and -8 which moved in the direction of the plasma membrane, as well as Vti1b, which shifted after a prolonged activation phase of 2 hours also towards the cell edge. In activated mast cells colocalization of SNAP-23, syntaxin-3 and -4, VAMP-7 and -8, as well as Vti1b could be demonstrated. Using co-immunoprecipitation complex formation of SNAP-23 with syntaxin-4, VAMP-7 and -8 was confirmed. For the elucidation of the functional significance of individual SNARE proteins, respective SNAREs have been turned off. After transfection of human intestinal mast cells by using different transfection reagents failed, transfection of mast cells with siRNA by electroporation succeeded. However, it turned out that the electrically treated cells lost their ability to be stimulated. In contrast, blocking of the SNARE proteins achieved by using inhibitory antibodies was successful with no deterioration of cells. Interestingly enough it was revealed that a variety of SNARE proteins participate in degranulation processes in mast cells. The release of the prestored beta-hexosaminidase was significantly reduced by antibodies against SNAP-23, syntaxin-3, -4 and -6, VAMP-7 and -8 and Vti1b after a stimulation time of one hour. It could be noted that VAMP-7 seemed to play no role anymore in the case of a prolonged activation time of six hours. The same SNAREs were also responsible for the release of LTC4. The liberation of the cytokines and chemokines IL-5, IL-6, IL-8, MCP-1, MIP-1-alpha and MIP-1-beta showed an inconsistent involvement of SNARE proteins. However, inhibition of SNAP-23, syntaxin-3 and Vti1b resulted in almost complete blockage of the release of all measured cytokines. Furthermore, antibodies against syntaxin-6 caused a significant reduction in the secretion of IL-5, IL-8 and MCP-1 and a trend towards a reduction of IL 6, MIP-1-alpha and MIP-1-beta. To different degrees both of the v-SNAREs VAMP-7 and -8 seemed to account individually for the release of different cytokines. Since release of the chemokine MIP-1-beta was not affected neither by the blockade of VAMP-7 nor by VAMP-8, another non-studied member of the VAMP family could be involved. Inhibition of syntaxin-4 led only for IL-8 to a marked and significant reduction in the release. In summary, it was shown that the release of different mediators in human intestinal mast cells is catalyzed by various different SNAREs and thus the existence of specific carriers and a targeted transport of mediators are very likely. One explanation for the involvement of a number of SNARE proteins in secretion processes may be that while some SNAREs are responsible for building up intracellular structures which are necessary for the compound exocytosis, some others are in charge of the docking of vesicles at the plasma membrane. A detailed understanding of the mechanisms and the involvement of specific SNARE proteins in the release of various mediators of human mast cells is the basis for new forms of treatments for allergies and asthma. New therapeutic approaches might aim through the temporary deactivation of certain SNARE proteins to achieve an intended reduction of mediator release.
Geschlechtsspezifische Unterschiede in der Entstehung von alkoholbedingten Lebererkrankungen
(2010) Wagnerberger, Sabine; Bode, Christiane
Women are assumed to have a higher susceptibility to alcohol-induced liver disease (ALD) than men. Gender-related differences in food preference were described in previous studies for several populations. As certain micronutrients are reported to take influence on the development of ALD in animal experiments, the hypothesis of the present retrospective cross-sectional study was that gender-dependent (micro-) nutrient intake in patients with ALD may cause the higher susceptibility of women to this disease. In 210 patients (male: 158, female: 52) with different stages of ALD (ALD1: mild stage of liver damage; ALD2: moderately severe changes of the liver with signs of hepatic inflammation; ALD3: severely impaired liver function) and in 336 controls (male: 208, female: 128), nutrient intake was determined by a computer-guided diet history and related to the severity of ALD in dependence on the sex of the patients. No significant differences between males and females with ALD were calculated for the intake (per kg body/day) of protein, carbohydrates, fat, and the intake (per kg body/day) of most micronutrients. In females with ALD, higher intake was found for vitamin C (ALD3), calcium (ALD2), iron (ALD1 and ALD2), and zinc (ALD1), but the consumption of none of these micronutrients seems to contribute to a higher susceptibility to ALD in females. In the present study, a higher activity of ?liver-specific? enzymes and a higher DeRitis quotient was measured in female patients with ALD despite equal or lower amounts of consumed alcohol. This may indicate a higher susceptibility to the development of ALD in women. However, the data of calculated daily macro- and micronutrient intake do not suggest any explicit influence of gender-specific nutrition in the development of ALD. In a chronic setting of alcohol intake, women and female rodents are more susceptible to alcohol-induced liver disease than men and male mice. Starting from this background, the purpose of the present study was to determine if female mice are also more susceptible to acute alcohol-induced steatosis than male mice and to investigate whether this is due to alterations in hepatic lipid export. Male and female C57/Bl6-mice received one single dose of ethanol (6 g/kg) or isocaloric maltose-dextrin solution (control) intragastrically. Hepatic triglycerides, lipid accumulation, mRNA expression of microsomal triglyceride transfer protein (MTP) and apolipoprotein (Apo) B, as well as MTP activity were measured 12, 24, and 48 h after alcohol intake. In both genders, acute alcohol ingestion markedly increased hepatic lipid and triglyceride levels; however, total lipid accumulation was ~2-fold higher and more persistent in livers of female than in male mice. Fourty-eight h after ethanol treatment hepatic triglyceride concentrations in male and female ethanol-treated mice were similar to those of controls. MTP activity was significantly increased only in male mice 12 h after ethanol ingestion; whereas expression of MTP mRNA was significantly reduced in female alcohol-treated animals compared to controls at this timepoint. Expression of ApoB was also reduced only in livers of female mice after 12 h; however, differences did not reach level of significance. The results of the present study suggest that the markedly more pronounced and more prolonged susceptibility to acute alcohol-induced liver steatosis of female mice results at least partly from a gender-specific regulation of hepatic lipid export. In our experiments, the selective estrogen recepor modulator (SERM) toremifen did not protect against alcohol-induced hepatic lipid accumulation. The liver plays an important role not only in the metabolism of ethanol but also in the immune system. Lymphatic NK cells are present at an unusually high frequency among liver-resident lymphocytes (30-50 %). By producing the pro-inflammatoric and anti-fibrotic cytokine IFN-g NK cells are involved in the development of liver diseases. Results of studies of our own working group indicate a decrease of IL-12-induced IFN-g production in NK-92 cells after treatment with ethanol for 6 h. The aim of the present study was to investigate whether male (testosterone) or female (estrogen, progesterone, FSH, LH) sex hormones influence the ethanol-induced immunosuppression in NK-92 cells. Therefore, NK-92 cells were incubated with different sex hormones for 19 h and were subsequently treated with ethanol (1-3 ?) and sex hormones for 18 h. Concentrations of IFN-g were determined by ELISA. According to previous studies ethanol treatment resulted in a significant decrease of released IFN-g in comparison to NK-92 cells that were not incubated with ethanol. However, treatment with male and female sex hormones did not affect IFN-g release in NK-92 cells. The results of the present study suggest that solely ethanol treatment but not incubation with sex hormones has an immun modulating effect on NK-92 cells.
Untersuchungen zur Bedeutung des Sulfonylharnstoffrezeptors 1 für die Modulation von Apoptose durch 17 beta-Estradiol in rekombinanten HEK (Human Embryonic Kidney) 293-Zellen und in pankreatischen beta-Zellen
(2009) Ackermann, Stefanie; Bode, Christiane
The sulfonylurea receptor (SUR) 1 forms the regulatory subunit of pancreatic ATP-sensitive potassium channels (KATP channels) which are essential for triggering insulin secretion in the beta-cell. Insulin secretion is modulated by additional KATP channel-independent pathways and by adaptive variation of beta-cell mass due to apoptosis, proliferation and/or neogenesis of beta-cells. Apoptosis of beta-cells is assumed to be involved in the pathophysiology of diabetes type 1 and 2. Previously it has been shown, that the insulinotropic sulfonylurea glibenclamide and the natural compound resveratrol can induce enhanced apoptosis and that this effect is specifically linked to the expression of SUR1. In the present work, it has been investigated whether there are substances that are more potent in inducing apoptosis than glibenclamide and resveratrol. Thereby the main focus was put on 17beta-estradiol which shows structural and functional analogies to the ?phytoestrogen? resveratrol. Like resveratrol, this naturally occurring estrogen is able to induce apoptosis in different experimental systems. Furthermore, it is known that 17beta-estradiol is able to decrease KATP channel activity in beta-cells acting as a KATP channel blocker. It is still discussed whether 17beta-estradiol directly interacts with KATP channels or whether it binds to so far unidentified ?non-classical? plasmalemmal estrogen receptors which are linked to KATP channels via an intracellular signaling cascade. In heterologous competition experiments, Ackermann et al. (2008) were able to show that 17beta-estradiol is a specific ligand of SUR like glibenclamide and resveratrol. Obviously SUR1 can act as a ?non-classical? estrogen receptor. In the present work it was investigated whether 17beta-estradiol induces apoptosis that is specifically linked to the expression of SUR1. Furthermore, the role of the SUR-isoforms SUR1 and SUR2 and of several SUR-mutants in the induction of apoptosis by 17beta-estradiol was investigated. Therefore, experiments were performed with recombinant HEK293-cells expressing the different isoforms of SUR. Cells that were transfected with empty pcDNA expression vector (pcDNA-cells) were used as control cells. By quantification of different apoptotic parameters such as cell detachment, changes in nuclear morphology as well as increased activity of caspase-3, it was shown that 17beta-estradiol induces specific apoptosis in cells expressing SUR1. In cells expressing the pancreatic isoform SUR1, treatment with 17beta-estradiol resulted in massive apoptosis while cells expressing the cardiac isoform SUR2A or the vascular isoform SUR2B as well as sham-transfected control-cells were less affected. Furthermore, 17beta-estradiol is more potent in inducing apoptosis in cells expressing SUR1 than glibenclamide or resveratrol. The pancreatic KATP channel consists of the regulatory subunit SUR1 and the pore-forming unit Kir6.2. In the present work, it has been proven that this SUR1-dependent effect of 17beta-estradiol was not significantly modified by coexpression with Kir6.2. These data show that apoptosis induced by 17beta-estradiol does not require the existence of functional pancreatic KATP-channels (formed by SUR1 and Kir6.2 subunits). These results provide evidence for an additional function of SUR1 apart from regulating electrical activity of the pancreatic KATP channels. SUR1 might be specifically involved in an adaptive change of the beta-cell mass and could contribute to the regulation of insulin secretion via influencing beta-cell mass. Additional experiments with cells from the clonal beta-cell lines HIT-T15 and RIN-m5F, endogenously expressing SUR1, showed that treatment with 17beta-estradiol can induce apoptosis in these cells. In pancreatic islet cells from mice aged 20-32 weeks, a clear induction of apoptosis after treatment with 17beta-estradiol was observed. Beta-cells of Langerhans also express SUR1 endogenously. Treatment of islet cells from wildtype mice with 17beta-estradiol resulted in intensive changes in nuclear morphology while islet cells from SUR1 knockout (SUR1KO) mice of the same age as well as untreated or solvent-treated islet cells from wildtype and SUR1KO mice did not show any marked signs of apoptosis. In contrast to islet cells from elderly mice aged 20-32 weeks (male or female), clear anti-apoptotic effects were detected in islet cells from young mice aged 5-7 weeks (male or female) after treatment with 17beta-estradiol. In untreated or solvent-treated islet cells from young mice (male or female) apoptosis was measured to a large extent, which was reduced by treatment with 17beta-estradiol. These results provide evidence that age is obviously an important factor which can influence the effect of 17beta-estradiol. The apoptotic effect of 17beta-estradiol in elderly mice as well as the anti-apoptotic effect in younger mice is specific to the expression of SUR1 as it was missing in experiments with islet cells from SUR1KO mice. During pregnancy, plasma concentrations of 17beta-estradiol in humans markedly increase. In the third trimester of pregnancy, 17beta-estradiol concentrations between approximately 50 and 100 nM can be readily achieved. At this point of time, the concentration of maternal serum 17beta-estradiol can be elevated up to more than 100 times compared to serum concentrations during the normal menstrual cycle (follicular phase: approx. 0.1-1.0 nM; luteal phase: approx. 0.5-2.0 nM). Changes in beta-cell mass mediated by 17beta-estradiol might contribute to the etiology of gestational diabetes mellitus (GDM). GDM is defined as glucose intolerance that appears or is first recognized during the last trimester of pregnancy. Estron, another endogenously occurring estrogen, also shows the ability to induce apoptosis in HEK293-cells expressing SUR1 as well as in cells from the SUR1 expressing clonal beta-cell lines HIT-T15 and RIN-m5F. However, the extent of apoptosis after treatment with estron is much lower than after treatment with 17beta-estradiol, although estron differs from 17beta-estradiol only in lacking one hydroxyl group. This hydroxyl group seems to be important for this pronounced SUR1-specific induction of apoptosis by 17beta-estradiol. In the present work, also the role of different SUR-mutants was examined. SUR1(M1289T) is a mutant, in which the amino acid methionine at position 1289 in transmembrane helix 17 (TM17) of SUR1 was exchanged by the corresponding amino acid of SUR2B (threonine). The experiments indicate that the amino acid methionine at position 1289 in TM17 obviously plays an important role in apoptosis which is induced by 17beta-estradiol and is specific for the expression of SUR1. This apoptotic effect after treatment with 17beta-estradiol is abolished by this single mutation. To investigate whether this apoptotic effect after treatment with 17beta-estradiol was linked to a correct function of the nucleotide binding folds, experiments with the mutants SUR1(R1379C) and SUR1(R1379L) were performed. Both mutations are located in the second nucleotide binding fold of SUR1 and result in an enhanced ATP-hydrolysis at the NBFs. These naturally occurring mutations were found in patients with neonatal diabetes with some of them showing a family history in adult-onset type 2 diabetes or GDM. The expression of these mutants in HEK293-cells leads to a much stronger induction of apoptosis than the expression of SUR1. The observation that apoptosis induced by 17beta-estradiol can be influenced by certain mutations of SUR could be of particular importance for the pathophysiology of diseases like cancer or diabetes. The enhanced activity of caspase-9 in cells expressing SUR1 after treatment with 17beta-estradiol suggests that the mitochondrial pathway might play a major role in this apoptotic process.
Bedeutung von Tumorantigenen bei Hauttumorpatienten
(2008) Wiedemann, Nicole; Eichmüller, Stefan
Essential prerequisites for the development of specific immunotherapies and procedures for diagnosis and prognosis of tumor diseases are the knowledge and characterization of tumor antigens. Potential antigens for immunotherapeutic strategies and diagnosis tools should be tumor-specific. Moreover, indications for immune responses in patients e.g. antibody or T cell responses should be available. In this thesis, antibody responses of patients of cutaneous T cell lymphoma (CTCL), melanoma (MM) and controls were investigated using two different methods. In addition, the tumor antigen GAGE-7b was characterized using a rabbit polyclonal antibody. In order to test antibody frequencies of various tumor antigens in parallel, a serum antibody detection array (SADA) was established. Testing 42 tumor antigens, antibodies against 14 antigens were only detectable in patients but not in controls. Serum antibody frequencies of antigens GBP-5ta, GBP-5a, HD-MM48 and HD-MM-19 were significantly higher in MM patients compared to controls. The same was true for antigens HD-MM-48 and HD-CL-02 comparing CTCL patients and controls. In summary, antibody frequencies were relatively low ranging most frequently from 0% to 10% which limits the use of the respective antigens as diagnostic targets. An additional method to measure immunoreactivities of 13 cancer testis and tumor-associated antigens in different groups named multiplex serology was used. Antibodies against LAGE-1a were detected significantly more frequently in MM patients in comparison to controls. In addition, this higher antibody frequency in MM patients was stage dependent. For antigens MAGE-A3, GAGE-7b and se57-1 a higher antibody frequency in MM patients was measured likewise. Antibody titers of MM and CTCL patients rarely changed over time, thus seroconversions were seen only sporadically. Significantly higher reactivities could be identified for MAGE-A1 and A9 in CTCL patients of different stages compared to controls. In future, analyzing patients immunized with interferon-g, tumor or dendritic cells or immunotherapy targeting specific tumor antigens would be of high interest. For the first time, a comprehensively characterized rabbit polyclonal antibody against GAGE-7b was generated and used in Western Blot and immunohistology. In Western Blot analysis, 59% of all MM cell lines tested expressed GAGE-7b, whereas no protein was detected in both CTCL cell lines and control tissues except testis. This expression pattern was confirmed by immunocytology. Immunohistological tests confirmed that 53% of MM tissues are positive for GAGE-7b protein expression. Moreover, antibodies against GAGE-7b could be identified in 6% of MM and CTCL patients whereas controls had no detectable antibody responses. On the basis of the achieved results, the GAGE-7b antibody recognizes probably all GAGE family members. Future analysis using this antibody may permit the elucidation of functional aspects of GAGE expression in tumor diseases.
Effect of low ethanol concentrations on the production and stability of Interferon gamma
(2008) Sauter, Senja; Bode, Christiane
Alcohol is known to modulate the immune system in a complex manner. The effects of alcohol on immune responses vary with acute and chronic exposure as well as depending on the history of alcohol consumption and the blood level of alcohol. The presence or absence of alcohol can affect the cytokine cascade in complex ways. In the current study the immunmodulatory capability of an acute, moderate (1 ?) to high amount (3 ?) of alcohol was tested on isolated Peripheral Blood Mononuclear Cells production of several proinflammatory and antiinflammatory cytokines after incubation for 12 to 72 hours. The most affected cytokine in our model system of isolated human PBMC treated with two different ethanol concentrations was IFN-γ. Its concentration decreased in a highly significant manner in PHA- as well as in LPS-stimulated PBMC when treated with 66 mM ethanol and in a significant manner in PHA-activated PBMC when treated with 22 mM ethanol. The fact that ethanol negatively affects IFN-γ production is supported by several in vivo and in vitro studies by Wagner et al., 1992, Chen et al., 1993, Laso et al., 1997 Waltenbaugh et al., 1998, Starkenburg et al., 2001, Szabo et al., 2001, Dokur et al., 2003. The reduced IFN-γ level observed might be a key factor in explaining comprised immunity seen after chronic alcohol abuse, since together with IL-12, IFN-γ is crucial for the innate and adaptive immune response to viral and bacterial infection (Vicente-Gutierrez et al., 1991, Windle et al., 1993, Szabo 1997, Szabo et al., 1999). As seen in isolated human Peripheral Blood Mononuclear Cells IFN-γ production by IL-12 stimulated NK-92 cells is significantly reduced in the presence of ethanol. However, this decrease did not correlate with decreased phosphorylation and nuclear translocation of STAT4, a central regulator of IFN-γ gene expression. These results indicated that acute alcohol treatment in vitro did not affect intracellular pathways leading to IFN-γ gene expression. These findings paralleled results indicating that the amount of mRNA for IFN-γ synthesis in NK-92 cells is not affected by the applied ethanol concentrations as well. Additionally it was shown within the current work, that the reduced IFN-γ production by NK-92 cells in the presence of ethanol might not be explained by an intracellular accumulation of the IFN-γ protein. The inhibitory action of ethanol on IFN-γ may rather be caused by posttranslational modification once IFN-γ is released by NK-92 cells, since the addition of recombinant human IFN-γ to the cell culture supernatants of ethanol-treated cells led to a decline in the amount of IFN-γ concentration. We therefore hypothesized that ethanol may cause the release of either an IFN-γ-binding or IFN-γ-degrading protein. An increase in soluble IFN-γ receptor as a result of ethanol treatment was not observed. But the addition of mixture of 5 commercially available protease inhibitors counteracted the effect of ethanol treatment, giving us a first hint of IFN-γ-modulatory mechanism, where IFN-γ released by NK-92 cells may be disintegrated by a protease released as consequence of ethanol incubation. To our best knowledge we are the first to demonstrate a posttranslational modification of IFN-γ as a consequence of ethanol incubation. In summary, the present results support the inhibitory role of ethanol on IFN-γ, but are too preliminary to explain the underlying immunmodulatory effect.
Wechselwirkungen zwischen humanen Darmmastzellen und humanen Darmfibroblasten
(2008) Montier, Yves; Bischoff, Stephan C.
Fibroblasts (FB) play a central role in the pathogenesis of fibrosis since they are the major source of extracellular matrix proteins. However, the regulation of extracellular matrix production in fibroblasts, the mechanisms that lead to loss of control of extracellular matrix homeostasis during chronic inflammation and the role of human intestinal mast cells are still not fully understood. Mast cells are key effector cells in allergic reactions but also involved in host defense and tissue remodeling processes such as wound healing, angiogenesis, and fibrogenesis. The group pf Prof. Bischoff has shown previously that human intestinal fibroblasts suppress apoptosis in human intestinal MC independent of the known human mast cell growth factors stem cell factor interleukin-3, interleukin-4, and nerve growth factor.In this work I could show that the effects of fibroblasts on mast cells are mediated by interleukin-6. The molecular crosstalk between human mast cells and human fibroblasts, both isolated and purified from intestinal tissue was analyzed. Mast cells survival in the presence of fibroblasts could be blocked using an anti-interleukin-6 antibody. Mast cells incubated with interleukin-6 survived for up to 3 weeks. Intestinal fibroblasts produced interleukin-6 upon direct stimulation by mast cells in co-culture or by mast cell mediators such as tumor necrosis factor alpha, interleukin-1 beta, tryptase or histamine. Moreover, fibroblasts stimulated by mast cell mediators produce the antifibrotic enzyme matrix metalloproteinase-1. Matrix metalloproteinase-1 should be considered as multifunctional molecule since it participates not only in the turnover of collagen fibrils in the extracellular space but also in the cleavage of a number of non-matrix substrates and cell surface molecules suggesting a role in the regulation of cellular behaviour. Noteworthy, fibroblasts co-cultured with mast cells or treated with matrix metalloproteinase-1 lost confluence. Matrix metalloproteinase-1 expression in fibroblasts triggered by mast cells was dependent on the MEK/ERK cascade as shown by inhibitor experiments. In conclusion, this study show that mast cells mediators stimulate fibroblasts to produce interleukin-6, and, vice versa, fibroblasts derived interleukin-6 supports mast cells survival. Furthermore, mast cell mediators induce expression of matrix metalloproteinase-1 in fibroblasts, a key enzyme in fibrolysis, which in turn leads to lost of confluence of cultured fibroblasts. Taken together the results of my work suggest that mast cells accumulating at sites of fibrosis rather limite than promote fibrogenesis.

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