Browsing by Person "Bode, Christiane"

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    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.
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    Einfluss der Ernährung und von Genussmitteln auf Risikofaktoren für das Auftreten von ischämischem Herzinfarkt und Schlaganfall
    (2005) Eckoldt, Joachim; Bode, Christiane
    Background and aims of the study: Arteriosclerotic changes of blood vessels which contribute to coronary heart disease (CHD) and ischemic stroke are influenced by risk factors like cigarette smoking, overweight, hypertension, diabetes mellitus, missing physical exercise and nutritional factors, such as alcohol consumption. Beyond this, the concentrations of serum lipids, antioxidants, coagulation factors or other risk factors, such as C-reactive protein, and homocysteine are considered to be additional factors that indicate an enhanced or lowered risk of atherosclerosis. In this study we examined the effect of nutritional factors, in particular alcohol consumption, on various plasma components that are believed to play a role in the pathogenesis of atherosclerosis. Multiple epidemiologic studies suggest that moderate alcohol consumption reduces the mortality from cardiovascular diseases and that this effect is chiefly mediated by elevation of high-density lipoprotein (HDL). This cross-sectional study assessed the effect of moderate alcohol consumption and other life-style factors on the composition of HDL in healthy working males. An additional goal of the present study was to find out whether there is an association between alcohol consumption and the concentration of vitamins and cardio-protective substances. Methods: We included a collective of healthy men (n = 284, age 23-66 years), investigated with respect to cardiovascular risk factors. The average daily alcohol consumption, nutrient intake, smoking and other life-style factors was assessed by a computer based questionnaire. Group 1 (n = 62) comprised subjects with an average daily alcohol consumption of 0-5g, group 2 (n = 175): 5-30g, and group 3 (n = 47): 30-70g. In addition, the study design made it possible to subdivide groups 2 and 3 in a so called ?beer drinker group 2+3? (> 80 % beer), a ?wine drinker group 2+3? (> 80 % wine) and persons without preference of a certain alcoholic beverage. Results: The alcohol groups showed no significant differences in the nutritional profile (nutrients, energy intake, and metabolic rate). The markers for regular, higher alcohol consumption (g-GT and MCV) were positive correlated to the amount of alcohol consumption. There was no correlation between the great number of clinical laboratory parameters and the amount or kind of alcohol consumption. Thereby the groups are comparable in view of these laboratory parameters. Besides, there were no indications for the existence of diseases, which might influence blood lipid and vitamin concentrations. Antioxidative substances in the blood: Dietary assessment: The intake of vitamin B2, B6, B12, folic acid, retinol, ß-carotene and other carotenoids, as assessed by the computer interview was comparable in groups 1-3. The subjects in group 1 had a higher supply of the vitamins C, B1 and a-tocopherol, ?beer drinker Gr. 2+3? had a higher intake of vitamin B2, B6 and folic acid. Blood measurements: Antioxidative vitamins: The vitamin B2 status in erythrocytes (EGRAC) was lower in group 3 (vs. group 1 and 2). The plasma level of ß-carotene and ß-cryptoxanthin was lower in group 3 than in group 1. Vitamins that influence homocysteine metabolism (including homocysteine): Influence of beer and wine: The status of vitamin B6 and the concentration of free plasma pydridoxal phosphate in group 3 was significantly higher than in group 1. These results cannot explain the postulated positive influence of moderate or higher alcohol consumption through improvements of the vitamin status and the concentration of vitamins in the blood. The vitamins in beer improved the vitamin status only in case of vitamin B6, no effect was calculated in case of vitamin B2 and folic acid. Higher alcohol consumption (group 3) made the vitamin status respectively the plasma concentration of vitamin B2, ß-carotene and ß-cryptoxanthin lower compared with group 1 ? in spite of comparable supply. Coagulation factors, markers of inflammation: The coagulation factors prothrombin time and fibrinogen as well as the ?newer? risk factors C-reactive protein and homocysteine were not correlated to the amount or the kind of alcohol. Lipoproteins: The serum concentration of total cholesterol, cholesterol ester, phospholipids, apolipoprotein A-1 and A-2 was higher in group 3. Moderate and higher alcohol consumption raises the concentration of cholesterol in the high-density lipoproteins (HDL) (including subfractions) ? independent of the sort of alcoholic beverage. The concentration of cholesterol in the low- (LDL) (including subfractions), very-low (VLDL) and intermediate-density lipoproteins (IDL) in the blood was not influenced by alcohol consumption. Composition of HDL: The induced increase of HDL cholesterol was lower in the subfraction HDL3 as in the subfractions HDL2b und HDL2a. Besides we found qualitative changes of the HDL-components: the phospholipid component increased more than the other HDL-components. This phenomenon might play a beneficial role in the mechanism of atherosclerosis. Conclusions: Vitamins: The changes of antioxidative vitamins and vitamins influencing homocysteine metabolism observed in persons with moderate and increased alcohol consumption do not explain the antiatherogenic effect of alcohol. On the other hand, our study confirmed a positive association of moderate alcohol consumption with HDL plasma levels ? independent of other nutritional factors. In addition, alcohol might induce qualitative alterations of HDL composition (more pronounced increased of HDL2 relative increase of the phospholipid component). The pathophysiological significance of this phenomenon remains unclear.
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    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.
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    Probiotic bacteria enhance the antibacterial barrier of enterocytes: insights into their mechanism of action
    (2007) Schlee, Miriam; Bode, Christiane
    In the healthy intestine there is a stable balance of luminal bacteria and host factors to prevent infections or inflammatory bowel diseases (IBD). A complex network of environmental, genetic, and immunoregulatory factors may precipitate the onset of ulcerative colitis (UC) and Crohn's disease (CD), the primary manifestations of inflammatory bowel disease (IBD). It is currently believed that IBD results from an aberrant immune response of the intestinal mucosa towards the normal commensal bacterial flora. Alternatively, a primary defect in the mucosal barrier might permit bacterial invasion and trigger inflammation. In our research group the hypothesis was proposed that the defective barrier in Crohn´s disease may be due to a lack of defensins which form a chemical barrier against luminal bacteria. A major gut defensin is the human beta defensin-2 (hBD-2) which is an inducible antimicrobial peptide synthesized and secreted by the epithelium to counteract bacterial adherence and invasion. Proinflammatory cytokines, as well as certain bacterial strains, have been identified as potent endogenous inducers. In recent studies, Fellermann et al demonstrated that the defective expression of hBD-2 which was measured in the gut mucosa of patients with Crohn´s disease was due to a reduced copy number of the hBD-2 gene. In patients with ulcerative colitis beta-defensin expression is low in the colon during remission, but readily inducible during inflammation. Probiotic bacteria might act beneficially in the human gut by inducing the expression of defensins and thereby reinforcing the mucosal barrier. Recently, our group has been the first to describe hBD-2 induction by the probiotic strain E. coli Nissle (Mutaflor®) which is an effective treatment for ulcerative colitis during remission. The aim of the present work was to determine the underlying molecular mechanisms. We determined a time- and dose-dependent expression pattern of hBD-2 in Caco-2 cells upon stimulation with IL-1 beta;, E. coli Nissle culture supernatant and diverse other probiotic strains. We further investigated the transcriptional regulation of hBD-2 expression mediated by probiotics. The hBD-2 promoter contains several elements known to be involved in transcriptional upregulation such as the NF-kappa B element, which is believed to be one of the main regulators of the hBD-2 gene expression. However, for certain signals, the expression of the hBD-2 gene has been reported to depend on the activation of a second transcription factor, such as AP-1. Most importantly, E. coli Nissle was shown to shed or secrete factors, contained in the bacterial supernatant, which were sufficient to trigger activation of NF-kappa B and AP-1 and to induce hBD-2. Our results indicated further that the supernatant-induced activation of the MAP kinase pathways ERK½, JNK, and p38 may be directly responsible for the probiotic supernatant-induced activation of the transcription factors AP-1 and NF-kappa B and subsequent synthesis of hBD-2. A further aim of the present study was to identify and isolate the bacterial components which are responsible for E. coli Nissle mediated hBD-2 induction. As E. coli Nissle culture supernatant was found to be a more potent stimulant than the bacterial pellet, we investigated the characteristics of the unknown soluble or shed molecules in the bacterial culture media. The first analysis revealed the factor as a heat resistant and proteinase sensitive molecule. Both, E. coli Nissle specific lipopolysaccharide (LPS) and bacterial DNA, which might contain immunostimulatory DNA motifs, failed to trigger hBD-2 expression. Based on the knowledge of the surface composition several E. coli Nissle deletion mutants were constructed and tested for their ability to induce hBD-2 expression in Caco-2 cells. Deletion mutants for flagellin, the flagella filament protein, specifically exhibited an impaired immunostimulatory capacity. Reinsertion of the flagellin gene restored the induction capacity to normal levels. Next, we isolated flagellins from different bacteria strains (Salmonella enterica serovar Enteritidis, E. coli ATCC 25922, E. coli Nissle and the uropathogenic E. coli strain CFT073 Delta hly, whose genome structure resembles closely that of E. coli Nissle). In the Western blot anti-H1 flagellin displayed immunoreactivity against the different types of flagellins, due to the highly conserved central region of the flagellin filament structure. Incubation of Caco-2 cells with isolated E. coli Nissle flagellin (molecular size 60.81 kDa) induced hBD-2 promoter activation in a dose-dependent manner. The induction of hBD-2 expression by flagellin was confirmed with a positive control (Salmonella flagellin). Interestingly, the serotype-identical CFT073 Delta hly flagellin expressed only moderate hBD-2 inducing ability compared to E. coli Nissle flagellin. Thus, differences in extracellular matrix e.g. the glycosilation degree might underlie the differentially modulated hBD-2 response of Caco-2 cells by the two flagellins. H1 flagellin antiserum abrogated hBD-2 expression induced by flagellin as well as E. coli Nissle supernatant, confirming that flagellin is the major stimulatory factor of E. coli Nissle. In conclusion, flagellin of E. coli Nissle provides reinforcement of mucosal antimicrobial function, apparently without inducing inflammation. This might explain the beneficial effects of E. coli Nissle on remission maintenance in ulcerative colitis. In patients with Crohn´s disease there is evidence against a therapeutic effect of probiotics and this may be explained by a defective defensin system. Future investigations about strain-specific beneficial functions might contribute to the therapeutic application of science-based probiotic products.
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    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.