Juergen Schrezenmeir

Guidance for Substantiating the Evidence for Beneficial Effects of Probiotics: Prevention and Management of Infections by Probiotics

The rationale for the use of probiotics in the management of infectious diseases is supported by their potential to influence and stabilize the composition of gut microbiota, enhance colonization resistance, and modulate immune function parameters. A literature review was conducted to determine the efficacy of using probiotics in selected infections: 1) infectious diarrhea in infants and children, 2) travelers diarrhea, 3) necrotizing enterocolitis in infants, 4) Helicobacter pylori infection, 5) respiratory tract infections in adults and children, 6) ear, nose, and throat infections, and 7) infectious complications in surgical and critically ill patients. The different types of infections that have been subject to clinical studies with different probiotics obviously prevent any generic conclusions. Furthermore, the lack of consistency among studies focusing on 1 specific infection, in study design, applied probiotic strains, outcome parameters, and study population, along with the still limited number of studies, preclude clear and definite conclusions on the efficacy of probiotics and illustrate the need for better-aligned study designs and methodology. Exceptions were the management of infectious diarrhea in infants and travelers diarrhea, antibiotic-associated diarrhea, and necrotizing enterocolitis. Sufficient consistent data exist for these applications to conclude that certain probiotics, under certain conditions, and in certain target populations, are beneficial in reducing the risk of infection. In addition, some evidence exists, although conclusions are premature, for the management of Helicobacter pylori infection and possible reduction of treatment side effects. Certain probiotics may also reduce the risk of various symptoms of respiratory tract infections in adults and children, including ear, nose, and throat infections, although data are currently far too limited to distill any clinical recommendations in this area. Positive but also negative results have been obtained in prevention of infectious complications in surgical and critically ill patients. For future studies it is recommended that researchers provide adequate power, identify pathogens, and report both clinical outcomes and immune biomarkers relating to putative underlying mechanisms.

Effects of probiotic bacteria and their genomic DNA on TH1/TH2-cytokine production by peripheral blood mononuclear cells (PBMCs) of healthy and allergic subjects

Among the factors potentially involved in the increased prevalence of allergic diseases, modification of the intestinal flora or lack of microbial exposure during childhood has been proposed. T(H)2-cytokines increase the production of IgE and stimulate mast cells and eosinophils, whereas T(H)1-cytokines, such as IFN-gamma, may suppress IgE synthesis and stimulate the expression of the secretory piece of IgA. Thus, a dysregulation in the expression of T(H)1- and T(H)2-cytokines may contribute to the initiation and maintenance of allergic diseases. Lactobacilli belonging to the natural intestinal microflora were reported to reduce the incidence of atopic dermatitis and the severity of allergic manifestations and to modulate T(H)1/T(H)2 responses. The mechanisms still remain to be elucidated. We sought to assess the effect of different probiotics, Lactobacillus rhamnosus GG, Lactobacillus gasseri (PA16/8), Bifidobacterium bifidum (MP20/5), and Bifidobacterium longum (SP07/3), on the T(H)1 and T(H)2 responses of peripheral blood mononuclear cells (PBMCs) from healthy subjects and from patients with allergy against house dust mite to Staphylococcus enterotoxin A (SEA) and Dermatophagoides pteronyssinus (Dpt). To elucidate the molecular basis of these effects, the effects of bacterial genomic DNA were compared with the effects of viable bacteria. PBMCs from allergic patients and from healthy donors were incubated for 24 or 48 h, respectively, with or without SEA and Dpt allergens. The effects of preincubation with live probiotic bacteria and the effect of their genomic DNA, added simultaneously to cultures and incubated for 24h, were assessed by measuring T(H)1/T(H)2-cytokine production. The tested live Gram-positive probiotic bacteria and their genomic DNA inhibited SEA- and Dpt-stimulated secretion of T(H)2-cytokines (IL-4 and IL-5) and enhanced the stimulation of IFN-gamma. This effect was dose-dependent with a dosage-optimum, which was identical for all lactic acid producing bacteria (LAB) tested (10 bacteria per PBMC) and their DNA (75 ng/ml). Based on the maximal effects achieved with LAB and their DNA, more than 50% of the effects seem to be contributed by DNA. No significant effect was induced by the control, Gram-negative Escherichia coli TG1. Lactobacilli and bifidobacteria reduced SEA-stimulated IL-4 and IL-5 production more effectively in PBMCs from healthy subjects than from allergic patients. In contrast to this, inhibition of Dpt-stimulated IL-4- and IL-5-secretion was more pronounced in cells from allergic subjects. Compared with living LAB, bacterial DNA inhibited IL-4- and IL-5-secretion in a similar manner. SEA- and even more so Dpt-stimulated IFN-gamma stimulation by living LAB was less pronounced in allergic than in healthy subjects, whereas IFN-gamma stimulation by their DNA was more pronounced in allergic subjects. The tested probiotic bacteria as well as their genomic DNA modulated the T(H)1/T(H)2 response to some allergens dose-dependently. DNA seems to contribute to 50% of the effect exerted by living bacteria in this in vitro model. The magnitude of the probiotic effects differed between healthy and allergic subjects. Whether the modulation found for the tested strains might be useful for the prevention and treatment of allergic diseases has to be assessed in clinical trials.

A Genome-wide Association Study Identifies LIPA as a Susceptibility Gene for Coronary Artery Disease

Background— eQTL analyses are important to improve the understanding of genetic association results. We performed a genome-wide association and global gene expression study to identify functionally relevant variants affecting the risk of coronary artery disease (CAD). Methods and Results— In a genome-wide association analysis of 2078 CAD cases and 2953 control subjects, we identified 950 single-nucleotide polymorphisms (SNPs) that were associated with CAD at P<10−3. Subsequent in silico and wet-laboratory replication stages and a final meta-analysis of 21 428 CAD cases and 38 361 control subjects revealed a novel association signal at chromosome 10q23.31 within the LIPA (lysosomal acid lipase A) gene (P=3.7×10−8; odds ratio, 1.1; 95% confidence interval, 1.07 to 1.14). The association of this locus with global gene expression was assessed by genome-wide expression analyses in the monocyte transcriptome of 1494 individuals. The results showed a strong association of this locus with expression of the LIPA transcript (P=1.3×10−96). An assessment of LIPA SNPs and transcript with cardiovascular phenotypes revealed an association of LIPA transcript levels with impaired endothelial function (P=4.4×10−3). Conclusions— The use of data on genetic variants and the addition of data on global monocytic gene expression led to the identification of the novel functional CAD susceptibility locus LIPA, located on chromosome 10q23.31. The respective eSNPs associated with CAD strongly affect LIPA gene expression level, which was related to endothelial dysfunction, a precursor of CAD.

Effect of natural commensal‐origin DNA on toll‐like receptor 9 (TLR9) signaling cascade, chemokine IL‐8 expression, and barrier integritiy of polarized intestinal epithelial cells

Background and Aim: The intestinal epithelium is constantly exposed to high levels of genetic material like bacterial DNA. Under normal physiological conditions, the intestinal epithelial monolayer as a formidable dynamic barrier with a high‐polarity structure facilitates only a controlled and selective flux on components between the lumen and the underlining mucosa and even is able to facilitate structure‐based macromolecules movement. The aim of this study was to test the effect of natural commensal‐origin DNA on the TLR9 signaling cascade and the barrier integrity of polarized intestinal epithelial cells (IECs). Methods: Polarized HT‐29 and T84 cells were treated with TNF‐&agr; in the presence or absence of DNA from Lactobacillus rhamnosus GG (LGG) and Bifidobacterium longum. TLR9 and interleukin‐8 (IL‐8) mRNA expression was assessed by semiquantitative and TaqMan real‐time reverse‐transcription polymerase chain reaction. Expression of TLR9 protein, degradation of inhibitor of kappa B alpha (I&kgr;B&agr;), and p38 mitogen‐activated protein kinase (p38 MAP) phosphorylation were assessed by Western blotting. To further reveal the role of TLR9 signaling, the TLR9 gene was silenced by siRNA. IL‐8 secretion was measured by an enzyme‐linked immunosorbent assay. Nuclear factor‐kappa B (NF‐&kgr;B) activity was assessed by the electrophoretic mobility shift assay (EMSA) and NF‐&kgr;B‐dependent luciferase reporter gene assays. As an indicator of tight junction formation and monolayer integrity of epithelial cell monolayers, transepithelial electrical resistance (TER) was repetitively monitored. Transmonolayer movement of natural commensal‐origin DNA across monolayers was monitored using qRT‐PCR and nested PCR based on bacterial 16S rRNA genes. Results: In response to apically applied natural commensal‐origin DNA, polarized HT‐29 and T84 cells enhanced expression of TLR9 in a specific manner, which was subsequently associated with attenuation of TNF‐&agr;‐induced NF‐&kgr;B activation and NF‐&kgr;B‐mediated IL‐8 expression. TLR9 silencing abolished this inhibitory effect. Apically applied LGG DNA attenuated TNF‐&agr;‐enhanced NF‐&kgr;B activity by reducing I&kgr;B&agr; degradation and p38 phosphorylation. LGG DNA did not decrease the TER but rather diminished the TNF‐&agr;‐induced TER reduction. Translocation of natural commensal‐origin DNA into basolateral compartments did not occur under tested conditions. Conclusions: Our study indicates that TLR9 signaling mediates, at least in part, the anti‐inflammatory effects of natural commensal‐origin DNA on the gut because TLR9 silencing abolished the inhibitory effect of natural commensal‐origin DNA on TNF‐&agr;‐induced IL‐8 secretion in polarized IECs. The nature of the TLR9 agonist, the polarity of cells, and the tight junction integrity of IECs has to be taken into account in order to predict the outcome of TLR9 signaling. (Inflamm Bowel Dis 2010)

Lactic acid bacteria enhance autophagic ability of mononuclear phagocytes by increasing Th1 autophagy-promoting cytokine (IFN-γ) and nitric oxide (NO) levels and reducing Th2 autophagy-restraining cytokines (IL-4 and IL-13) in response to Mycobacterium tuberculosis antigen

BACKGROUND AND OBJECTIVES Control of the intracellular Mycobacterium tuberculosis (Mtb), mainly requires an appropriate ratio of Th1/Th2 cytokines to induce autophagy, a physiologically, and immunologically regulated process that has recently been highlighted as an innate defense mechanism against intracellular pathogens. Current vaccines/adjuvants induce both protective Th1 autophagy-promoting cytokines, such as IFN-gamma, and immunosuppressive Th2 autophagy-restraining cytokines, such as IL-4 and IL-13. TB infection itself is also characterized by relatively high levels of Th2 cytokines, which down-regulate Th1 responses and subsequently subvert adequate protective immunity, and a low ratio of IFN-gamma/IL-4. Therefore, there is a need for a safe and non-toxic vaccine/adjuvant that will induce Th1 autophagy-promoting cytokine (IFN-gamma) secretion and suppress the pre-existing subversive Th2 autophagy-restraining cytokines (IL-4 and IL-13). As lactic acid bacteria (LAB) belonging to the natural intestinal microflora and their components have been shown to shift immune responses against other antigens from Th2-type cytokines toward Th1-type cytokines like IFN-gamma, we investigated whether LAB can improve the polarization of Th1/Th2 cytokines and autophagic ability of mononuclear phagocytes in response to Mtb antigen. METHODS Peripheral blood mononuclear cells (PBMCs), which are a part of the mononuclear phagocyte system and source of crucial macrophage activators in the in vivo situation, and human monocyte-derived macrophages (HMDMs) were treated with Mtb antigen in the presence or absence of two strains of LAB, L. rhammosus GG (LGG) and Bifidobacterium bifidum MF 20/5 (B.b). PBMCs cell culture supernatants were analyzed for the production of the autophagy-promoting factors IFN-gamma, and nitric oxide (NO) and the autophagy-restraining cytokines IL-4 and IL-13, using ELISA and Griess assays to detect the production of cytokines and NO, respectively. In HMDMs, expression of microtubule-associated protein 1 light chain 3 (LC3-I), membrane-associated (LC3-II) forms of LC3 protein and Beclin-1, as hallmarks of autophagy, were assessed using Western blot to detect the autophagy markers. The secreted interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin (IL)-12 and transformig growth factor-beta (TGF-beta), and chemokine (C-C motif) ligand 18 (CCL18) from HMDMs were determined by ELISA. Also, reverse transcription polymerase chain reaction (RT-PCR) analysis was used to assess the mRNA expressions of CCL18 in HMDMs. RESULTS Treatment of PBMCs with either Mtb antigen or with LAB significantly increased the IFN-gamma and NO production. Combination of Mtb antigen and LAB led to synergistic increase in IFN-gamma, and an additive increase in NO. Treatment with Mtb antigen alone significantly increased the IL-4 and IL-13 production. LAB significantly decreased IL-4 and IL-13 secretion in both unstimulated and Mtb antigen-stimulated PBMCs. The IFN-gamma/IL-4+IL-13 ratio was enhanced, indicating Th1/Th2 polarization. Treatment of macrophages with combined use of Mtb antigen and LAB led to an additive increase in Beclin-1, LC3-II expression, as well as in synergistic increase in IL-12 production. Treatment of macrophages with combined use of Mtb antigen and LAB led to a decrease in IL-6, IL-10, and CCL18 secretion. LAB inhibited the secretion of TGF-beta by Mtb-stimulated macrophages, however not significantly. Treatment of macrophages with combined use of Mtb antigen and LAB led to a decrease in CCL18 mRNA expression. CONCLUSION Our study implies that LAB may reinforce the response of the mononuclear phagocytes to Mtb antigen by inducing production of the autophagy-promoting factors IFN-gamma and NO, while decreasing the Th2 autophagy-restraining cytokines IL-4 and IL-13. Hence, combination of Mtb antigen and LAB may perhaps be safer in more efficacious TB vaccine formulation.

The phenomenon of a high triglyceride response to an oral lipid load in healthy subjects and its link to the metabolic syndrome

Excessive postprandial triglyceride (TG) responses despite normal fasting TG levels have been described in single cases within small groups of healthy subjects and in patients with obesity or precocious atherosclerosis, known to be associated with high insulin fasting levels. To clarify this association, fasting and postprandial TG and insulin levels were studied in 113 healthy young (25.7 +/- 2.6 years), normal weight (body mass index 20.8 +/- 2.3 kg/m2) male subjects who were selected from among 117 subjects on the basis of TG fasting levels < 200 mg/dl. After a 12-hour fast a standardized liquid lipid load was administered containing 58 g mainly saturated fat and 1,017 kcal energy. Both fasting TG values and postprandial TG peak values showed bimodal frequency distributions. Statistical analysis of fasting TG discriminated two groups: a low fasting TG group with normally distributed values < 150 mg/dl (mean +/- SEM: 79.5 +/- 2.7 mg/dl; n = 104) and a high fasting TG group > 150 mg/dl (194.5 +/- 7.2 mg/dl; n = 13). Likewise, two groups could be differentiated according to their maximal postprandial TG response (TG max) to the lipid load: (1) normal responders with TG max < 260 mg/dl (mean +/- SEM: 123 +/- 4.8 mg/dl; n = 96) and (2) high responders with TG max > 260 mg/dl (272.5 +/- 20.5 mg/dl; n = 17). Fasting TG and TG max were highly correlated (r = 0.745; p < 0.0001). However, 9 of 17 (53%) high responders had fasting TG < 150 mg/dl, which means that the prediction of high response is only 47.0% based on fasting TG values. Fasting insulin levels were significantly higher in high responders than in normal responders, whereas they did not differ between the low and high fasting TG group. In conclusion, the bimodal frequency distribution of TG max after a lipid load permitted the differentiation of two groups, normal responders and high responders, with higher fasting insulin levels, which might indicate a link to the metabolic syndrome.

Epigenetic imprinting by commensal probiotics inhibits the IL-23/IL-17 axis in an in vitro model of the intestinal mucosal immune system

The pathophysiology of IBD is characterized by a complex interaction between genes and the environment. Genetic and environmental differences are attributed to the heterogeneity of the disease pathway and to the epigenetic modifications that lead to altered gene expression in the diseased tissues. The epigenetic machinery consists of short interfering RNA, histone modifications, and DNA methylation. We evaluated the effects of Bifidobacterium breve (DSMZ 20213) and LGG (ATCC 53103), as representatives of commensal probiotics on the expression of IL‐17 and IL‐23, which play an important role in IBD, and on the epigenetic machinery in a 3D coculture model composed of human intestinal HT‐29/B6 or T84 cells and PBMCs. The cells were treated with LPS in the presence or absence of bacteria for 48 h, and the expression of IL‐17, IL‐23, and CD40 at the mRNA and protein levels was assessed using TaqMan qRT‐PCR and ELISA, respectively. Western blotting was used to assess the expression of the MyD88, the degradation of IRAK‐1 and IκBα, the expression of the NF‐κB p50/p65 subunits, the p‐p38 MAPK and p‐MEK1, as well as histone modifications. NF‐κB activity was assessed by NF‐κB‐dependent luciferase reporter gene assays. The accumulation of Ac‐H4 and DNA methylation was quantitatively assessed using colorimetric assays. B. breve and LGG diminished the LPS‐induced expression of IL‐17, IL‐23, CD40, and histone acetylation, while slightly enhancing DNA methylation. These effects were paralleled by a decrease in the nuclear translocation of NF‐κB, as demonstrated by a decrease in the expression of MyD88, degradation of IRAK‐1 and IκBα expression of the nuclear NF‐κB p50/p65 subunits, p‐p38 MAPK and p‐MEK1, and NF‐κB‐dependent luciferase reporter gene activity in LPS‐stimulated cells. B. breve and LGG may exert their anti‐inflammatory effects in the gut by down‐regulating the expression of the IBD‐causing factors (IL‐23/IL‐17/CD40) associated with epigenetic processes involving the inhibition of histone acetylation and the optimal enhancement of DNA methylation, reflected in the limited access of NF‐κB to gene promoters and reduced NF‐κB‐mediated transcriptional activation. We describe a new regulatory mechanism in which commensal probiotics inhibit the NF‐κB‐mediated transcriptional activation of IBD‐causing factors (IL‐23/IL‐17/CD40), thereby simultaneously reducing histone acetylation and enhancing DNA methylation.

Postprandial Pattern of Triglyceride Rich Lipoprotein in Normal-Weight Humans after an Oral Lipid Load: Exaggerated Triglycerides and Altered Insulin Response in Some Subjects

In 13 healthy, male nonsmokers (mean age: 25.7 +/- 2.4 years) with normal fasting triglycerides we investigated postprandial changes of triglycerides in several lipoprotein fractions. After a 12-hour overnight fast they ingested a standardized lipid load (1,017 kcal) including 30,000 IU retinyl palmitate. Postprandially, total triglycerides increased significantly (p < 0.001) to a peak value of 221 +/- 81 mg/dl at 5 h. Two subjects had an exceptionally strong triglyceride response (peak values: 363 and 390 mg/dl). They had the highest levels of retinyl palmitate in the chylomicron and the nonchylomicron fraction, and one of them showed elevated intermediate-density lipoprotein values throughout the test period. In addition, they showed an altered early and an increased late postprandial insulin response. Thus, our data provide evidence that an exaggerated postprandial triglyceride response may point to an increased atherogenic risk even in healthy subjects with normal fasting triglycerides.

s-ICAM-1 and s-VCAM-1 in healthy men are strongly associated with traits of the metabolic syndrome, becoming evident in the postprandial response to a lipid-rich meal

BackgroundThe importance of the postprandial state for the early stages of atherogenesis is increasingly acknowledged. We conducted assessment of association between postprandial triglycerides, insulin and glucose after ingestion of a standardized lipid-rich test meal, and soluble cellular adhesion molecules (sCAM) in young healthy subjects.MethodsMetabolic parameters and sICAM-1, sVCAM-1 and E-selectin were measured before and hourly until 6 hours after ingestion of a lipid-rich meal in 30 healthy young men with fasting triglycerides <150 mg/dl and normal fasting glucose levels. Subjects were classified as either normal responders (NR) (postprandial triglyceride maxima < 260 mg/dl) or high responders (HR) (postprandial triglyceride maxima > 260 mg/dl). Levels of CAM were compared in HR and NR, and correlation with postprandial triglyceride, insulin and glucose response was assessed.ResultsFasting sICAM-1 and sVCAM-1 levels were significantly higher in HR as compared to NR (p = 0.046, p = 0.03). For sE-selectin there was such a trend (p = 0.05). There was a strong positive and independent correlation between sICAM-1 and postprandial insulin maxima (r = 0.70, p < 0.001). sVCAM-1 showed significant correlation with postprandial triglycerides (AUC) (r = 0.37, p = 0.047). We found no correlation between sCAMs and fasting insulin or triglyceride concentrations.ConclusionThis independent association of postprandial triglycerides with sICAM-1 may indicate a particular impact of postprandial lipid metabolism on endothelial reaction.

A common functional exon polymorphism in the microsomal triglyceride transfer protein gene is associated with type 2 diabetes, impaired glucose metabolism and insulin levels

AbstractThe microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apolipoprotein B-containing lipoproteins. Emerging evidence has indicated that the functional MTP exon polymorphism I128T is associated with dyslipidemia and other traits of the insulin-resistance syndrome, and the T128 variant seems to confer a reduced stability of MTP, resulting in reduced binding of LDL particles. The aim of the study was to elucidate the association of this MTP polymorphism with parameters of postprandial metabolism. A total of 716 male subjects from a postprandially characterized cohort (MICK) and a nested case-control study (EPIC) of 190 incident type 2 diabetes cases and 380 sex- or age-matched controls were genotyped for the I128T exon polymorphism. In comparison to homozygote subjects of the wild allele, carriers of the less common allele of the MTP T128 genotype showed significantly lower postprandial insulin levels (P=0.017), lower diastolic blood pressure (P=0.049) and had a lower prevalence of impaired glucose metabolism and diabetes type 2 (P=0.03) in the MICK. Consistent with this, we found a lower incidence of type 2 diabetes in male subjects of the nested case-control study in the T128 genotype (P=0.007). These results suggest that the rare allele of the MTP I128T polymorphism may be protective against impaired glucose tolerance, type 2 diabetes and other parameters of the metabolic syndrome.