A protocol for PCR in situ hybridization of hyphomycetes

A protocol for application of Polymerase Chain Reaction (PCR) in situ hybridization for the detection of hyphomycetes is presented. The experiments are exemplary carried out with strains of the genera Penicillium and Cladosporium. The small ribosomal subunit is amplified in situ by PCR using fungal specific primers. The amplicon is used as target region for a fluorescein-marked probe. The permeability of the fungal cell wall for the primers and the probe can be successfully achieved by enzymatic treatment with ß-glucanase. The protocol can be used as a basis for further development of in situ hybridization with taxon specific probe

Throwing the dice for the diagnosis of vaginal complaints?

BACKROUND: Vaginitis is among the most common conditions women are seeking medical care for. Although these infections can easily be treated, the relapse rate is high. This may be due to inadequate use of the diagnostic potential. METHODS: We evaluated the misjudgement rate of the aetiology of vaginal complaints. A total of 220 vaginal samples from women with a vaginal complaint were obtained and analysed for numbers of total lactobacilli, H(2)O(2)-producing lactobacilli, total aerobic cell counts and total anaerobic cell counts including bifidobacteria, Bacteroides spp., Prevotella spp. Additionally, the presence of Atopobium vaginae, Gardnerella vaginalis, Candida spp. and Trichomonas vaginalis was evaluated by DNA-hybridisation using the PCR and Affirm VPIII Microbial Identification Test, respectively. RESULTS: The participating physicians diagnosed Bacterial vaginosis (BV) as origin of discomfort in 80 cases, candidiasis in 109 cases and mixed infections in 8 cases. However, a present BV, defined as lack of H(2)O(2)-lactobacilli, presence of marker organisms, such as G. vaginalis, Bacteroides spp. or Atopobium vaginae, and an elevated pH were identified in only 45 cases of the women examined. Candida spp. were detected in 46 cases. Interestingly, an elevated pH corresponded solely to the presence of Atopobium vaginae, which was detected in 11 cases. CONCLUSION: Errors in the diagnosis of BV and candida vulvovaginitis (CV) were high. Interestingly, the cases of misjudgement of CV (77%) were more numerous than that of BV (61%). The use of Amsel criteria or microscopy did not reduce the number of misinterpretations. The study reveals that the misdiagnosis of vaginal complaints is rather high

Adjunctive use of essential oils following scaling and root planing: a randomized clinical trial

Background: Hitherto no study has been published on the effect of the adjunctive administration of essential oils following scaling and root planing (SRP). This study describes the effect of a mouthrinse consisting of essential oils (Cymbopogon flexuosus, Thymus zygis and Rosmarinus officinalis) following SRP by clinical and microbiological variables in patients with generalized moderate chronic periodontitis. Methods: Forty-six patients (aged 40–65 years) with moderate chronic periodontitis were randomized in a double-blind study and rinsed their oral cavity following SRP with an essential oil mouthrinse (n = 23) or placebo (n = 23) for 14 days. Probing depth (PD), attachment level (AL), bleeding on probing (BOP) and modified sulcus bleeding index (SBI) were recorded at baseline and after 3 and 6 months. Subgingival plaque was taken for assessment of major bacteria associated with periodontitis. Results: AL, PD, BOP and SBI were significantly improved in both groups after three (p < 0.001) and 6 months (p ≤ 0.015). AL improved significantly better in the test than in the control group after 3 and 6 months (p < 0.001), so did PD after three months in the tendency (p = 0.1). BOP improved better in the test group after 3 months (p = 0.065). Numbers of Treponema denticola (p = 0.044) and Fusobacterium nucleatum (p = 0.029) decreased more in the test than in the control group after 3 months, those of Tannerella forsythia after 6 months (p = 0.039). Prevotella micra (p < 0.001,p = 0.035) and Campylobacter rectus (p = 0.002,p = 0.012) decreased significantly in both groups after 3 months. Conclusions: The adjunctive use of a mouthrinse containing essential oils following SRP has a positive effect on clinical variables and on bacterial levels in the subgingival biofilm

Short-chain fatty acids and intestinal inflammation in multiple sclerosis: modulation of female susceptibility by microbial products?

Background: Multiple Sclerosis (MS) is an autoimmune-mediated disease of the central nervous system. Experi mental data suggest a role of intestinal microbiota and microbial products such as short-chain fatty acids (SCFAs) in the pathogenesis of MS. A recent clinical study reported benefcial efects (mediated by immunomodulatory mecha nisms) after oral administration of the SCFA propionate in MS patients. Based on available evidence, we investigated whether SCFAs and the fecal infammation marker calprotectin are altered in MS. Methods: 76 subjects (41 patients with relapsing–remitting MS and 35 age-matched controls) were investigated in this case–control study. All subjects underwent clinical assessment with established clinical scales and provided fecal samples for a quantitative analysis of fecal SCFA and fecal calprotectin concentrations. Fecal markers were com pared between MS patients and controls, and were analyzed for an association with demographic as well as clinical parameters. Results: Median fecal calprotectin concentrations were within normal range in both groups without any group-spe cifc diferences. Fecal SCFA concentrations showed a non-signifcant reduction in MS patients compared to healthy subjects. Female subjects showed signifcantly reduced SCFA concentrations compared to male subjects. Conclusions: In our cohort of MS patients, we found no evidence of an active intestinal infammation. Yet, the vast majority of the investigated MS patients was under immunotherapy which might have afected the outcome meas ures. The sex-associated diference in fecal SCFA concentrations might at least partially explain female predominance in MS. Large-scale longitudinal studies including drug-naïve MS patients are required to determine the role of SCFAs in MS and to distinguish between disease-immanent efects and those caused by the therapeutic regime

Bacterial Species Associated with Highly Allergenic Plant Pollen Yield a High Level of Endotoxins and Induce Chemokine and Cytokine Release from Human A549 Cells

none10siSensitization to pollen allergens has been increasing in Europe every year. Most studies in this field are related to climate change, phenology, allergens associated with different pollens, and allergic disorders. As a plant microhabitat, pollen is colonized by diverse microorganisms, including endotoxin-producing bacteria which may contribute to pollen allergy (pollinosis). Therefore, bacteria isolated from high allergenic and low allergenic plant pollen, as well as the pollen itself with all microbial inhabitants, were used to assess the effect of the pollen by measuring the endotoxins lipopolysaccharides (LPS) and lipoteichoic acid (LTA) concentrations and their effect on chemokine and cytokine release from transwell cultured epithelial A549 cells as a model of epithelial lung barrier. High allergenic pollen showed a significantly higher level of bacterial endotoxins; interestingly, the endotoxin level found in the bacterial isolates from high allergenic pollen was significantly higher compared to that of bacteria from low allergenic pollen. Moreover, bacterial LPS concentrations across different pollen species positively correlated with the LPS concentration across their corresponding bacterial isolates. Selected bacterial isolates from hazel pollen (HA5, HA13, and HA7) co-cultured with A549 cells induced a potent concentration-dependent release of the chemokine interleukin-8 and monocyte chemotactic protein-1 as well as the cytokine TNF-alpha and interleukin-2 to both apical and basal compartments of the transwell model. This study clearly shows the role of bacteria and bacterial endotoxins in the pollen allergy as well as seasonal allergic rhinitis.Ambika Manirajan, Binoy; Hinrichs, Ann-Kathrin; Ratering, Stefan; Rusch, Volker; Schwiertz, Andreas; Geissler-Plaum, Rita; Eichner, Gerrit; Cardinale, Massimiliano; Kuntz, Sabine; Schnell, SylviaAmbika Manirajan, Binoy; Hinrichs, Ann-Kathrin; Ratering, Stefan; Rusch, Volker; Schwiertz, Andreas; Geissler-Plaum, Rita; Eichner, Gerrit; Cardinale, Massimiliano; Kuntz, Sabine; Schnell, Sylvi

Dietary Wheat Amylase Trypsin Inhibitors Impact Alzheimer’s Disease Pathology in 5xFAD Model Mice

Wheat amylase trypsin inhibitors (ATIs) represent a common dietary protein component of gluten-containing cereals (wheat, rye, and barley). They act as toll-like receptor 4 ligands, and are largely resistant to intestinal proteases, eliciting a mild inflammatory response within the intestine after oral ingestion. Importantly, nutritional ATIs exacerbated inflammatory bowel disease and features of fatty liver disease and the metabolic syndrome in mice. For Alzheimer’s disease (AD), both inflammation and altered insulin resistance are major contributing factors, impacting onset as well as progression of this devastating brain disorder in patients. In this study, we evaluated the impact of dietary ATIs on a well-known rodent model of AD (5xFAD). We assessed metabolic, behavioral, inflammatory, and microbial changes in mice consuming different dietary regimes with and without ATIs, consumed ad libitum for eight weeks. We demonstrate that ATIs, with or without a gluten matrix, had an impact on the metabolism and gut microbiota of 5xFAD mice, aggravating pathological hallmarks of AD. If these findings can be translated to patients, an ATI-depleted diet might offer an alternative therapeutic option for AD and warrants clinical intervention studies

Metabolic phenotyping of diet and dietary Intake

Nutrition provides the building blocks for growth, repair, and maintenance of the body and is key to maintaining health. Exposure to fast foods, mass production of dietary components, and wider importation of goods have challenged the balance between diet and health in recent decades, and both scientists and clinicians struggle to characterize the relationship between this changing dietary landscape and human metabolism with its consequent impact on health. Metabolic phenotyping of foods, using high-density data-generating technologies to profile the biochemical composition of foods, meals, and human samples (pre- and postfood intake), can be used to map the complex interaction between the diet and human metabolism and also to assess food quality and safety. Here, we outline some of the techniques currently used for metabolic phenotyping and describe key applications in the food sciences, ending with a broad outlook at some of the newer technologies in the field with a view to exploring their potential to address some of the critical challenges in nutritional science

Studies on the human gut microbiota : Relevance for maturation, inflammation and nutritional processes

In this postdoctoral qualification thesis the effects and significance of the human microbiota and its members on the maturation, inflammation and nutritional processes in humans were investigated. In order to understand and exploit the impact of the gut microbes on human health and well-being, it is necessary to gain insight into the succession of the gut microbiota during aging

The Gut Microbiota and their Metabolites: Potential Implications for the Host Epigenome

The gut microbiota represents a metabolically active biomass of up to 2 kg in adult humans. Microbiota-derived molecules significantly contribute to the host metabolism. Large amounts of bacterial metabolites are taken up by the host and are subsequently utilized by the human body. For instance, short chain fatty acids produced by the gut microbiota are a major energy source of humans. It is widely accepted that microbiota-derived metabolites are used as fuel for beta-oxidation (short chain fatty acids) and participate in many metabolic processes (vitamins, such as folic acid). Apart from these direct metabolic effects, it also becomes more and more evident that these metabolites can interact with the mammalian epigenetic machinery. By interacting with histones and DNA they may be able to manipulate the host's chromatin state and functionality and hence its physiology and health. In this chapter, we summarize the current knowledge on possible interactions of different bacterial metabolites with the mammalian epigenetic machinery, mostly based on in vitro data. We discuss the putative impact on chromatin marks, for example histone modifications and DNA methylation. Subsequently, we speculate about possible beneficial and adverse consequences for the epigenome, the physiology and health of the host, as well as plausible future applications of this knowledge for in vivo translation to support personal health