Heyndrickxia coagulans strain SANK70258 suppresses symptoms of upper respiratory tract infection via immune modulation: a randomized, double-blind, placebo-controlled, parallel-group, comparative study.

Probiotic consumption strongly influences local intestinal immunity and systemic immune status. Heyndrickxia coagulans strain SANK70258 (HC) is a spore-forming lactic acid bacterium that has immunostimulatory properties on peripheral tissues. However, few reports have examined the detailed effectiveness of HC on human immune function and its mechanism of action. Therefore, we conducted a randomized, double-blind, placebo-controlled, parallel-group study to comprehensively evaluate the effects of HC on immunostimulatory capacity, upper respiratory tract infection (URTI) symptoms, and changes in intestinal organic-acid composition. Results of a questionnaire survey of URTI symptoms showed that runny nose, nasal congestion, sneezing, and sore throat scores as well as the cumulative number of days of these symptoms were significantly lower in the HC group than in the placebo group during the study period. Furthermore, the salivary secretory immunoglobulin A (sIgA) concentration was significantly higher, and the natural killer (NK) cell activity tended to be higher in the HC group than in the placebo group. In addition, we performed an exposure culture assay of inactivated influenza virus on peripheral blood mononuclear cells (PBMCs) isolated from the blood of participants in the HC and placebo groups. Gene-expression analysis in PBMCs after culture completion showed that IFNα and TLR7 expression levels were significantly higher in the HC group than in the placebo group. In addition, the expression levels of CD304 tended to be higher in the HC group than in the placebo group. On the other hand, the HC group showed a significantly higher increase in the intestinal butyrate concentration than the placebo group. HC intake also significantly suppressed levels of IL-6 and TNFα produced by PBMCs after exposure to inactivated influenza virus. Collectively, these results suggest that HC activated plasmacytoid dendritic cells expressing TLR7 and CD304 and strongly induced IFNα production, subsequently activating NK cells and increasing sIgA levels, and induced anti-inflammatory effects via increased intestinal butyrate levels. These changes may contribute to the acquisition of host resistance to viral infection and URTI prevention.

Development of Oral Care Chip, a novel device for quantitative detection of the oral microbiota associated with periodontal disease.

Periodontal disease, the most prevalent infectious disease in the world, is caused by biofilms formed in periodontal pockets. No specific bacterial species that can cause periodontitis alone has been found in any study to date. Several periodontopathic bacteria are associated with the progress of periodontal disease. Consequently, it is hypothesized that dysbiosis of subgingival microbiota may be a cause of periodontal disease. This study aimed to investigate the relationship between the subgingival microbiota and the clinical status of periodontal pockets in a quantitative and clinically applicable way with the newly developed Oral Care Chip. The Oral Care Chip is a DNA microarray tool with improved quantitative performance, that can be used in combination with competitive PCR to quantitatively detect 17 species of subgingival bacteria. Cluster analysis based on the similarity of each bacterial quantity was performed on 204 subgingival plaque samples collected from periodontitis patients and healthy volunteers. A significant difference in the number of total bacteria, Treponema denticola, Campylobacter rectus, Fusobacterium nucleatum, and Streptococcus intermedia bacteria in any combination of the three clusters indicated that these bacteria gradually increased in number from the stage before the pocket depth deepened. Conversely, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Streptococcus constellatus, which had significant differences only in limited clusters, were thought to increase in number as the pocket depth deepened, after periodontal pocket formation. Furthermore, in clusters where healthy or mild periodontal disease sites were classified, there was no statistically significant difference in pocket depth, but the number of bacteria gradually increased from the stage before the pocket depth increased. This means that quantitative changes in these bacteria can be a predictor of the progress of periodontal tissue destruction, and this novel microbiological test using the Oral Care Chip could be effective at detecting dysbiosis.

Method for absolute quantification of microbial communities by using both microarrays and competitive PCR.

Methods for the robust quantification of bacterial communities are still under development. In this context, the present study aimed to evaluate a method combining competitive PCR (cPCR) and microarray assays for the determination of absolute content of total bacteria and individual bacterial species in samples. For this, a competitor DNA for cPCR and microarrays containing three types of DNA probes was prepared. A calibration curve was generated with genomic DNA samples as standards, which was then utilized for cPCR-based determination of the total number (in moles) of 16S rRNA genes in other bacterial samples. Moreover, scatter plots of species-specific probes versus total bacteria probe for each genomic DNA of known concentration was fit to the regression model, and the obtained slope value was defined as the hybridization affinity ratio. The cPCR assay was performed for both a commercially available mixed genomic DNA sample and human oral bacterial DNA samples, and the total number of moles of 16S rRNA genes was determined. These values were distributed among each species on the basis of the signal intensities of species-specific probes and the hybridization affinity ratio. The total number of bacterial genomes and those of individual species were determined by dividing the copy number of 16S rRNA genes per genome. The obtained results were confirmed by quantitative real-time PCR (qPCR). For values of >1 × 102 copies determined by qPCR, the ratio of the values measured by DNA chips to by qPCR was 1.53-fold on average and <2.6-fold for all data. These results show that the combined method of cPCR and microarray is useful to quantify the absolute numbers of several types of bacteria in a sample at one time.

Gene expression analysis of the liver and skeletal muscle of psyllium-treated mice.

Psyllium, a dietary fibre rich in soluble components, has both cholesterol- and TAG-lowering effects. Many studies have verified these actions using liver samples, whereas little information is available on the effects of psyllium treatment on other organs. The purpose of the present study was to evaluate the possible beneficial effects of psyllium. We investigated the gene expression profiles of both liver and skeletal muscle using DNA microarrays. C57BL/6J mice were fed a low-fat diet (LFD; 7 % fat), a high-fat diet (HFD; 40 % fat) or a HFD with psyllium (40 % fat+5 % psyllium; HFD+Psy) for 10 weeks. Body weights and food intake were measured weekly. After 10 weeks, the mice were killed and tissues were collected. Adipose tissues were weighed, and plasma total cholesterol and TAG blood glucose levels were measured. The expression levels of genes involved in glycolysis, gluconeogenesis, glucose transport and fatty acid metabolism were measured by DNA microarray in the liver and skeletal muscle. In the HFD+Psy group, plasma total cholesterol, TAG and blood glucose levels significantly decreased. There was a significant reduction in the relative weight of the epididymal and retroperitoneal fat tissue depots in mice fed the HFD+Psy. The expression levels of genes involved in fatty acid oxidation and lipid transport were significantly up-regulated in the skeletal muscle of the HFD+Psy group. This result suggests that psyllium stimulates lipid transport and fatty acid oxidation in the muscle. In conclusion, the present study demonstrates that psyllium can promote lipid consumption in the skeletal muscle; and this effect would create a slightly insufficient glucose state in the liver.

The hepatic circadian clock is preserved in a lipid-induced mouse model of non-alcoholic steatohepatitis.

Recent studies have correlated metabolic diseases, such as metabolic syndrome and non-alcoholic fatty liver disease, with the circadian clock. However, whether such metabolic changes per se affect the circadian clock remains controversial. To address this, we investigated the daily mRNA expression profiles of clock genes in the liver of a dietary mouse model of non-alcoholic steatohepatitis (NASH) using a custom-made, high-precision DNA chip. C57BL/6J mice fed an atherogenic diet for 5 weeks developed hypercholesterolemia, oxidative stress, and NASH. DNA chip analyses revealed that the atherogenic diet had a great influence on the mRNA expression of a wide range of genes linked to mitochondrial energy production, redox regulation, and carbohydrate and lipid metabolism. However, the rhythmic mRNA expression of the clock genes in the liver remained intact. Most of the circadianly expressed genes also showed 24-h rhythmicity. These findings suggest that the biological clock is protected against such a metabolic derangement as NASH.

Quantitative assessment of RUNX3 methylation in neoplastic and non-neoplastic gastric epithelia using a DNA microarray.

Silencing of the RUNX3 gene by hypermethylation of its promoter CpG island plays a major role in gastric carcinogenesis. To quantitatively evaluate RUNX3 methylation, a fiber-type DNA microarray was used on which methylated and unmethylated sequence probes were mounted. After bisulfite modification, a part of the RUNX3 promoter CpG island, at which methylation is critical for gene silencing, was amplified by polymerase chain reaction using a Cy5 end-labeled primer. Methylation rates (MR) were calculated as the ratio of the fluorescence intensity of a methylated sequence probe to the total fluorescence intensity of methylated and unmethylated probes. Five gastric cancer cell lines were analyzed, as well as 26 primary gastric cancers and their corresponding non-neoplastic gastric epithelia. MR in four of the cancer cell lines that lost RUNX3 mRNA ranged from 99.0% to 99.7% (mean, 99.4%), whereas MR in the remaining cell line that expressed RUNX3 mRNA was 0.6%. In primary gastric cancers and their corresponding non-neoplastic gastric epithelia, MR ranged from 0.2% to 76.5% (mean, 22.7%) and from 0.7% to 25.1% (mean, 5.5%). Ten (38.5%) of the 26 gastric cancers and none of their corresponding non-neoplastic gastric epithelia had MR >30%. Most of the samples with MR >10% tested methylation-positive by conventional methylation-specific polymerase chain reaction (MSP). This microarray-based methylation assay is a promising method for the quantitative assessment of gene methylation.

Multiple tumor suppressor genes are increasingly methylated with age in non-neoplastic gastric epithelia.

A number of tumor suppressor and tumor-related genes are silenced by promoter hypermethylation in gastric cancer. Hypermethylation is not restricted to cancer cells, but is also present in non-neoplastic cells during aging. Such age-related methylation in non-neoplastic gastric epithelia is postulated to constitute a field defect that increases the risk for development of gastric cancer. To quantitatively evaluate age-related methylation in non-neoplastic gastric epithelia, we used a fiber-type DNA microarray on which methylated and unmethylated sequence probes were mounted. After bisulfite modification, a part of the promoter CpG island of four tumor suppressor genes, lysyl oxidase (LOX), p16, RUNX3 and tazarotene-induced gene 1 (TIG1), were amplified by PCR using Cy5 end labeled primers. Methylation rates (MRs) were calculated as the ratio of the fluorescence intensity of a methylated sequence probe to the total fluorescence intensity of methylated and unmethylated probes. Non-neoplastic gastric mucosa was obtained from 24 non-cancer-bearing stomachs at autopsy. MRs ranged from 0.0% to 77.2% (mean, 15.8%) for LOX, 0.0% to 45.8% (mean, 10.0%) for p16, 0.0% to 83.8% (mean, 9.0%) for RUNX3, and 0.0% to 46.1% (mean, 6.6%) for TIG1, and significantly correlated with aging (P < 0.01). The regression curves were: y = 0.013x(2) - 0.6184x + 4.0512, R(2) = 0.5728 (P < 0.001) for LOX; y = 0.0107x(2) - 0.6055x + 5.2943, R(2) = 0.7891 (P < 0.00001) for p16; y = 0.0182x(2) - 1.2234x + 11.566, R(2) = 0.5595 (P < 0.001) for RUNX3; and y = 0.0068 x(2) - 0.3586 x + 2.4306, R(2) = 0.4670 (P < 0.01) for TIG1. Thus, our present results are consistent with the notion that age-related methylation is associated with cancer susceptibility in the elderly. Quantitative analysis of DNA methylation using DNA microarrays is a promising method for risk assessment in the development of gastric cancer.

Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays.

Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.