Immunoglobulin A-specific deficiency induces spontaneous inflammation specifically in the ileum.

OBJECTIVE: Although immunoglobulin A (IgA) is abundantly expressed in the gut and known to be an important component of mucosal barriers against luminal pathogens, its precise function remains unclear. Therefore, we tried to elucidate the effect of IgA on gut homeostasis maintenance and its mechanism. DESIGN: We generated various IgA mutant mouse lines using the CRISPR/Cas9 genome editing system. Then, we evaluated the effect on the small intestinal homeostasis, pathology, intestinal microbiota, cytokine production, and immune cell activation using intravital imaging. RESULTS: We obtained two lines, with one that contained a <50 base pair deletion in the cytoplasmic region of the IgA allele (IgA tail-mutant; IgAtm/tm) and the other that lacked the most constant region of the IgH α chain, which resulted in the deficiency of IgA production (IgA-/-). IgA-/- exhibited spontaneous inflammation in the ileum but not the other parts of the gastrointestinal tract. Associated with this, there were significantly increased lamina propria CD4+ T cells, elevated productions of IFN-γ and IL-17, increased ileal segmented filamentous bacteria and skewed intestinal microflora composition. Intravital imaging using Ca2+ biosensor showed that IgA-/- had elevated Ca2+ signalling in Peyer's patch B cells. On the other hand, IgAtm/tm seemed to be normal, suggesting that the IgA cytoplasmic tail is dispensable for the prevention of the intestinal disorder. CONCLUSION: IgA plays an important role in the mucosal homeostasis associated with the regulation of intestinal microbiota and protection against mucosal inflammation especially in the ileum.

Long-term administration of Tetragenococcus halophilus No. 1 over generations affects the immune system of mice.

Japanese people have been consuming miso soup over generations; it is beneficial for health and longevity. In this study, Tetragenococcus halophilus No. 1 in miso was found to possess salient immunomodulatory functions. Recently, we also demonstrated its effect on boosting immunological robustness. Although the consumption of miso is suggested to affect health over generations, such a long-term experiment has not been conducted until now. Thus, we evaluated the effects of miso-derived T. halophilus No. 1 over generations on the immune system of mice. As the generations increase, the proportion of germinal center B cells tends to increase. Furthermore, we found that CD4+ T cells expressing CD69, an activation marker, were increased in the third generation of mice. In addition, the proportion of follicular helper T cells and regulatory T cells tended to increase. Among the subsets of CD4+ T cells in the fourth generation, effector T cells and effector memory T cells tended to increase. In contrast, central memory T cells and naive T cells decreased. Moreover, autoimmunity was suppressed by long-term administration of T. halophilus No. 1. Based on these findings, we believe that the long-term administration of T. halophilus No. 1 over generations promotes immune activation and tolerance and enhances immunological robustness.

Ingestion of miso regulates immunological robustness in mice.

In Japan, there is a long history of consumption of miso, a fermented soybean paste, which possesses beneficial effects on human health. However, the mechanism behind these effects is not fully understood. To clarify the effects of miso on immune cells, we evaluated its immunomodulatory activity in mice. Miso did not alter the percentage of B and T cells in the spleen; however, it increased CD69+ B cells, germinal center B cells and regulatory T cells. Anti-DNA immunoglobulin M antibodies, which prevent autoimmune disease, were increased following ingestion of miso. Transcriptome analysis of mouse spleen cells cultured with miso and its raw material revealed that the expression of genes, including interleukin (IL)-10, IL-22 and CD86, was upregulated. Furthermore, intravital imaging of the small intestinal epithelium using a calcium biosensor mouse line indicated that miso induced Ca2+ signaling in a manner similar to that of probiotics. Thus, ingestion of miso strengthened the immune response and tolerance in mice. These results appear to account, at least in part, to the salubrious effects of miso.

Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method.

Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) from the hyperthermophile Thermotoga maritima was biochemically characterized with the aim of establishing a colorimetric assay for inorganic pyrophosphate (PPi). When heterologously expressed in Escherichia coli, T. maritima PPDK (TmPPDK) was far more stable any other PPDK reported so far: it retained >90% of its activity after incubation for 1 h at 80°C, and >80% of its activity after incubation for 20 min at pHs ranging from 6.5 to 10.5 (50°C). In contrast to PPDKs from protozoa and plants, this TmPPDK showed very long-term stability at low temperature: full activity was retained even after storage for at least 2 years at 4°C. TmPPDK was successfully applied to a novel colorimetric PPi assay, which employed (i) a PPi cycling reaction using TmPPDK and nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) from Saccharomyces cerevisiae and (ii) a NAD cycling reaction to accumulate reduced nitroblue tetrazolium (diformazan). This enabled detection of 0.2 µM PPi, making this method applicable for preliminary measurement of PPi levels in PCR products in an automatic clinical analyzer.

Isolation of food-derived bacteria inducing interleukin-22 in B cells.

Recently, we found a novel function of the lactic acid bacterium Tetragenococcus halophilus derived from miso, a fermented soy paste, that induces interleukin (IL)-22 production in B cells preferentially. IL-22 plays a critical role in barrier functions in the gut and skin. We further screened other bacteria species, namely, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Weissella, Pediococcus, and Bacillus, in addition to Tetragenococcus and found that some of them possessed robust IL-22-inducible function in B cells in vitro. This process resulted in the augmented expression of activation markers CD86 and CD69 on B and T cells, respectively. However, these observations were not correlated with IL-22 production. We isolated Bacillus coagulans sc-09 from miso and determined it to be the best strain to induce robust IL-22 production in B cells. Furthermore, feeding B. coagulans sc-09 to mice augmented the barrier function of the skin regardless of gut microbiota.

Propolis induces Ca2+ signaling in immune cells.

Propolis possesses several immunological functions. We recently generated a conditional Ca2+ biosensor yellow cameleon (YC3.60) transgenic mouse line and established a five-dimensional (5D) (x, y, z, time, and Ca2+ signaling) system for intravital imaging of lymphoid tissues, including Peyer's patches (PPs). To assess the effects of propolis on immune cells, we analyzed Ca2+ signaling in vitro and in vivo using CD11c-Cre/YC3.60flox transgenic mice, in which CD11c+ dendritic cells (DCs) specifically express YC3.60. We found that propolis induced Ca2+ signaling in DCs in the PPs. Intravital imaging of PPs also showed that an intraperitoneal injection of propolis augmented Ca2+ signaling in CD11c+ cells, suggesting that propolis possesses immune-stimulating activity. Furthermore, CD11c+ cells in PPs in mice administrated propolis indicated an increase in Ca2+ signaling. Our results indicate that propolis induces immunogenicity under physiological conditions.

Isolation of immune-regulatory Tetragenococcus halophilus from miso.

Tetragenococcus halophilus is a halophilic lactic acid bacterium that exists in the traditional Japanese seasoning miso-a fermented soy paste. Considering the popularity of miso as a component of healthy diet, we attempted to evaluate the immunoregulatory functions of T. halophilus spices isolated from miso. We screened 56 strains that facilitated the upregulation of activation markers such as CD86 and CD69 on B cells and T cells in vitro. Of these, 7 strains (Nos. 1, 3, 13, 15, 19, 30, and 31) were found to preferentially induce the CD86 expression on B cells. Furthermore, DNA microarray analysis revealed that T. halophilus strain No. 1 significantly augmented the gene expressions of CD86, CD70, IL-10, INF-γ, and IL-22 in B cells. We confirmed these results at the protein level by flow cytometry. Mice feeding diet containing 1% T. halophilus No. 1 exhibited significantly greater IgA production in the serum. Furthermore, a diet containing 1% T. halophilus No. 1 augmented ovoalbumin (OVA)-specific IgG titer in mice upon OVA/alum immunization. Thus, we demonstrated that T. halophilus No. 1 is a strong immunomodulatory strain with potential as a probiotic.

Hypothyroidism caused by phenobarbital affects patterns of estrous cyclicity in rats.

We found that repeated treatment with phenobarbital (PB), a thyroid modulator, resulted in a persistent estrous stage in the present study. Although the effects of PB in blocking the surge release of luteinizing hormone (LH), inducing anovulation and prolonging the diestrous period has been well established, there is still no research describing the appearance of persistent estrous states in normal cycling rats dosed with PB. To further study this phenomenon, female rats exhibiting regular estrous cycle were administered an oral dose of PB for 14 consecutive days. Consecutively, vaginal smears were observed and rats from all the groups were sacrificed and serum hormone levels for prolactin, progesterone, estradiol, triiodothyronin (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) were measured. Pituitary, thyroid, liver, uteri and ovaries were excised, weighed and further subjected to histological observations. We found that PB induced irregular estrous cycles, especially persistent estrus in rats. Histopathologically, the persistent estrous stages are characterized by persistent vaginal cornification in the vagina, cystic follicles and anovulation in the ovaries. Endocrinologically, serum T3 and T4 levels were significantly lower, and TSH was higher in treated-female rats compared to control females. The serum estradiol level and the estradiol/progesterone ratio tend to increase in treated-females. Furthermore, PB-treated animals with irregular estrous cycle were reduced by T4 replacement. Our data indicate that treatment with PB resulted in hypothyroidism and irregular estrous cycle, particularly a persistent estrous stage in normal cycling female rats.

The alpha-glucosidase inhibitor miglitol delays the development of diabetes and dysfunctional insulin secretion in pancreatic beta-cells in OLETF rats.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model of type 2 diabetes, exhibits obesity, hyperglycemia and hyperlipidemia, with late onset of chronic and slowly progressive hyperinsulinemia. In this study, we examined effects of long-term dietary supplementation with the alpha-glucosidase inhibitor miglitol on the development of diabetes and the reduction of beta-cells in the pancreas of OLETF rats. The OLETF rats were fed a control diet or a diet containing 800 ppm miglitol (miglitol diet) for 65 weeks from pre-onset stage (5 weeks old). The non-fasting blood glucose concentrations gradually increased in OLETF rats fed the control diet and, at week 64, were significantly higher than those in OLETF rats fed the miglitol diet and age-matched Long-Evans Tokushima Otsuka (LETO) rats, which are control, non-diabetic, non-obese rats of the same strain. Oral glucose tolerance tests revealed that OLETF rats fed the control diet showed pronounced impaired glucose tolerance, but those fed the miglitol diet did not. Furthermore, insulin concentrations after glucose-loading were significantly lower in OLETF rats fed the control diet than in those fed the miglitol diet. The islets of 65-week-old OLETF rats fed the control diet showed significant fibrosis and loss of beta-cells, while those of age-matched control LETO rats had a normal appearance. Feeding OLETF rats a miglitol diet reduced fibrosis and the loss of beta-cells. Our results suggest that dietary supplementation with miglitol from pre-onset stage in OLETF rats delays the onset and development of diabetes and preserves the insulin secretory function of pancreatic islets.

Collaborative work on evaluation of ovarian toxicity by repeated-dose and fertility studies in female rats.

The National Institute of Health Sciences (NIHS) and 18 pharmaceutical companies of the Japan Pharmaceutical Manufacturers Association (JPMA) have conducted a validation study intended to evaluate whether a 2-week repeated general toxicity period with histopathological examination is sufficient to detect ovarian toxicity or not. The current repeated dose general toxicity study is considered to be insufficient in terms of evaluating female reproductive function due to a lack of evidence indicating that it is adequate. Evaluation of ovarian toxicity by comprehensive histopathological examination of the female reproductive organs based on the underlying morphology of a normal cycle of the reproductive tract including the ovary and additional immunohistochemical staining with proliferative cell nuclear antigen (PCNA) to identify small follicles may be a good tool to assess female reproductive function. In the collaborative study, 2- or 4-week repeated dose toxicity studies with ovarian histopathological examinations were conducted. A female fertility study was also conducted to compare the results with those of the ovarian histopathological findings. A total of 17 test substances were evaluated and categorized into hormone analogues, primordial follicle damaging agents, metabolite imbalance inducers, and endocrine imbalance inducers. Based on the results, ovarian toxicity could be detected by a careful histopatholgical examination. A 2-week dosing period may be sufficient for the evaluation of ovarian toxicity, except for cytotoxic compounds such as alkylating agents. The pathological findings of ovarian toxicity (decreases in follicles, increases in atretic follicles, increases in currently formed corpora lutea, etc) reflected the female fertility parameters (irregular estrous cycle, pre-implantation loss).

Collaborative work on evaluation of ovarian toxicity. 15) Two- or four-week repeated-dose studies and fertility study of bromocriptine in female rats.

The main focus of this study is to determine the optimal administration period concerning toxic effects on ovarian morphological changes in a repeated-dose toxicity study. To assess morphological and functional changes induced in the ovary by bromocriptine, the compound was administered to female rats at dose levels of 0, 0.08, 0.4 and 2 mg/kg for the 2- or 4-week repeated-dose toxicity study, and for the female fertility study from 2 weeks prior to mating to day 7 of gestation. In the 2-week repeated-dose toxicity study, increase of ovarian weights was observed at 2 mg/kg. In the 4-week repeated-dose toxicity study, ovarian weights were increased at 0.4 and 2 mg/kg. The number of corpora luteum was increased in the 0.4 and 2 mg/kg groups of the 2- and 4-week repeated-dose toxicity studies by histopathological examination of the ovaries. Bromocriptine did not affect estrous cyclicity in 2- and 4-week repeated dosing. In the female fertility study, although animals in any groups mated successfully, no females in 0.4 and 2 mg/kg groups were pregnant. There were no adverse effects on reproductive performance in the 0.08 mg/kg group. Based on these findings, the histopathological changes in the ovary are considered important parameters for evaluation of drugs including ovarian damage. We conclude that a 2-week administration period is sufficient to detect ovarian toxicity of bromocriptine in a repeated-dose toxicity study.

Effects of miglitol, an alpha-glucosidase inhibitor, on glycaemic status and histopathological changes in islets in non-obese, non-insulin-dependent diabetic Goto-Kakizaki rats.

Miglitol, a 1-deoxynojirimycin derivative, is an alpha-glucosidase inhibitor. In the present study, the effects of acute (single-dose) and chronic (8-week) oral administration of miglitol in Goto-Kakizaki (GK) rats, an animal model of type 2 diabetes, were investigated. Dose-dependent decreases in incremental blood glucose concentrations integrated over a period of 2 h (deltaAUC0-2 h) for values of blood glucose after sucrose-loading in miglitol-treated GK rats were observed following an acute oral administration of miglitol (1, 3 or 10 mg/kg body weight). At 10 mg/kg, the deltaAUC0-2 h of blood glucose was decreased by 45 % compared with the control group. Following the oral administration of miglitol in a dietary mixture (10 mg, 20 mg or 40 mg miglitol/100 g control diet) for 8 weeks, the ratio of HbA1c at 8 weeks compared with 0 weeks in GK rats treated with 40 mg miglitol/100 g control diet miglitol was significantly decreased compared with control GK rats without changes in body weight. In oral glucose tolerance testing, miglitol caused a slight decrease in the deltaAUC0-2 h of plasma glucose concentration. In addition, miglitol treatment slightly inhibited the reduction in beta-cell mass, and lessened the irregular contours and fibrosis of the islets in GK rats. These results indicate that miglitol ameliorates the hyperglycaemic state of GK rats and the impaired function of the pancreatic islets, as well as preventing the degeneration of islets in GK rats.

Mode of action of a germination-specific cortex-lytic enzyme, SleC, of Clostridium perfringens S40.

The hydrolysis of the bacterial spore peptidoglycan (cortex) is a crucial event in spore germination. It has been suggested that SleC and SleM, which are conserved among clostridia, are to be considered putative cortex-lytic enzymes in Clostridium perfringens. However, little is known about the details of the hydrolytic process by these enzymes during germination, except that SleM functions as a muramidase. Muropeptides derived from SleC-digested decoated spores of a Bacillus subtilis mutant that lacks the enzymes, SleB, YaaH and CwlJ, related to cortex hydrolysis were identified by amino acid analysis and mass spectrometry. The results suggest that SleC is most likely a bifunctional enzyme possessing lytic transglycosylase activity and N-acetylmuramoyl-L-alanine amidase activity confined to cross-linked tetrapeptide-tetrapeptide moieties of the cortex structure. Furthermore, it appears that during germination of Clostridium perfringens spores, SleC causes merely small and local changes in the cortex structure, which are necessary before SleM can function.

Effects of soybean food pellets on m-CPP-induced anxiety model of mice.

In this study, we gave the soybean powder-added food pellets (soybean pellets) to investigate anti-anxious effects of soybean in male mice. Twenty eight days after feeding control pellets or soybean pellets, we observed the behavioral changes in the elevated plus maze. There was no significant difference on the time spent in the open arms (%) between mice fed the control and soybean pellets. When we administered m-chlorophenylpiperazine (m-CPP, 2.5 mg/kg, i.p.) to mice, the mice fed control pellets showed the decrease in the time spent in the open arms, suggesting that anxiety-like behavior was induced by m-CPP. On the other hand, we could not observe the m-CPP-induced anxiety-like behavior in mice fed soybean pellets in this test. These results suggest that soybean pellets may attenuate anxiety-like behavior in mice.