Unkeito Suppresses RANKL-Mediated Osteoclastogenesis via the Blimp1-Bcl6 and NF-κB Signaling Pathways and Enhancing Osteoclast Apoptosis.

Osteoporosis is a common bone disease, particularly in menopausal women. Herein, we screened four Kampo medicines (Unkeito (UKT), Kamishoyosan (KSS), Kamikihito (KKT), and Ninjinyoeito (NYT)), frequently used to treat menopausal syndromes, for their effects on receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclast differentiation in RAW 264 cells. Considering that UKT exhibited the most potent effect, we examined its effect on RANKL-induced osteoclastogenesis, the induction of osteoclast apoptosis, and the mechanisms underlying its effects. UKT inhibits RANKL-induced osteoclast differentiation in the early stage and decreases osteoclast-related genes, including tartrate-resistant acid phosphatase (Trap), dendritic cell-specific transmembrane protein (Dcstamp), matrix metalloproteinase-9 (Mmp9), and cathepsin K (Ctsk). Specifically, UKT inhibits the nuclear factor of activated T cells 1 (NFATc1), which is essential for osteoclastogenesis. UKT increases Bcl6, which antagonizes NFATc1 and Dc-stamp, thereby blocking the progression of osteoclasts to maturation. UKT also decreased nuclear translocation by downregulating the activity of p65/NF-κB. In addition, UKT enhances mononuclear osteoclast apoptosis via activation of caspase-3. Herein, we demonstrate that UKT suppresses RANKL-mediated osteoclastogenesis via the Blimp1-Bcl6 and NF-κB signaling pathways and enhances mononuclear osteoclast apoptosis. Furthermore, UKT prevents bone loss in OVX mice. Thus, UKT might be a potential therapeutic agent for postmenopausal osteoporosis.

Induction of γδT cells from HSC-enriched BMCs co-cultured with iPSC-derived thymic epithelial cells.

T cells bearing γδ antigen receptors have been investigated as potential treatments for several diseases, including malignant tumours. However, the clinical application of γδT cells has been hampered by their relatively low abundance in vivo and the technical difficulty of inducing their differentiation from hematopoietic stem cells (HSCs) in vitro. Here, we describe a novel method for generating mouse γδT cells by co-culturing HSC-enriched bone marrow cells (HSC-eBMCs) with induced thymic epithelial cells (iTECs) derived from induced pluripotent stem cells (iPSCs). We used BMCs from CD45.1 congenic C57BL/6 mice to distinguish them from iPSCs, which expressed CD45.2. We showed that HSC-eBMCs and iTECs cultured with IL-2 + IL-7 for up to 21 days induced CD45.1+ γδT cells that expressed a broad repertoire of Vγ and Vδ T-cell receptors. Notably, the induced lymphocytes contained few or no αßT cells, NK1.1+ natural killer cells, or B220+ B cells. Adoptive transfer of the induced γδT cells to leukemia-bearing mice significantly reduced tumour growth and prolonged mouse survival with no obvious side effects, such as tumorigenesis and autoimmune diseases. This new method suggests that it could also be used to produce human γδT cells for clinical applications.

Coal burning-derived SO2 and traffic-derived NO2 are associated with persistent cough and current wheezing symptoms among schoolchildren in Ulaanbaatar, Mongolia.

BACKGROUND: Children in Ulaanbaatar are exposed to air pollution, but few epidemiological studies have been conducted on the effects of environmental risk factors on children's health. Also, no studies have yet examined the prevalence of respiratory symptoms in children in suburban areas, where air quality-monitoring stations have not yet been installed. This cross-sectional study evaluated the associations between outdoor air pollution and respiratory symptoms among schoolchildren in urban and suburban districts of Ulaanbaatar. METHODS: The ATS-DLD-78 C questionnaire was used to investigate the respiratory symptoms of schoolchildren aged 6-12 years (n = 1190) who lived in one of three urban districts or a suburban district of Ulaanbaatar. In each district, the outdoor concentrations of nitrogen dioxide (NO2) and sulfur dioxide (SO2) were measured at two sites (at ≤100 m and > 100 m from the nearest major road) in the 2-year period from 2015 to 2016. The associations between health outcomes and exposure to air pollutants were estimated using the multinomial logistic regression method. RESULTS: The outdoor concentration of SO2 was significantly associated with persistent cough symptom (OR = 1.12, 95% CI 1.04-1.22). Furthermore, the outdoor concentration of NO2 was significantly associated with the current wheezing symptom (OR = 1.33, 95% CI 1.01-1.75) among children in urban and suburban. CONCLUSIONS: The prevalence of persistent cough symptom was markedly high among the schoolchildren in urban/suburban districts of Ulaanbaatar. Overall, the increases in the prevalence of respiratory symptoms among children might be associated with ambient air pollution in Ulaanbaatar.

The in vitro estrogenic activity of the crude drugs found in Japanese herbal medicines prescribed for menopausal syndrome was enhanced by combining them.

BACKGROUND: Japanese herbal medicines can be used as alternatives to estrogen therapy and are sometimes prescribed for menopausal syndrome because they have fewer side effects and are associated with better compliance than estrogen therapy, but little is known about the pharmacological mechanisms of such treatments. This study aimed to explore the mechanisms responsible for the estrogen-like effects of five widely prescribed Japanese herbal medicines (unkeito, kamishoyosan, nyoshinsan, keishibukuryogan, and tokishakuyakusan). METHODS: We evaluated the estrogenic activity of these five Japanese herbal medicines and their metabolites using an estrogen receptor (ER)-dependent cell proliferation bioassay and an ER-dependent reporter assay. We also investigated the estrogenic activity of the crude drugs within the medicines and attempted to detect inter-crude drug synergistic effects using the ER-dependent reporter assay. RESULTS: We found that unkeito, kamishoyosan, and nyoshinsan exhibited estrogenic activity, and they displayed stronger estrogenic activity after being metabolized. Then, we focused on investigating the estrogenic activity of the crude drugs present within unkeito. We found that glycyrrhizae radix, cinnamomi cortex, evodiae fructus, and zingiberis rhizoma demonstrated ERß-dependent estrogenic activity. The combined use of evodiae fructus and glycyrrhizae radix, or evodiae fructus and cinnamomi cortex produced synergistic ERß-dependent estrogenic activity. CONCLUSION: It was suggested that unkeito, kamishoyosan, and nyoshinsan exert estrogenic activity, and hence, might be useful for treating menopausal syndrome. Furthermore, synergistic estrogenic effects were detected between some of the crude drugs present within unkeito.

A modified seated side tapping test in which the arms are crossed also reflects gait function in community-dwelling elderly.

[Purpose] Seated side tapping test (SST) performance is associated with mobility impairment in the elderly. Although this test was developed to assess trunk function, interpretation of its results may be confounded by the upper-limb movements in its protocol. Here, this study aimed to validate the association between trunk function and gait function by means of the Arms Crossed SST (AC-SST), a modified version of the SST in which the arms are crossed over the chest, to exclude the effects of the upper limbs. [Subjects and Methods] A total of 116 community-dwelling elderly people were enrolled in the study (mean age: 75.1 ± 5.5 yrs). Measurement categories were gait function (gait speed and TUG), lower extremity strength (knee extension and flexion strength), trunk muscle endurance (trunk extension and flexion endurance), and trunk function (SST and AC-SST). [Results] AC-SST performance significantly correlated with gait function items, as did SST performance. Moreover, AC-SST was one of the significant predictor variables of gait function selected in stepwise multiple regressions. [Conclusion] Gait function associated with performance on the AC-SST, a test of trunk function in which the effects of upper limb function were excluded, reinforcing the importance of trunk function to elderly mobility.

Effect of combination of dietary fish protein and fish oil on lipid metabolism in rats.

This study examined the effects of fish protein in combination with fish oil on rat lipid metabolism. Male Wistar rats were divided into four groups and fed an AIN93G-based diet with casein (20%) + soybean oil (7%), casein (10%) + fish protein (10%) + soybean oil (7%), casein (20%) + soybean oil (5%) + fish oil (2%), and casein (10%) + fish protein (10%) + soybean oil (5%) + fish oil (2%) for 4 weeks. The dietary combination of fish protein and fish oil decreased the contents of serum triacylglycerol, serum cholesterol, liver triacylglycerol and liver cholesterol in addition to altering liver lipid fatty acid composition. These effects are partly due to the increase in fecal cholesterol, bile acid excretion, and increased enzyme activities of fatty acid ß-oxidation in the liver. These data suggest that combined intake of fish protein and fish oil lead to both hypocholesterolemic and hypotriglyceridemic in serum and the liver, while sole intake of fish protein or fish oil decrease only cholesterol and triglyceride levels, respectively. These results suggest that combined intake of fish protein and fish oil may play beneficial roles in the prevention of lifestyle-related diseases as compared with sole fish protein intake.

Diagnosis of active tuberculosis using MPB64, a specific antigen of Mycobacterium bovis.

Because the incidence of tuberculosis (TB) is still high in developing countries, an inexpensive and rapid diagnostic test for this infection is needed. To develop a screening test for TB, MPB64 antigen was produced by recombinant technology and purified with a polyhistidine tag. Next, serum and urine samples from patients with TB and uninfected individuals were examined by the dot-blot assay method using this purified antigen. Serum samples from patients with TB reacted more strongly with MPB64 antigen than did those from uninfected individuals. In addition, serum samples from TB patients with active infection reacted more strongly with the antigen than did samples from patients with inactive TB. When urine samples were assessed using this assay, similar results were obtained. Correlations between the data obtained from serum and urine samples were analyzed for all subjects, including uninfected individuals, and a strong positive correlation between the results of serum and urine tests (n = 36, r = 0.672) was found. The sensitivity and specificity of this assay for serum samples was 85.7 % and 85.0 %, and for urine samples 75.0 % and 85.0 %, respectively. These results suggest that dot-blot assay with MPB64 antigen could be a useful screening test for active TB. Because urine samples can be obtained more easily than serum samples and because urine is less contagious, urine testing should probably be employed for screening purposes.

Dietary Alaska Pollock Protein Attenuates the Experimental Colitis Induced by Dextran Sulfate Sodium via Regulation of Gut Microbiota and Its Metabolites in Mice.

Protein derived from fish has not only nutritional properties but also health-promoting properties. Few studies have examined the effect of dietary Alaska pollock protein (APP) on the anticolitis effect reported to be associated with metabolic syndrome (MetS). This study investigated the effect of APP intake on colitis symptoms, gut microbiota, and its metabolites in the experimental colitis mouse model induced by dextran sulfate sodium (DSS). Male C57BL/6J mice were divided into three groups: (1) DSS-untreated mice fed an American Institute of Nutrition (AIN) 93G diet (protein source is casein), (2) DSS-treated mice fed an AIN93G diet, and (3) DSS-treated mice fed an APP diet. After the mice were fed the diets for 21 days, experimental colitis was induced by three cycles of 2% DSS administration for 5 days followed by washouts over the course of 5 days. APP-reduced body weight loss increased the disease activity index, and elevated spleen weight and alleviated colon length shortening and colonic tissue damage. Furthermore, APP altered the structure and composition of the microbiota and short-chain fatty acids in feces. Since APP intake alleviates experimental colitis induced by DSS administration through alterations in the gut microbiota and its metabolites, we deduced that APP would inhibit MetS progression via colitis suppression.

Reduction in Anti-Dengue Virus IgG Antibody Levels with the Use of a Larvicide for Vector Control in Rural Lao People's Democratic Republic.

The Lao People's Democratic Republic is an endemic area of dengue, with cases reported in urban and rural areas every year. In this study, we indirectly evaluated the efficacy of a larvicide (SumiLarvTM 2MR discs) that was used for vector control against Aedes mosquitoes. Villages in a rural area of Lao PDR were selected as study areas, non-intervention and intervention villages. At the intervention village, the larvicide was used to treat refillable water containers for 27 months (October 2017 to February 2020), while at the non-intervention villages were no treatment. The serum samples of villagers from both villages were randomized to collect in the pre-intervention and in post-intervention periods. An enzyme-linked immunosorbent assay (ELISA) was used to examine anti-dengue virus (DENV) IgG antibody levels in serum samples. Recombinant DENV serotype 2 non-structural protein1 was used as an antigen for the ELISA, the optical density (OD) values were analyzed for comparison. The results showed that the OD values decreased significantly (p < 0.01) between the pre-intervention and post-intervention periods at the intervention site. The treatment of water storage containers in rural areas with SumiLarvTM 2MR discs may help to protect residents from Aedes mosquito bites, and hence, reduce DENV infections.

Dietary Oyster (Crassostrea gigas) Extract Ameliorates Dextran Sulfate Sodium-Induced Chronic Experimental Colitis by Improving the Composition of Gut Microbiota in Mice.

Previously, we have reported that the intake of oyster extract (OE), prepared from Pacific oysters (Crassostrea gigas), can attenuate symptoms of dextran sulfate sodium (DSS)-induced acute experimental colitis in mice. Herein, we aimed to evaluate whether OE intake ameliorates chronic experimental colitis induced by repeated DSS administration in mice. Male C57BL/6J (4-week-old) mice were fed either the standard diet AIN93G (control diet) or the control diet containing 5.0% (w/w) OE (OE diet). After 21 days of diet feeding, chronic experimental colitis was induced by three cycles of 2.0% (w/w) DSS solution administration (5 days), followed by distilled water (5 days). Mice fed OE alleviated the shortened colonic length, increased the relative weight of the spleen, colonic histopathological score (regeneration), and blood in the stool score compared with mice fed control diet. A tendency to improve the α-diversity of fecal microbiota, which was exacerbated by colitis, was observed in mice fed OE. Correlation analysis suggested that the anti-colitis effect of OE intake could be related to the valeric acid content and relative abundances of Ruminococcus and Enterococcus in the feces. In conclusion, OE could ameliorate DSS-induced chronic experimental colitis by improving the gut environment, including the microbiota community and SCFA composition.

Oyster (Crassostrea gigas) Extract Attenuates Dextran Sulfate Sodium-Induced Acute Experimental Colitis by Improving Gut Microbiota and Short-Chain Fatty Acids Compositions in Mice.

Drugs for inflammatory bowel diseases can be associated with serious side effects, and the development of alternative candidate resources derived from natural products has attracted considerable attention. Oyster extract (OE) derived from Crassostrea gigas contains glycogen, taurine, and amino acids, and has been assigned diverse health-promoting properties. This study investigated the anti-colitis effect of OE intake on fecal microbiota and its metabolites of acute experimental colitis mouse model induced by dextran sulfate sodium (DSS). C57BL/6J mice (male) were divided into three groups: (1) American Institute of Nutrition (AIN) 93G diet + DSS-untreated, (2) AIN93G diet + DSS-treated, and (3) 5% OE diet + DSS-treated. Mice were fed each diet for 21 days, and then administered 2.5% DSS solution to induce acute colitis for 7 days. In DSS-induced colitis mice, OE decreased body weight loss and increased disease activity index during the DSS-induced period. In addition, OE tended to decrease the colon length shortening and the relative spleen weight and alleviated colonic tissue damage. Moreover, OE improved fecal short-chain fatty acids compositions and altered the structure of fecal microbiota. These results provide insight into the health-promoting property of OE in alleviating DSS-induced acute colitis, providing a basis for the development and use of functional foods.

Comparison of the Cholesterol-Lowering Effect of Scallop Oil Prepared from the Internal Organs of the Japanese Giant Scallop (Patinopecten yessoensis), Fish Oil, and Krill Oil in Obese Type II Diabetic KK-A y Mice.

Due to the growing demand of n-3 polyunsaturated fatty acids (PUFA) as supplements and pharmaceutical products worldwide, there are concerns about the exhaustion of n-3 PUFA supply sources. We have successfully prepared high-quality scallop oil (SCO), containing high eicosapentaenoic acid and phospholipids contents, from the internal organs of the Japanese giant scallop (Patinopecten yessoensis), which is the largest unutilized marine resource in Japan. This study compared the cholesterol-lowering effect of SCO with fish oil (menhaden oil, MO) and krill oil (KO) in obese type II diabetic KK-A y mice. Four-week-old male KK-A y mice were divided into four groups; the control group was fed the AIN93G-modified high-fat (3 wt% soybean oil + 17 wt% lard) diet, and the other three groups (SCO, MO, and KO groups) were fed a high-fat diet, in which 7 wt% of the lard in the control diet was replaced with SCO, MO, or KO, respectively. After the mice were fed the experimental diet for 42 days, their serum, liver, and fecal lipid contents as well as their liver mRNA expression levels were evaluated. The SCO group had significantly decreased cholesterol levels in the serum and liver; this decrease was not observed in the MO and KO groups. The cholesterol-lowering effect of SCO was partly mediated by the enhancement of fecal total sterol excretion and expression of liver cholesterol 7α-hydroxylase, a rate-limiting enzyme for bile acid synthesis. These results indicate that dietary SCO exhibits serum and liver cholesterol-lowering effects that are not found in dietary MO and KO and can help prevent lifestyle-related diseases.

Dietary Eicosapentaenoic Acid and Docosahexaenoic Acid Ethyl Esters Influence the Gut Microbiota and Bacterial Metabolites in Rats.

Dietary fish oil containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been reported to affect the diversity and composition of gut microbiota and bacterial metabolites. However, few reports have focused on the effects of EPA and DHA on gut microbiota diversity and bacterial metabolites. This study evaluated the effects of dietary EPA-ethyl ester (EE) and DHA-EE on steroid metabolism, gut microbiota, and bacterial metabolites in Wistar rats. Male rats were fed the experimental diets containing 5% (w/w) soybean oil-EE (SOY diet), EPA-EE (EPA diet), and DHA-EE (DHA diet) for four weeks. The lipid contents in the serum and liver, mRNA expression levels in the liver, and the diversity, composition, and metabolites of the gut microbiota were evaluated. The EPA and DHA diets decreased serum and liver cholesterol contents compared to the SOY diet. In addition, there were no significant changes in gene expression levels related to steroid metabolism in the liver between the EPA and DHA groups. Rats fed the DHA diet had lower microbiota diversity indices, such as Simpson and Shannon indices, than rats fed the SOY and EPA diets. In addition, rats fed EPA and DHA had significant differences in the relative abundance of microbiota at the genus level, such as Phascolarctobacterium, Turicibacter, and [Eubacterium]. Therefore, it was concluded that EPA and DHA have different effects on the diversity and composition of gut microbiota under the experimental conditions employed herein.

Phenotypic and haplotypic profiles of insecticide resistance in populations of Aedes aegypti larvae (Diptera: Culicidae) from central Lao PDR.

BACKGROUND: Aedes aegypti, which is widely distributed in the Lao People's Democratic Republic (PDR), is the primary vector of arboviral diseases. Chemical insecticides have been intensively used to eliminate mosquito-borne diseases, resulting in the development of insecticide resistance. However, little is known about the insecticide resistance of mosquito populations in Lao PDR and the mechanisms responsible for it, which have important implications for vector management programs. Here, we examined the phenotypic and haplotypic profiles of insecticide resistance in populations of Ae. aegypti larvae from central Lao PDR. METHODS: Ae. aegypti larvae were collected from four sites in Lao PDR, and their susceptibility to temephos, deltamethrin, permethrin, and Bacillus thuringiensis israelensis (Bti) was tested using larval bioassays. Synergistic tests were also conducted to evaluate the activity of insecticide-metabolizing enzymes in the larvae. Deltamethrin-resistant and Deltamethrin-susceptible larvae were then genotyped for knockdown resistance (kdr) mutations to determine the associations between each genotype and resistance. RESULTS: Ae. aegypti larvae from central Lao PDR were considered to be "resistant" (<98% mortality) to organophosphates and pyrethroids. The bio-insecticide Bti remains effective against such larvae. The resistance mechanisms of Ae. aegypti larvae were found to vary among populations, especially for pyrethroid resistance. Kdr mutations were significantly associated with deltamethrin resistance in Ae. aegypti from the Xaythany population. In contrast, synergist assays with piperonyl butoxide suggested that cytochrome P450 monooxygenases played an important role in the resistance seen in the Khounkham and Thakhek populations. CONCLUSION: This study obtained information that will aid the design and implementation of insecticide-based vector management of Ae. aegypti in central Lao PDR. Ae. aegypti larvae from central Lao PDR were highly susceptible to Bti, while they were resistant to temephos at a diagnostic dose of 0.0286 mg/L. Given the limited number of insecticides that are approved for vector control, it is important to alternate between temephos and other larvicides, such as Bti and pyriproxyfen. The differences in pyrethroid resistance mechanisms seen among the Ae. aegypti populations highlight the need to tailor vector-control strategies to each region to increase the success of dengue control in Lao PDR.

Aedes mosquito surveillance and the use of a larvicide for vector control in a rural area of the Lao People's Democratic Republic.

BACKGROUND: Refillable water containers are commonly used in rural areas of Lao PDR, and they act as Aedes mosquito breeding sites. Aedes aegypti and Ae. albopictus mosquitos are transmission vectors for the dengue virus, which causes dengue fever. METHODS: Two isolated rural villages in the central part of Lao PDR were selected as study sites. In the intervention village, domestic water containers were continuously treated with a long-lasting matrix release formulation, containing pyriproxyfen, named SumiLarv®2MR. In the control village, entomological activity was monitored, but no intervention was performed. Baseline data were collected in both villages during the late rainy season (October 2017) then distributed SumiLarv®2MR disks in intervention village. This data was compared with data collected during the intervention periods in the dry season (February 2018), rainy season (July 2018 and 2019), and late rainy season (September 2018) in the region. RESULTS: Compared with the baseline data (20.24%), the percentage of water containers infested with Ae. aegypti larvae was significantly decreased in the treated village, especially in the rainy seasons in July 2018 (4.11%; P < 0.001) and July 2019 (2.46%; P < 0.001), while the percentage of water containers infested with Ae. albopictus larvae did not decrease significantly in prevalence. No reduction in the frequency of Aedes species was seen in the control village. The Ae. albopictus liked to breed in small habitats (the median water volume of its habitats was 5 L and 10 L in the control and treated village, respectively, while the equivalent values for Ae. aegypti were 30 L and 50 L, respectively). CONCLUSION: The treatment of refillable water storage containers in a rural village with SumiLarv®2MR disks led to significant reductions in the Ae. aegypti population. However, the Ae. albopictus population did not decrease in either the control or treated village. This discrepancy was due to differences in habitat-seeking behaviors and preferred breeding sites such as types of water, water container, and water volume, then led to the differences in results of mosquito prevalence after SumiLarv®2MR disk treatments. The SumiLarv®2MR disk treatment was proven to be effective against the primary dengue-virus vector mosquitoes, Ae. aegypti.

Correction to: Multiple arboviral infections during a DENV-2 outbreak in Solomon Islands.

[This corrects the article DOI: 10.1186/s41182-020-00217-8.].

Multiple arboviral infections during a DENV-2 outbreak in Solomon Islands.

BACKGROUND: Solomon Islands, a country made up of tropical islands, has suffered cyclic dengue fever (DF) outbreaks in the past three decades. An outbreak of dengue-like illness (DLI) that occurred in April 2016 prompted this study, which aimed to determine the population's immunity status and identify the arboviruses circulating in the country. METHODS: A household survey, involving 188 participants in two urban areas (Honiara and Gizo), and a parallel hospital-based clinical survey were conducted in April 2016. The latter was repeated in December after a surge in DLI cases. Arbovirus IgG ELISA were performed on the household blood samples to determine the prevalence of arboviruses in the community, while qPCR testing of the clinical samples was used to identify the circulating arboviruses. Dengue virus (DENV)-positive samples were further characterized by amplifying and sequencing the envelope gene. RESULTS: The overall prevalence rates of DENV, Zika virus, and chikungunya virus were 83.4%, 7.6%, and 0.9%, respectively. The qPCR positivity rates of the clinical samples collected in April 2016 were as follows: DENV 39.6%, Zika virus 16.7%, and chikungunya virus 6.3%, which increased to 74%, 48%, and 20% respectively in December 2016. The displacement of the circulating serotype-3, genotype-1, with DENV serotype 2, genotype cosmopolitan was responsible for the outbreak in 2016. CONCLUSIONS: A DENV outbreak in Solomon Islands was caused by the introduction of a single serotype. The high prevalence of DENV provided transient cross-protection, which prevented the introduction of a new serotype from the hyperendemic region for at least 3 years. The severe outcomes seen in the recent outbreak probably resulted from changes in the causative viruses and the effects of population immunity and changes in the outbreak pattern. Solomon Islands needs to step up surveillance to include molecular tools, increase regional communication, and perform timely interventions.

Dietary Alaska pollock protein alters insulin sensitivity and gut microbiota composition in rats.

Fish protein is not only nutritional but also promotes health by improving insulin sensitivity and hypercholesterolemia. Few studies have examined the relationship between gut microbiota and the enhanced insulin sensitivity due to the intake of Alaska pollock protein (APP). Hence, we assessed the glycolytic enzyme inhibitory activity of APP in in vitro study and the alteration of blood glucose level in insulin tolerance test (ITT) and glucose tolerance test (GTT) and gut microbiota following APP intake in the in vivo study. In initial experiments, the glycolytic enzyme (α-amylase, α-glucosidase, and sucrase) inhibitory activities of APP and its digest were not drastically altered compared with that of casein and its digests. In further experiments, rats fed an AIN-93G diet containing 20% (w/w) casein or APP for 8 weeks, and the composition of fecal microbiota analyzed by 16S rRNA amplicon sequence analysis. In addition, at 6 and 7 weeks of administration of experimental diet, insulin and glucose tolerance tests were evaluated, respectively. Compared with dietary casein, dietary APP has blood glucose-lowering activity as evident in the ITT and GTT. Moreover, APP group altered the structure of fecal microbiota, and area under the curves of the ITT and GTT and the relative abundance of Blautia, which is associated with glucose metabolism, tended to be positively correlated (P = 0.08 and 0.10, respectively). This study illustrates a novel finding that APP intake could alter the composition of gut microbiota and improve insulin sensitivity. PRACTICAL APPLICATION: Studies in animals and humans have shown that Alaska pollock protein (APP) intake improves insulin sensitivity, allowing the body to utilize blood glucose more effectively, thereby keeping blood sugar levels under control. Microorganisms residing in the human gut are associated with glucose metabolism. This study shows that the relative APP intake alters the composition of these gut microorganisms, more than casein intake and therefore might prevent hyperglycemia and type 2 diabetes.

Increased nitric oxide production by T- and B-cells in idiopathic nephrotic syndrome.

The pathogenesis of idiopathic nephrotic syndrome (INS) remains unclear. To study the role of nitric oxide (NO) in INS, we measured intracellular NO produced by T- and B-cells using a novel fluorescent indicator. Twelve children with INS (mean age 7.3 years; group A-1: in relapse, group A-2: in remission) were enrolled in the study together with 16 children with other renal diseases (9.5 years; group B) and 42 healthy control children (7.7 years; group C). The amount of NO produced by CD3+ cells (T-cells) and CD19+ cells (B-cells) and of plasma NO(x) was measured by flow cytometry and colorimetry, respectively. The average amount of NO produced by CD3+ and CD19+ cells in group A-1 subjects was significantly higher than that produced by these cells in group A-2 and B patients and the healthy controls (group C), respectively (P < 0.01), and it decreased after the patients achieved remission (P < 0.01). Plasma NO(x) levels in group A-1 patients was also highest among the different groups (P < 0.01). There were no significant differences in intracellular NO and plasma NO(x) among group A-2, B, and C subjects (P > 0.05). A significant correlation between plasma NO(x) and urinary protein excretion was found only in group A patients and not in group B patients. We conclude that an aberrant immune system may exist not only in T-cells but also in B-cells, and NO may play some role in INS.

Shared expression of mucin12 in Ascaris lumbricoides and the human small intestine.

This study focuses on the host-parasite relationship of human Ascaris lumbricoides, which is a parasite of the small intestine and is also one of the commonest parasites worldwide. As part of this investigation, we examined the host-parasite relationship assuming that there is a common antigenicity, shared protein between A. lumbricoides and human small intestinal mucosa, using molecular techniques. We obtained three DNA clones from human colon cDNA library by screening for anti-A. lumbricoides polyclonal antibodies. The transmembrane mucin12 gene was identified after sequencing analysis of these clones. Specific signals of immunostaining with polyclonal anti-mucin12 antibodies were observed in the mucous secretory organs, epidermis, and intestinal canal of A. lumbricoides. These signals disappeared when immunohistochemistry was performed using pre-absorbed polyclonal antibodies with a specific peptide. These results suggest that mucin12 is localized in the mucous secretory organs in the epidermis of A. lumbricoides. Furthermore, we examined the site of mucin12 localization in the host; specific mucin12 signals were observed on the mucosal epithelia present around intestinal crypts and villi of the small intestine. Therefore, we suggest that mucin12 is a protein that shows common antigenicity in both A. lumbricoides and its host. It is presumed that adult A. lumbricoides live in their preferred environment, which is the small intestine, by secreting mucin12 to avoid being attacked by the host immune system.