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Currently, four kinds of phosphorodiamidate morpholino oligomers (PMOs), such as viltolarsen, have been approved for the treatment of Duchenne muscular dystrophy (DMD); however, it is unclear whether human efficacy can be estimated using plasma concentrations. This study summarizes the tissue distribution of viltolarsen in mice and cynomolgus monkeys and evaluates the relationship between exposure and efficacy based on exon skipping. In the tissue distribution studies, all muscles in DMD-model mice showed higher concentrations of viltolarsen than those in wild-type mice and cynomolgus monkeys, and the concentrations in skeletal muscle were correlated with the exon-skipping efficiency in mice and cynomolgus monkeys. In addition, a highly sensitive bioanalytical method using liquid chromatography with tandem mass spectrometry shows promise for determining plasma concentrations up to a later time point, and the tissue (muscle)/plasma concentration ratio (Kp) in DMD-model mice was shown to be useful for predicting changes in pharmacodynamic (PD) markers in humans. Our results suggest that pharmacokinetic (PK)/PD analysis can be conducted by using the human PK profile or Kp values and skipping efficiency in DMD-model mice. This information will be useful for the efficient and effective development of PMOs as therapeutic agents. SIGNIFICANCE STATEMENT: We evaluated the relationship between the plasma or tissue concentrations and the efficiency of exon skipping for viltolarsen as an example phosphorodiamidate morpholino oligomers in the skeletal and cardiac muscle of mice and cynomolgus monkeys for pharmacokinetic/pharmacodynamic (PK/PD) analysis. The results suggest that PK/PD analysis can be conducted by using the human PK profile or tissue (muscle)/plasma concentration ratios and skipping efficiency in DMD-model mice., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Human pregnane X receptor (PXR) is a major nuclear receptor that upregulates the expression of drug-metabolizing enzymes such as CYP3A4. In our recent study, it was revealed that PXR interacts with DAZ-associated protein 1 (DAZAP1), which is an essential component of the paraspeckle, a membraneless nuclear body, and the interaction was disassociated by rifampicin, a ligand of PXR. The purpose of this study was to clarify the roles of paraspeckles in PXR-mediated transcriptional regulation. Immunoprecipitation assays using PXR-overexpressing HepG2 (ShP51) cells revealed that PXR interacts with not only DAZAP1 but also NEAT1_2, a long noncoding RNA included in the paraspeckle, and that the interaction between PXR and NEAT1_2 was disassociated by rifampicin. These results suggest that PXR is trapped in paraspeckles and that the activation of PXR by its ligands facilitates its disassociation from paraspeckles. Induction of CYP3A4 by rifampicin was significantly enhanced by the knockdown of NEAT1_2 or DAZAP1 in ShP51 cells and their parental HepG2 cells. A luciferase assay using a plasmid containing the PXR response elements of CYP3A4 revealed that the increased CYP3A4 induction by siNEAT1_2 or siDAZAP1 was due to the increased transactivation by PXR. These results suggest that paraspeckles play a role in trapping nuclear PXR in the absence of the ligand to negatively regulate transactivation of its downstream gene. Collectively, this is the first study to demonstrate that the paraspeckle components NEAT1_2 and DAZAP1 negatively regulate CYP3A4 induction by PXR. SIGNIFICANCE STATEMENT: This study revealed that PXR interacts with paraspeckle components NEAT1_2 and DAZAP1 to suppress CYP3A4 induction by PXR, and the interaction is dissociated by PXR ligands. This finding provides a novel concept that paraspeckles formed by liquid-liquid phase separation potentially affect drug metabolism via negative regulation of PXR function., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Postprandial hyperglycemia is a known risk factor for the development of several health disorders, including type 2 diabetes, obesity and cardiovascular disease. This study aimed to investigate the acute effect of Undaria pinnatifida (Wakame), a discriminative constituent of the Japanese diet, on postprandial blood glucose and insulin levels. The study was conducted using a crossover method among 26 subjects. Blood was sampled for glucose and insulin measurements at 0, 30, 60, 90 and 120 min after a subject consumed either 200 g of rice or 200 g rice with 4 g of dried wakame. Blood glucose and insulin levels were significantly lower at 30 min after consuming rice with wakame than after consuming rice alone. Moreover, the incremental areas under the curves for glucose and insulin were lower when wakame was included. Wakame intake can improve postprandial glucose homeostasis. Wakame intake may offer a simple behavioural strategy that can reduce glycemic excursions in prediabetes. This study was registered with the UMIN Clinical Trial Registry (UMIN000031050).

A simple microfluidic system has been presented to perform continuous two-parameter cell sorting based on size and surface markers. Immunomagnetic bead-conjugated cells are initially sorted based on size by utilizing the hydrodynamic filtration (HDF) scheme, introduced into individual separation lanes, and simultaneously focused onto one sidewall by the hydrodynamic effect. Cells are then subjected to magnetophoretic separation in the lateral direction, and finally they are individually recovered through multiple outlet branches. We successfully demonstrated the continuous sorting of JM (human lymphocyte cell line) cells using anti-CD4 immunomagnetic beads and confirmed that accurate size- and surface marker-based sorting was achieved. In addition, the sorting of cell mixtures was performed at purification ratios higher than 90%. The proposed system enables two-dimensional cell sorting without necessitating complicated setups and operations, and thus, it can be a useful tool for general biological experiments including cell-based disease diagnosis, stem cell engineering, and cellular physiological studies.

Type-1 immunity plays a crucial role in host defense against various tumors and infectious diseases. Here, we first demonstrated that extract of Larix Leptolepis (ELL), one of the most popular timbers at Hokkaido area in Japan, strongly activated Type-1 immunity. ELL induced production of Type-1 cytokines such as IL-12 and TNF-α from bone marrow-derived dendritic cells (BMDCs) in TLR2- and TLR4-dependent manner and remarkably up-regulated the expression of MHC and co-stimulatory molecules. In addition, antigen-specific CTLs were significantly augmented by the combined administration of ELL, antigen and BMDCs. Finally, we revealed that combination therapy using ELL, antigen and BMDCs significantly inhibited the growth of established tumor in mouse model. Thus, these findings suggested that ELL would be a novel adjuvant for inducing an activation of Type-1-dependent immunity including activation of BMDCs and induction of tumor-specific CTLs, which is applicable to the therapy of cancer and infectious diseases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Type-1 immunity has an essential role for our host defenses against cancer and outer pathogens such as bacteria and virus. We demonstrated here that the edible plant extract of Chrysanthemum coronarium L. (C. coronarium) remarkably activates Type-1 immunity in a Toll-like receptor (TLR)2-, TLR4-, and TLR9-dependent manner. In the present experiments, the extract of C. coronarium significantly induces interferon (IFN)-γ production by mouse spleen cells. In addition, the IFN-γ production by spleen cells was completely blocked by the addition of anti-Interleukin (IL)-12 monoclonal antibodies. We confirmed that NK1.1(+) natural killer (NK) cells, NKT cells, and CD11c(+) dendritic cells (DC) were immediately activated after the stimulation with the extract of C. coronarium and the IFN-γ production was abolished in NK1.1(+) cell-depleted spleen cells. The stimulation with the extract of C. coronarium caused DC maturation involving with up-regulations of surface expression levels of MHC class I, MHC class II, CD40, and CD86 as well as induction of IL-12 production. The IFN-γ production induced by the extract was significantly reduced in the spleen cells depleted CD11c(+) cells. Furthermore, the IFN-γ production after the stimulation was strongly reduced in TLR4- and partially in TLR2- and TLR9-deficient spleen cells. Thus, we demonstrated the cellular mechanism for the activation of Type-1 immunity via NK cells, NKT cells, and DC by the extract of C. coronarium. These findings strongly suggest that C. coronarium would be a promising immuno-improving adjuvant, which might be useful for prevention of infectious, cancer, and allergic diseases through the activation of Type-1 immunity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

During the search for immuno-improving foods, we found that a variety of the Japanese soybean, Glycine max cv. Kurosengoku (Kurosengoku), which activated Type-1 immunity in a Toll-like receptor (TLR)4- and TLR2-dependent manner. Namely, the extract of Kurosengoku first caused production of IL-12 from DC and sequentially induced IFN-γ production by NK1.1(+) NK cells and NKT cells. The IFN-γ production was significantly blocked by neutralizing mAb against IL-12 or TLR4- and TLR2-deficient condition, indicating that TLR4- and TLR2-dependent activation of DC to produce IL-12 was essential for the production of IFN-γ from spleen cells by Kurosengoku. Moreover, the extract of Kurosengoku also enhanced production of IFN-γ from human PBMC by co-stimulation with anti-CD3 mAb in a TLR2- and TLR4-dependent manner. Thus, our findings strongly suggest that Kurosengoku might a novel immuno-improving food, which would be a useful tool for preventing the tip of immune balance in developed countries., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Activation of type 1 immunity plays a critical role in host defense mechanisms against infectious disease and tumor. Lactic acid bacteria, existing in the gastrointestinal tract, are one of the powerful tools to induce a type-1-dominant immunity, which may improve Th2-dependent allergic diseases. In the present work, we found that an oral intake of Lactobacillus pentosus strain, S-PT84 into mice significantly enhanced NK activity of spleen cells in vivo. We further revealed that NK1.1 positive NK cells and NKT cells are responsible cells for producing IFN-gamma after stimulation with S-PT84 in vitro. S-PT84 induced IFN-gamma-producing cells through activation of IL-12 production by CD11c(+)DCs in Toll-like receptor (TLR) 2- and/or TLR4-dependent manner. Interestingly, direct interaction between DCs and NK1.1(+) cells was also essential for the IFN-gamma production in response to the S-PT84 stimulation. Therefore, we concluded that S-PT84 effectively promoted type 1 immunity through IL-12 and IFN-gamma which were produced by DCs and NK1.1(+) cells, respectively. Thus, S-PT84 would be a nice immune modulator for improving immunobalance, which plays a pivotal role for controlling allergy, infectious diseases and tumor.

Background: Vitamin D (VD) and estrogen deficiencies impair Ca absorption and bone mineralization, and the relevance of the interaction between these factors has not been fully understood., Aim of the Study: The aim of the present study was to clarify the effects of a nondigestible saccharide, difructose anhydride III (DFAIII), on the interaction of VD and estrogen deficiencies involved in Ca malabsorption by assessing changes in intestinal Ca absorption and bone mineralization by feeding of DFAIII in rats with VD or estrogen deficiency or with a combined loss of VD and estrogen., Methods: Three-week-old female Sprague-Dawley rats were divided into four groups: two groups were ovariectomized (OVX) and two were laparotomized (sham). One group each of OVX and sham rats were fed an AIN93G-based normal diet, and the other groups were fed a VD-deficient diet for 8-weeks. Rats from the four groups were divided into two subgroups and fed the normal or VD-deficient diet with or without DFAIII for next 4-weeks., Results: VD deficiency decreased Ca absorption and bone mineralization with reductions in duodenal calbindin D9k mRNA and serum Ca levels. There were no additional reductions in these parameters in the OVX. The reductions in Ca absorption and femoral Ca were restored partially or fully by DFAIII. Recovery of Ca absorption rate by DFAIII was greater in the OVX than in the sham showing an interaction between OVX and VD deficiency in, at least, the DFAIII-fed groups. The cecal pH was lower and the level of short-chain fatty acids in the cecal contents was higher in all the DFAIII groups than those in the control groups., Conclusions: VD deficiency impaired Ca absorption and bone mineralization, and feeding DFAIII partially restored Ca malabsorption and fully recovered bone Ca in VD-deficient rats. No additional reductions in these parameters with a combination of VD deficiency and OVX were noted. However, interactions were found between these factors in the DFAIII-induced increase in Ca absorption.

This study demonstrates that feeding difructose anhydride III (DFAIII) improves bone strength and femoral mineral concentrations in a rat model of oestrogen deficiency. We showed the relationship between Ca, Mg and P absorption and bone characteristics in rats. Two groups of female Sprague-Dawley rats (6 weeks old) underwent bilateral ovariectomy (ovariectomized rats, OVX rats) or bilateral laparotomy (sham rats). At 10 weeks old, OVX and sham rats were divided into three subgroups and fed a control, 1.5 % DFAIII or 3 % DFAIII diet for 8 weeks, respectively. Ca but not Mg absorption rates were lowered by ovariectomy; however, ingestion of the 1.5 % and 3 % DFAIII diets similarly restored the reduced Ca absorption in OVX rats at 4 and 8 weeks after feeding of the test diets. DFAIII increased Mg absorption dose-dependently in sham and OVX rats. The bone strength, femoral Ca and Mg concentrations, and distal bone mineral density in the 3 % DFAIII group were higher than those in the control group in OVX rats. The absorption rates of Ca and Mg were significantly correlated with femoral Ca and Mg concentrations and strength, which suggests that increasing both Ca and Mg absorption improves bone characteristics in OVX rats. There were no differences in any of the variables in the femur between the 1.5 % and 3 % DFAIII groups in OVX rats. In conclusion, feeding of a low dose of DFAIII increased intestinal Ca and Mg absorption, and the promotive effect of DFAIII persisted for over 8 weeks. This effect was associated with prevention of ovariectomy-induced osteopenia.

We examined the effects of feeding raffinose on intestinal calcium absorption in ovariectomized rats by two separate experiments. In experiment 1, female Sprague-Dawley rats (6 wk old) were divided into two groups: sham operation and ovariectomy, and fed diets with or without raffinose (30 g/kg diet) for 4 wk. In experiment 2, ovariectomized rats with cecocolonectomy or transsection and reanastomosis (sham) were divided into two groups as in experiment 1 and fed the same diets for 3 wk. In experiment 1, calcium absorption was lower in the ovariectomized rats than in the sham rats but calcium absorption in rats fed the raffinose diet was higher than that in rats fed the raffinose-free diet. In experiment 2, increased calcium absorption in the raffinose group was abolished by cecocolonectomy. The impaired absorption in ovariectomized rats was restored by feeding raffinose. The large intestine is involved in the beneficial effects of raffinose.

This study examined the effects of feeding water-soluble soybean fiber (WSSF), a highly fermentable dietary fiber with low viscosity, on intestinal calcium (Ca) absorption, bone mineral content, and serum total cholesterol levels using female Sprague-Dawley rats (5 weeks old) with or without ovariectomy (OVX). The rats in each group were fed a WSSF (50 g/kg of diet) or WSSF-free diet for 4 weeks. Ca absorption was higher in OVX rats fed the WSSF diet than in OVX rats fed the WSSF-free diet. Femoral Ca content in both sham and OVX rats fed the WSSF diet was higher than that in rats fed the WSSF-free diet. The serum total cholesterol levels were elevated after OVX compared with that in the sham rats, and this elevation was prevented by the WSSF diet. In conclusion, ingestion of WSSF prevented osteopenia and hypercholesterolemia induced by ovariectomy in rats.

In three separate experiments, we examined the effects of feeding difructose anhydride III (DFAIII) on intestinal calcium (Ca) absorption using female Sprague-Dawley rats (6 wk old) with or without ovariectomy (OVX). In Experiment 1, we showed that Ca absorption was lower in OVX rats fed the 2.0 and 3.0 g Ca/kg diets, but not the 1.0 g Ca/kg diet, than in sham-operated rats during a 3-wk test period. In Experiment 2, we demonstrated that Ca absorption rate in sham and OVX rats fed a diet containing 3% DFAIII was higher than that in rats fed a DFAIII-free diet 4 wk after consuming the test diets. Absorptive activities of everted sacs of the colon, but not of the duodenum, in rats fed DFAIII diet for 4 wk were higher than those in rats fed the control diet. In Experiment 3, we determined which of the small and large intestines is responsible for the effects of DFAIII on Ca absorption using OVX rats with cecocolonectomy or transsection and reanastomosis (sham). Both the sham and cecocolonectomized rats were divided into four subgroups and fed a control, polyethylene glycol (PEG), 1.5% DFAIII or 3% DFAIII diet. We set up the PEG group as another control group to observe the effects of shortening transit time of the small intestine in the DFAIII groups. Promotive effects of DFAIII on Ca absorption were abolished by cecocolonectomy. However, in the cecocolonectomized rats, the Ca absorption rate was still higher in the 1.5 and 3% DFAIII groups than in the PEG group. In conclusion, ovarian hormone deficiency impaired Ca absorption, but the reduction of Ca absorption was restored by feeding DFAIII.

To investigate hormonal changes before the onset of female puberty, we measured LH and FSH in serum samples drawn every 20 min for 24 h and measured testosterone and estradiol hourly for 24 h. Seventeen girls (13 prepubertal and 4 early pubertal) of short stature, from 5.1-11.4 yr of age, participated in this study. LH and FSH were measured using a time-resolved immunofluorometric assay, and testosterone and estradiol were measured using a sensitivity RIA capable of detecting testosterone and estradiol concentrations of 10 and 2 pg/mL, respectively. Diurnal rhythms of LH, FSH, and testosterone were apparent in all subjects, including those aged 5-6 yr. Serum LH and FSH concentrations showed night-day variation in a pulsatile fashion. The serum testosterone concentration was elevated in the early morning in all subjects. The serum estradiol concentration was elevated in the early morning in 4 of 13 prepubertal subjects and all 4 early pubertal subjects. The diurnal pattern of the serum estradiol concentration was similar to that of the serum testosterone concentration. Mean 24-h LH and testosterone concentrations in prepubertal subjects who did not attain puberty for at least 1 yr were 0.07 U/L and 65 pg/mL, respectively, whereas those in prepubertal subjects who attained puberty within 1 yr (0.14 U/L and 106 pg/mL, respectively) were significantly higher. Furthermore, mean 24-h LH, FSH, testosterone, and estradiol concentrations increased with the onset of puberty. In conclusion, the diurnal rhythms of LH, FSH, and testosterone already exist at 5-6 yr of age, and serum LH and testosterone levels increase before the onset of puberty. These results suggest that preparation for the onset of female puberty may begin in 5- to 6-yr-old girls.

To investigate hormonal change before the onset of male puberty, we measured LH and FSH in serum samples drawn every 20 min for 24 h and measured testosterone hourly for 24 h. Forty-six boys (32 prepubertal and 14 pubertal) of short stature, between 4.4-19.3 yr of age, participated in this study. LH and FSH were measured using a time-resolved immunofluorometric assay, and testosterone was measured using high sensitivity RIA capable of detecting a testosterone concentration of 0.01 ng/mL. Diurnal rhythms of LH, FSH, and testosterone were apparent in all subjects, including those aged 4-5 yr. Serum LH and FSH concentrations showed night-day variation in a pulsatile fashion. The serum testosterone concentration was elevated at early morning in all subjects. Mean 24-h LH, FSH, and testosterone concentrations of prepubertal subjects who did not attain puberty for at least 3 yr were 0.10 U/L, 0.63 U/L, and 0.06 ng/mL, respectively, whereas those of prepubertal subjects who attained puberty within 1 yr (0.54 U/L, 1.68 U/L, and 0.10 ng/mL, respectively) were significantly higher. Furthermore, mean 24-h LH, FSH, and testosterone concentrations increased with developing puberty. All of the 46 subjects showed positive cross-correlation between the LH and testosterone time series. The mean lag time from the LH to the testosterone time series in the prepubertal subjects who attained puberty within 1 yr (4.7 +/- 2.4 h, mean +/- SD) was shorter than that in the prepubertal subjects who attained puberty after at least 3 yr (7.3 +/- 2.2 h). This lag time decreased with developing puberty, plateauing at 1.4 +/- 0.9 h at midpuberty. Thus, the diurnal rhythms of LH, FSH, and testosterone already exist at 4-5 yr of age; serum LH, FSH, and testosterone levels increase before the onset of puberty; and a time delay is observed between the LH and testosterone time series that decreases before the onset of puberty.

In an infant with transient neonatal diabetes mellitus, control of the blood glucose concentration was attained with ultralente insulin treatment, without any episodes of hypoglycemia. We recommend subcutaneous injection of ultralente insulin, rather than lente or isophane (NPH) insulin, to avoid hypoglycemia during the treatment of transient neonatal diabetes mellitus.

A 15-year-old girl is described with encephalopathy associated with Hashimoto thyroiditis which subsequently developed into autoimmune thyrotoxicosis (hashitoxicosis) and distal renal tubular acidosis at age 5 years, pernicious anemia at age 9, and encephalopathy at age 12. Thyroid hormone levels were within the normal ranges at the time of neurologic presentation. Serum IgG concentration and oligoclonal IgG bands in cerebrospinal fluid closely correlated with the severity of neurologic symptoms. Treatment with glucocorticoids improved the level of consciousness but her mental state continued to deteriorate. Repeated cranial computed tomographic scans revealed progressive symmetric atrophy in the gray matter without infarction. These findings suggest that encephalopathy associated with Hashimoto thyroiditis is caused by an antineuronal antibody-mediated reaction.

The growth hormone (GH) secretory capacities in patients with Turner syndrome aged 5.1-15.9 years and those with constitutional short stature (CSS) aged 5.2-14.2 years were evaluated by pharmacological and physiological means. The GH response to hypoglycemia in the patients with Turner syndrome was lower than that in the patients with CSS. However, the GH response to arginine was not significantly different between the two patient groups. For the physiological test, the integrated concentration of GH (ICGH), the number of episodic peaks and their mean height were evaluated using blood obtained from the patients every 20 minutes for a period of 24 hours. The ICGH and the mean height of the episodic peaks in the patients with Turner syndrome were significantly lower than those in the CSS patients during the night but not during the day. Negative correlation between the bone age and the night-time values of ICGH was observed in the patients with Turner syndrome. Such correlation was not observed in the CSS patients. The patients with CSS showed a significant day-night difference in the ICGH and the mean height of the episodic peaks, but the patients with Turner syndrome did not show any significant day-night difference in either the ICGH or the mean height of episodic peaks. In conclusion, the GH secretory capacity in patients with Turner syndrome is lower than that in CSS patients.

We have attempted to facilitate differential diagnosis of etiological types of congenital hypothyroidism using real-time ultrasonography. Sonography of the thyroid gland was performed on 418 normal children, and 23 patients with congenital hypothyroidism and hyperthyrotropinemia. The thyroid gland was imaged by transverse scanning at the neck; the maximum thickness and the maximum width of the right and left lobe were measured. On the basis of the normal thyroid gland size obtained from normal children, the thyroid gland image of the patients were classified into 4 types: large image, normal image, small image, and no image of the thyroid gland at the neck: no image of the thyroid gland indicated agenesis or ectopia; large thyroid gland image indicated goitorous hypothyroidism. On the other hand, normal or small thyroid gland image probably included mild or transient forms of hypothyroidism, and transient hyperthyrotropinemia; these 2 types required further examination to complete the diagnosis. We concluded that real-time ultrasonography of the thyroid gland was a useful diagnostic imaging technic for patients who revealed elevated serum thyrotropin on neonatal mass-screening.