Butyrate, Valerate, and Niacin Ameliorate Anaphylaxis by Suppressing IgE-Dependent Mast Cell Activation: Roles of GPR109A, PGE2, and Epigenetic Regulation.

Short-chain fatty acids (SCFAs) are produced by the intestinal microbiota during the fermentation of dietary fibers as secondary metabolites. Several recent studies reported that SCFAs modulate the development and function of immune-related cells. However, the molecular mechanisms by which SCFAs regulate mast cells (MCs) remain unclear. In the current study, we analyzed the function and gene expression of mouse MCs in the presence of SCFAs in vitro and in vivo. We found that the oral administration of valerate or butyrate ameliorated passive systemic anaphylaxis and passive cutaneous anaphylaxis in mice. The majority of SCFAs, particularly propionate, butyrate, valerate, and isovalerate, suppressed the IgE-mediated degranulation of bone marrow-derived MCs, which were eliminated by the Gi protein inhibitor pertussis toxin and by the knockdown of Gpr109a. A treatment with the HDAC inhibitor trichostatin A also suppressed IgE-mediated MC activation and reduced the surface expression level of FcεRI on MCs. Acetylsalicylic acid and indomethacin attenuated the suppressive effects of SCFAs on degranulation. The degranulation degree was significantly reduced by PGE2 but not by PGD2. Furthermore, SCFAs enhanced PGE2 release from stimulated MCs. The SCFA-mediated amelioration of anaphylaxis was exacerbated by COX inhibitors and an EP3 antagonist, but not by an EP4 antagonist. The administration of niacin, a ligand of GPR109A, alleviated the symptoms of passive cutaneous anaphylaxis, which was inhibited by cyclooxygenase inhibitors and the EP3 antagonist. We conclude that SCFAs suppress IgE-mediated activation of MCs in vivo and in vitro involving GPR109A, PGE2, and epigenetic regulation.

Cooperative Regulation of the Mucosal Mast Cell-Specific Protease Genes Mcpt1 and Mcpt2 by GATA and Smad Transcription Factors.

Mouse mast cell proteases (mMCP)-1 and -2 are specifically expressed in mucosal mast cells (MCs). However, the transcriptional regulation mechanism of the Mcpt1 and Mcpt2 genes induced in mucosal MCs is largely unknown. In the current study, we found that TGF-ß stimulation drastically induced upregulation of Mcpt1 and Mcpt2 mRNA in mouse bone marrow-derived MCs (BMMCs). TGF-ß-induced expression of Mcpt1 and Mcpt2 was markedly suppressed by transfection with small interfering RNA targeting Smad2 or Smad4 and moderately reduced by Smad3 small interfering RNA. We next examined the roles of the hematopoietic cell-specific transcription factors GATA1 and GATA2 in the expression of Mcpt1 and Mcpt2 and demonstrated that knockdown of GATA1 and GATA2 reduced the mRNA levels of Mcpt1 and Mcpt2 in BMMCs. The recruitment of GATA2 and acetylation of histone H4 of the highly conserved GATA-Smad motifs, which were localized in the distal regions of the Mcpt1 and Mcpt2 genes, were markedly increased by TGF-ß stimulation, whereas the level of GATA2 binding to the proximal GATA motif was not affected by TGF-ß. A reporter assay showed that TGF-ß stimulation upregulated GATA2-mediated transactivation activity in a GATA-Smad motif-dependent manner. We also observed that GATA2 and Smad4 interacted in TGF-ß-stimulated BMMCs via immunoprecipitation and Western blotting analysis. Taken together, these results demonstrate that TGF-ß induced mMCP-1 and -2 expression by accelerating the recruitment of GATA2 to the proximal regions of the Mcpt1 and Mcpt2 genes in mucosal MCs.

Suppressive role of PPARγ in the IgE-dependent activation of mast cells.

Mast cells (MCs) play a central role in IgE-dependent immune responses. PPARγ is a nuclear receptor that is essential for adipocyte differentiation and insulin sensitivity. Although PPARγ is expressed in activated MCs, the effect of PPARγ suppression in IgE-mediated activation of MCs is largely unknown. In the current study, we evaluated the effect of PPARγ knockdown on the function of IgE plus antigen (Ag)-stimulated MCs using siRNA-transfected bone marrow-derived MCs (BMMCs). We found that the mRNA expression level of cytokines in IgE/Ag-stimulated BMMCs was significantly increased in PPARγ knockdown BMMCs, and IgE/Ag-mediated degranulation and the protein production level of TNF-α was moderately increased by PPARγ knockdown, whereas the cell surface expression level of FcεRI was not affected by PPARγ knockdown. Oral administration of pioglitazone (PPARγ agonist) significantly suppressed body temperature change of mice in passive systemic anaphylaxis, supporting the inhibitory functions of PPARγ in IgE/Ag-dependent activation of MCs in vivo. IgE-mediated up-regulation of mRNA levels of Ptgs2 (encoding COX-2) was drastically enhanced in PPARγ knockdown BMMCs. Although several prostaglandin (PG) derivatives are known to be ligands for PPARγ, treatment with a COX inhibitor, acetyl salicylic acid, up-regulated the IgE-mediated increase of Il13, Tnf and Ptgs2 mRNA levels in a synergistic manner with PPARγ siRNA. Knockdown of COX-1 and/or COX-2 by siRNA showed that suppression of IgE/Ag-mediated activation was mainly dependent on COX-1. Taken together, these results indicate that PPARγ suppresses IgE/Ag-induced transactivation of cytokine genes and the Ptgs2 gene in MCs in a manner distinguishable from that of PGs.

Blockade of TNF receptor superfamily 1 (TNFR1)-dependent and TNFR1-independent cell death is crucial for normal epidermal differentiation.

BACKGROUND: A delicate balance between cell death and keratinocyte proliferation is crucial for normal skin development. Previous studies have reported that cellular FLICE (FADD-like ICE)-inhibitory protein plays a crucial role in prevention of keratinocytes from TNF-α-dependent apoptosis and blocking of dermatitis. However, a role for cellular FLICE-inhibitory protein in TNF-α-independent cell death remains unclear. OBJECTIVE: We investigated contribution of TNF-α-dependent and TNF-α-independent signals to the development of dermatitis in epidermis-specific Cflar-deficient (CflarE-KO) mice. METHODS: We examined the histology and expression of epidermal differentiation markers and inflammatory cytokines in the skin of CflarE-KO;Tnfrsf1a+/- and CflarE-KO;Tnfrsf1a-/- mice. Mice were treated with neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand to block TNF-α-independent cell death of CflarE-KO;Tnfrsf1a-/- mice. RESULTS: CflarE-KO;Tnfrsf1a-/- mice were born but experienced severe dermatitis and succumbed soon after birth. CflarE-KO;Tnfrsf1a+/- mice exhibited embryonic lethality caused by massive keratinocyte apoptosis. Although keratinocytes from CflarE-KO;Tnfrsf1a-/- mice still died of apoptosis, neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand substantially prolonged survival of CflarE-KO;Tnfrsf1a-/- mice. Expression of inflammatory cytokines, such as Il6 and Il17a was increased; conversely, expression of epidermal differentiation markers was severely downregulated in the skin of CflarE-KO;Tnfrsf1a-/- mice. Treatment of primary keratinocytes with IL-6 and, to a lesser extent, IL-17A suppressed expression of epidermal differentiation markers. CONCLUSION: TNF receptor superfamily 1 (TNFR1)-dependent or TNFR1-independent apoptosis of keratinocytes promotes inflammatory cytokine production, which subsequently blocks epidermal differentiation. Thus blockade of both TNFR1-dependent and TNFR1-independent cell death might be an alternative strategy to treat skin diseases when treatment with anti-TNF-α antibody alone is not sufficient.

Critical Contribution of Nuclear Factor Erythroid 2-related Factor 2 (NRF2) to Electrophile-induced Interleukin-11 Production.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays a crucial role in protection of cells from electrophile-induced toxicity through up-regulating phase II detoxifying enzymes and phase III transporters. We previously reported that oxidative stress induces up-regulation of interleukin-11 (IL-11), a member of the IL-6 family that ameliorates acetaminophen-induced liver toxicity. However, a role for IL-11 in protection of cells from electrophile-induced toxicity remains unclear. Here we show that an environmental electrophile, 1,2-naphthoquinone (1,2-NQ), but not 15d-prostaglandin J2 (PGJ2) or tert-butylhydroxyquinone (tBHQ), induced IL-11 production. Consistent with a crucial role for prolonged ERK activation in H2O2-induced IL-11 production, 1,2-NQ, but not 15d-PGJ2 or tBHQ, elicited prolonged ERK activation. Conversely, inhibition of the ERK pathway by a MEK inhibitor completely blocked 1,2-NQ-induced IL-11 production at both protein and mRNA levels, further substantiating an intimate cross-talk between ERK activation and 1,2-NQ-induced IL-11 production. Promoter analysis of the Il11 gene revealed that two AP-1 sites were essential for 1,2-NQ-induced promoter activities. Among various members of the AP-1 family, Fra-1 was up-regulated by 1,2-NQ, and its up-regulation was blocked by a MEK inhibitor. Although NRF2 was not required for H2O2-induced IL11 up-regulation, NRF2 was essential for 1,2-NQ-induced IL11 up-regulation by increasing Fra-1 proteins possibly through promoting mRNA translation of FOSL1 Finally, intraperitoneal administration of 1,2-NQ induced body weight loss in wild-type mice, which was further exacerbated in Il11ra1-/- mice compared with Il11ra1+/- mice. Together, both Fra-1 and NRF2 play crucial roles in IL-11 production that protects cells from 1,2-NQ intestinal toxicity.

Anti-tumor activity of heat-killed Lactobacillus plantarum BF-LP284 on Meth-A tumor cells in BALB/c mice.

Probiotics exert numerous effects on human well-being. Here, heat-killed Lactobacillus plantarum BF-LP284 (H-Lp) was isolated as a potent immuno-modulator among 15 strains of lactobacilli in terms of TNF-α induction ability in peritoneal macrophages. In vitro TNF-α and IFN-γ induction in Peyer's patch (PP) cells was higher when incubated with H-Lp than with live L. plantarum BF-LP284 (L-Lp). Suppression of syngeneic Meth-A tumors in a murine model by oral administration of H-Lp was also greater than that of L-Lp and of controls. H-Lp stimulated IFN-γ production in spleen cells, which displayed inhibited tumor growth in Winn assays when treated with H-Lp. Moreover, H-Lp increased the ratio of CD3(+ )cells among peripheral blood mononuclear cells in Meth-A tumor-bearing mice, suggesting an H-Lp-mediated anti-tumor mechanism whereby immune cells that are activated by H-Lp in PP and acquire anti-tumor activity in the spleen migrate to tumor sites through lymphocyte homing to inhibit tumor growth.

Involvement of PU.1 in NFATc1 promoter function in osteoclast development.

BACKGROUND: The transcription factors NFATc1 and PU.1 play important roles in osteoclast development. NFATc1 and PU.1 transactivate osteoclast-specific gene expression and a deficiency in NFATc1 or PU.1 genes causes osteopetrosis due to an insufficient development of osteoclasts. However, the existence of cross-regulation between NFATc1 and PU.1 is largely unknown. In the present study, the role of PU.1 in NFATc1 expression was investigated. METHODS: Osteoclasts were generated from mouse bone marrow cells. PU.1 knockdown was performed with siRNA introduction. The mRNA levels in siRNA-introduced cells were determined by quantitative RT-PCR. The involvement of PU.1 in the NFATc1 promoter was analyzed by using a chromatin immunoprecipitation (ChIP) assay and a reporter assay. Retrovirus vector was used for enforced expression of PU.1. RESULTS: Introduction of PU.1 siRNA into bone marrow-derived osteoclasts resulted in a decrease in NFATc1 mRNA level. A ChIP assay showed that PU.1 bound to the NFATc1 promoter in osteoclasts. NFATc1 promoter activity was reduced in PU.1 knockdown cells as assessed by a reporter assay. PU.1 siRNA introduction also downregulated the expression of osteoclast-specific genes and tartrate resistant acid phosphatase (TRAP) activity. Enforced expression of PU.1 using a retrovirus vector increased NFATc1 expression and TRAP activity. When NFATc1 expression was knocked down by using siRNA, the induction of osteoclast-specific genes and TRAP-positive cells was suppressed without affecting the expression level of PU.1. CONCLUSIONS: These results indicate that PU.1 is involved in osteoclast development by transactivating NFATc1 expression via direct binding to the NFATc1 promoter.

Methodologic comparison of left ventricular stroke volumes in the early neonatal period by echocardiography.

Several methods for evaluating left ventricular stroke volume (SV) in neonates using echocardiography have been reported. However, no studies on methodologic comparison of SV with three-dimensional (3D) echocardiography are available. This is the first detailed report on a methodologic comparison of SV in the early neonatal period. The study group included 70 normal neonates (35 boys and 35 girls). An iE33 echocardiograph and Q-LAB supplied by Philips Electronics were used to examine and calculate volumes. Comparisons of SV were performed using Teichholz (T), the velocity time integral (VTI), Pombo (P), modified Simpson (MS), and 3D methods with normal neonates who had no persistent ductus arteriosus less than 7 days after birth. The mean SVs were 33.7 mL/m(2) (T), 30.6 mL/m(2) (VTI), 22.0 mL/m(2) (P), 17.5 mL/m(2) (3D), and 14.9 mL/m(2) (MS) using Haycock's formula of body surface area. The stroke volumes differed significantly depending on the different methods. The correlation coefficient was highest between the MS and 3D methods. The SVs of the T and VTI methods were significantly greater than those previously reported, and it seemed inappropriate to evaluate volumes in neonates. The 3D and MS methods were appropriate for measuring SV in neonates during the early neonatal period.

Trends in Anti-Influenza Drug Prescription and Adverse Drug Reaction Reporting After the Lifting of Oseltamivir Prescribing Restrictions in Pediatric Outpatients: An Ecological Study Using the MDV Analyzer® And the Japanese Adverse Drug Event Report Database.

BACKGROUND: Abnormal behavior after oseltamivir administration has been reported in the media; in 2007, the package insert for oseltamivir phosphate was revised to restrict its administration to individuals aged over 10 years. However, in 2018, the age limitation specified in the package insert was removed. Here, we evaluated the trends in anti-influenza drug prescription and adverse drug reactions (ADRs) reported in pediatric outpatients after revising the oseltamivir package insert as an ecological study. METHODS: Anti-influenza drug prescriptions for pediatric outpatients with influenza aged 0-19 years were downloaded from the acute Diagnosis Procedure Combination hospital databases using the MDV analyzer®. ADR reports on anti-influenza drug prescription among patients aged 0-20 years in the Japanese Adverse Drug Event Report database were downloaded from the Pharmaceutical and Medical Devices Agency website. Data were collected during the 2016/2017 and 2019/2020 influenza seasons. RESULTS: During the influenza epidemic season (January-March), the percentage of oseltamivir prescriptions for patients with influenza aged 10-19 years tripled after the revision of the oseltamivir package insert (9.3% during the 2016/2017 season and 29.2% during the 2019/2020 season); however, reports of abnormal behavior did not increase (two during the 2016/2017 season and none during the 2019/2020 season). CONCLUSIONS: The number of oseltamivir-related ADR reports among minors over 10 years of age did not increase although the proportion of oseltamivir prescriptions increased after the revision of the oseltamivir package insert.

Prescription of renin-angiotensin system inhibitors in Japan during the COVID-19 pandemic: interrupted time series study.

We surveyed changes in angiotensin-converting enzyme inhibitor (ACEIs) and angiotensin II receptor blocker (ARBs) prescription trends during the coronavirus disease 2019 (COVID-19) pandemic in Japan. Data of 1,605,708 outpatients with hypertension were extracted from the Medical Data Vision database. Trends for prescription of ACEIs and ARBs were assessed by analyzing the proportion of these prescriptions in each month, between April 2018 and November 2020. The proportion of ARBs prescriptions changed significantly in trend between the peri-pandemic and pre-pandemic periods (-0.05%/month, P = 0.012). In contrast, the proportion of ACEIs prescriptions did not change significantly in trend in the peri-pandemic period (0.01%/month, P = 0.189). There was no suggestion that the prescribing of ACEIs and ARBs was affected by the COVID-19 pandemic.

Fetal growth restriction followed by early catch-up growth impairs pancreatic islet morphology in male rats.

Fetal growth restriction (FGR), followed by postnatal early catch-up growth, is associated with an increased risk of metabolic dysfunction, including type 2 diabetes in humans. This study aims to determine the effects of FGR and early catch-up growth after birth on the pathogenesis of type 2 diabetes, with particular attention to glucose tolerance, pancreatic islet morphology, and fibrosis, and to elucidate its mechanism using proteomics analysis. The FGR rat model was made by inducing mild intrauterine hypoperfusion using ameroid constrictors (ACs). On day 17 of pregnancy, ACs were affixed to the uterine and ovarian arteries bilaterally, causing a 20.9% reduction in birth weight compared to sham pups. On postnatal day 4 (P4), the pups were assigned to either the good nutrition (GN) groups with 5 pups per dam to ensure postnatal catch-up growth or poor nutrition groups with 15 pups per dam to maintain lower body weight. After weaning, all pups were fed regular chow food ad libitum (P21). Rats in both FGR groups developed glucose intolerance; however, male rats in the FGR good nutrition (FGR-GN) group also developed hypertriglyceridemia and dysmorphic pancreatic islets with fibrosis. A comprehensive and functional analysis of proteins expressed in the pancreas showed that FGR, followed by early catch-up growth, severely aggravated cell adhesion-related protein expression in male offspring. Thus, FGR and early catch-up growth caused pancreatic islet morphological abnormalities and fibrosis associated with the disturbance of cell adhesion-related protein expressions. These changes likely induce glucose intolerance and dyslipidemia in male rats.

Altered offspring neurodevelopment in an L-NAME-induced preeclampsia rat model.

Introduction: To investigate the mechanism underlying the increased risk of subsequent neurodevelopmental disorders in children born to mothers with preeclampsia, we evaluated the neurodevelopment of offspring of a preeclampsia rat model induced by the administration of N-nitro-L-arginine methyl ester (L-NAME) and identified unique protein signatures in the offspring cerebrospinal fluid. Methods: Pregnant rats received an intraperitoneal injection of L-NAME (250 mg/kg/day) during gestational days 15-20 to establish a preeclampsia model. Behavioral experiments (negative geotaxis, open-field, rotarod treadmill, and active avoidance tests), immunohistochemistry [anti-neuronal nuclei (NeuN) staining in the hippocampal dentate gyrus and cerebral cortex on postnatal day 70], and proteome analysis of the cerebrospinal fluid on postnatal day 5 were performed on male offspring. Results: Offspring of the preeclampsia dam exhibited increased growth restriction at birth (52.5%), but showed postnatal catch-up growth on postnatal day 14. Several behavioral abnormalities including motor development and vestibular function (negative geotaxis test: p < 0.01) in the neonatal period; motor coordination and learning skills (rotarod treadmill test: p = 0.01); and memory skills (active avoidance test: p < 0.01) in the juvenile period were observed. NeuN-positive cells in preeclampsia rats were significantly reduced in both the hippocampal dentate gyrus and cerebral cortex (p < 0.01, p < 0.01, respectively). Among the 1270 proteins in the cerebrospinal fluid identified using liquid chromatography-tandem mass spectrometry, 32 were differentially expressed. Principal component analysis showed that most cerebrospinal fluid samples achieved clear separation between preeclampsia and control rats. Pathway analysis revealed that differentially expressed proteins were associated with endoplasmic reticulum translocation, Rab proteins, and ribosomal proteins, which are involved in various nervous system disorders including autism spectrum disorders, schizophrenia, and Alzheimer's disease. Conclusion: The offspring of the L-NAME-induced preeclampsia model rats exhibited key features of neurodevelopmental abnormalities on behavioral and pathological examinations similar to humans. We found altered cerebrospinal fluid protein profiling in this preeclampsia rat, and the unique protein signatures related to endoplasmic reticulum translocation, Rab proteins, and ribosomal proteins may be associated with subsequent adverse neurodevelopment in the offspring.

Systemic administration of clinical-grade multilineage-differentiating stress-enduring cells ameliorates hypoxic-ischemic brain injury in neonatal rats.

Multilineage-differentiating stress-enduring (Muse) cells are endogenous reparative pluripotent stem cells present in the bone marrow, peripheral blood, and organ connective tissues. We assessed the homing and therapeutic effects of systemically administered nafimestrocel, a clinical-grade human Muse cell-based product, without immunosuppressants in a neonatal hypoxic-ischemic (HI) rat model. HI injury was induced on postnatal day 7 (P7) and was confirmed by T2-weighted magnetic resonance imaging on P10. HI rats received a single dose nafimestrocel (1 × 106 cells/body) or Hank's balanced salt solution (vehicle group) intravenously at either three days (on P10; M3 group) or seven days (on P14; M7 group) after HI insult. Radioisotope experiment demonstrated the homing of chromium-51-labeled nafimestrocel to the both cerebral hemispheres. The cylinder test (M3 and M7 groups) and open-field test (M7 group) showed significant amelioration of paralysis and hyperactivity at five weeks of age compared with those in the vehicle group. Nafimestrocel did not cause adverse events such as death or pathological changes in the lung at ten weeks in the both groups. Nafimestrocel attenuated the production of tumor necrosis factor-α and inducible nitric oxide synthase from activated cultured microglia in vitro. These results demonstrate the potential therapeutic benefits and safety of nafimestrocel.

Trends in the prescription of anti-seizure medicines for pregnant women outpatients with epilepsy during 2016-2020 in Japan.

PURPOSE: The temporal trends in prescribing anti-seizure medicines (ASMs) for pregnant women with epilepsy are unclear. In this study, we investigated the trends in ASM prescriptions in pregnant Japanese women with epilepsy. METHODS: Administrative data (as of December 2021), pertaining to Japanese pregnant outpatient women with epilepsy, aged 16-49 years, who visited hospitals between January 1, 2016 and December 31, 2020 were included in the study. Annual prescription trends in ASMs during this period were calculated based on the proportions. The Cochran-Armitage trend test was used to evaluate the proportion of prescriptions for each ASM. RESULTS: The numbers of pregnant women with epilepsy were 404, 421, 368, 378, 386 for the years 2016, 2017, 2018, 2019, and 2020, respectively. As of 2020, levetiracetam had the highest proportion of prescriptions, followed by lamotrigine and valproic acid. From 2016 to 2020, the proportions of levetiracetam and lamotrigine prescribed for pregnant women with epilepsy have increased significantly from 19.1% to 30.8% and from 12.1% to 18.4%, respectively. In contrast, there was no temporal change in the proportion of valproic acid prescribed, which was 12.4% in 2016 and 10.1% in 2020. CONCLUSION: Our findings suggest that the trends in the prescription of ASMs in Japanese pregnant women outpatients with epilepsy have shifted toward ASMs with a lower teratogenic risk.

Antihypertensive drug prescription trends for pregnant women with hypertension in acute hospitals in Japan.

Hypertensive disorders of pregnancy cause maternal organ damage. Therefore, appropriate management with antihypertensive medication is required from the first trimester. We aimed to clarify the antihypertensive drug prescription trends in pregnant women with hypertension in Japan. The administrative data of pregnant outpatients aged 16-49 years who visited acute hospitals between 2013 and 2020 were included. The annual antihypertensive drug prescription trends were evaluated based on their prescription proportions. The most prescribed drug in 2020 was nifedipine, followed by methyldopa and amlodipine. The proportion of nifedipine prescriptions significantly increased from 33.5 to 40.8% during the study period, whereas that of methyldopa significantly decreased from 16.6 to 11.6%. There was no change in the prescription trend of amlodipine. Dihydropyridine calcium channel blockers were the most commonly prescribed drug for pregnant women with hypertension.

Safety and tolerability of a multilineage-differentiating stress-enduring cell-based product in neonatal hypoxic-ischaemic encephalopathy with therapeutic hypothermia (SHIELD trial): a clinical trial protocol open-label, non-randomised, dose-escalation trial.

INTRODUCTION: Neonatal hypoxic-ischaemic encephalopathy (HIE) is an important illness associated with death or cerebral palsy. This study aims to assess the safety and tolerability of the allogenic human multilineage-differentiating stress-enduring cell (Muse cell)-based product (CL2020) cells in newborns with HIE. This is the first clinical trial of CL2020 cells in neonates. METHODS AND ANALYSIS: This is a single-centre, open-label, dose-escalation study enrolling up to 12 patients. Neonates with HIE who receive a course of therapeutic hypothermia therapy, which cools to a body temperature of 33°C-34°C for 72 hours, will be included in this study. A single intravenous injection of CL2020 cells will be administered between 5 and 14 days of age. Subjects in the low-dose and high-dose cohorts will receive 1.5 and 15 million cells per dose, respectively. The primary outcome is the occurrence of any adverse events within 12 weeks after administration. The main secondary outcome is the Bayley Scales of Infant and Toddler Development Third Edition score and the developmental quotient per the Kyoto Scale of Psychological Development 2001 at 78 weeks. ETHICS AND DISSEMINATION: This study will be conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. The Nagoya University Hospital Institutional Review Board (No. 312005) approved this study on 13 November 2019. The results of this study will be published in peer-reviewed journal and reported in international conferences. TRIAL REGISTRATION NUMBERS: NCT04261335, jRCT2043190112.

Drug prescription for attention deficit hyperactivity disorder drugs in pediatric outpatients: A retrospective survey of Japanese administrative data (2012-2018).

We aimed to clarify the prescription trend of ADHD drugs in Japanese pediatric outpatients. From January 2012 to December 2018, we evaluated the trends of prescribing methylphenidate-osmotic-controlled release oral delivery system (OROS), atomoxetine, and guanfacine as monotherapy. In boys, methylphenidate-OROS and atomoxetine prescriptions decreased from 46.5 % to 37.2 % and 18.6 % to 15.6 %, respectively. Prescriptions of guanfacine increased from 0.0 % to 12.3 %. In girls, the methylphenidate-OROS prescriptions was not significantly different (37.0 % to 26.4 %); however, atomoxetine decreased from 23.1 % to 16.3 %, and guanfacine increased from 0.0 % to 12.8 %. Methylphenidate-OROS and atomoxetine prescriptions changed to guanfacine between 2012 and 2018.

Trends in prescription of anti-seizure medicines for Japanese pediatric outpatients during 2013-2019.

Temporal trends in prescriptions of anti-seizure medicines (ASMs) for children, including newer ASMs, are unclear. We investigated ASM prescription trends for pediatric outpatients in Japan. The MDV analyzer® was used to analyze the MDV database containing de-identified hospital administrative data. Administrative data as of June 2021 including pediatric outpatients (0-17 years) with epilepsy, visiting 123 acute diagnostic procedure combination hospitals during 2013-2019, were used. Annual ASMs prescription trends were calculated, based on proportions. The Cochran-Armitage trend test evaluated the proportion of prescriptions for each ASM. ASMs most often prescribed were valproic acid, carbamazepine, and levetiracetam, regardless of sex. In girls, the proportion of valproic acid and carbamazepine prescriptions decreased from 37.93% to 26.84%, and from 24.80% to 15.78%, respectively (p < 0.0001). Conversely, the proportion of levetiracetam prescriptions increased from 6.40% to 28.18% (p < 0.0001). In boys, the proportion of valproic acid and carbamazepine prescriptions decreased, from 36.58% to 32.20% and from 26.42% to 16.85%, respectively (p < 0.0001). The proportion of levetiracetam prescriptions increased from 5.64% to 23.02% (p < 0.0001). Overall, the proportion of valproic acid and carbamazepine prescriptions declined, whereas levetiracetam prescriptions increased. Trends in ASM prescriptions among pediatric outpatients with epilepsy in Japan have shifted towards more recently available ASMs.

Addendum: A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs.

The cDNA sequence of human SMART described in this Article was misreported, as described in the accompanying Addendum. This error does not affect the results or any conclusion of the Article.

A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs.

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like (MLKL). While danger-associated molecular pattern (DAMP)s are involved in various pathological conditions and released from dead cells, the underlying mechanisms are not fully understood. Here we develop a fluorescence resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation by RIPK3 based on FRET). SMART is composed of a fragment of MLKL and monitors necroptosis, but not apoptosis or necrosis. Mechanistically, SMART monitors plasma membrane translocation of oligomerized MLKL, which is induced by RIPK3 or mutational activation. SMART in combination with imaging of the release of nuclear DAMPs and Live-Cell Imaging for Secretion activity (LCI-S) reveals two different modes of the release of High Mobility Group Box 1 from necroptotic cells. Thus, SMART and LCI-S uncover novel regulation of the release of DAMPs during necroptosis.