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Autophagy is known to play an essential role in intracellular quality control through the degradation of damaged organelles and components. We previously demonstrated that β-cell-specific autophagy deficient mice, which lack Atg7, exhibited impaired glucose tolerance, accompanied by the accumulation of sequestosome 1/p62 (hereafter referred to as p62). Whereas p62 has been reported to play essential roles in regulating cellular homeostasis in the liver and adipose tissue, we previously showed that β-cell-specific p62 deficiency does not cause any apparent impairment in glucose metabolism. In the present study, we investigated the roles of p62 in β cells under autophagy-deficient conditions, by simultaneously inactivating both Atg7 and p62 in a β-cell specific manner. Whereas p62 accumulation was substantially reduced in the islets of Atg7 and p62 double-deficient mice, glucose tolerance and insulin secretion were comparable to Atg7 single-deficient mice. Taken together, these findings suggest that the p62 accumulation appears to have little effect on β-cell function under conditions of autophagy inhibition.

Background Subclinical diastolic dysfunction in patients with type 1 diabetes mellitus (T1DM) caused by myocardial injury due to diabetic cardiomyopathy leads to a high risk of death and heart failure. This myocardial injury extends not only to the left ventricle (LV) but also to the left atrium (LA). However, LA function in children and young adults with T1DM has not been extensively studied. Objective Therefore, the aim of this study was to assess LA dysfunction in pediatric and adult patients with T1DM usingLA strain analysis withechocardiography. Subjects Fifty-three patients (median age: 23 [range: 5-41] years) with T1DM. Methods We dividedthe patients into three age groups (D1: 5-14 years, D2: 15-24 years, D3: 25-41 years);53 age- and sex-matched controls were divided into three corresponding groups (C1, C2, and C3). LA and LV functions were evaluated usingechocardiography. Results LA reservoir strain was lower in the D2 and D3 groups than in the C2 and C3 groups (p = 0.001, p = 0.004, respectively). LA conduit strain was lower in the D2 group thanin the C2 group (p = 0.002). LA stiffness wassignificantlygreater in the D3 groupthan in the C3 group (p Conclusions In patients with T1DM, LA phasic function decreased in adolescents and young adults, and LA stiffness increased in adult patients aged>30 years. LA phasic function and LA stiffness can be potentially used as early markers for diastolic dysfunction. This article is protected by copyright. All rights reserved.

Background Cholesterol metabolism has dramatically changed under hyperthyroid status. However, a combination of hyperthyroidism and cholecystolithiasis is very rare. Case presentation We report a case of cholelithiasis accompanied by hyperthyroidism in a 13-year-old girl who had recently lost 13 kg of weight (from 53 to 40 kg) in 1 month without loss of appetite. Ultrasonography showed multiple hyperechoic areas with acoustic shadowing in the gallbladder. Thyroid function tests showed that her serum free triiodothyronine (T3) and thyroxine (T4) levels were elevated and the thyroid-stimulating hormone level was decreased. In addition, serum thyrotropin receptor antibody and thyroid-stimulating antibody were detected. The final diagnosis was cholelithiasis with Graves’ disease. Thiamazole ingestion was started immediately after the diagnosis, and laparoscopic cholecystectomy was performed 33 days after hospitalization. Conclusions Massive and sudden weight loss could be a risk factor for gallstone formation in children. In addition, hyperthyroidism has the potential to promote cholelithiasis via cholesterol metabolism.

Autophagy is a mechanism of bulk protein degradation that plays an important role in regulating homeostasis in many organisms. Among several methods for evaluating its activity, a fluorescent reporter GFP-LC3-RFP-LC3ΔG, in which GFP-LC3 is cleaved by ATG4 following autophagic induction and degraded in lysosome, has been used for monitoring autophagic flux, which is the amount of lysosomal protein degradation. In this study, we modified this reporter by exchanging GFP for pHluorin, which is more sensitive to low pH, and RFP to mCherry, to construct pHluorin-LC3-mCherry reporter. Following starvation or mTOR inhibition, the increase of autophagic flux was detected by a decrease of the fluorescent ratio of pHluorin to mCherry; our reporter was also more sensitive to autophagy-inducing stimuli than the previous one. To establish monitoring cells for mouse genome-wide screening of regulators of autophagic flux based on CRISPR/Cas9 system, after evaluating knockout efficiency of clones of Cas9-expressing MEFs, we co-expressed our reporter and confirmed that autophagic flux was impaired in gRNA-mediated knockout of canonical autophagy genes. Finally, we performed genome-wide gRNA screening for genes inhibiting starvation-mediated autophagic flux and identified previously reported genes such as Atgs. Thus, we have successfully established a system for screening of genes regulating autophagic flux with our pHluorin-LC3-mCherry reporter in mice.

BACKGROUND There have been many reports of heart failure due to diabetic cardiomyopathy and decreased left ventricular (LV) function with increasing age in patients with type 1 diabetes mellitus (T1DM). Recently, although left atrial (LA) function has been occasionally reported to be a more useful prognostic indicator than LV function in acquired heart diseases, LA function in patients with T1DM has not yet been studied. PURPOSE To investigate LA dysfunction in patients with T1DM. METHODS Fifty patients with T1DM were recruited (age, 5–41 years). We excluded patients who had a history of heart disease, hypertension, and those taking cardioprotective agents. The patients and 50 age-matched controls were classified into 3 age groups (D1, C1, 5–14 years; D2, C2, 15–29 years; D3, C3, 30–41 years). The LA phasic function serving as the reservoir, conduit, and pump strains; the LA strain rate (SR) in the systole, early diastole, and late diastole; and the LV global longitudinal strain (LV-LS) as determined via 2-dimensional speckle tracking imaging were measured from the apical four- and two-chamber views. We also calculated the LA stiffness index as the ratio of E/e’ to the LA reservoir strain. RESULTS There was no significant difference in left ventricular ejection fraction in each age group. The LA reservoir strains in D2 and D3 were significantly lower than those in C2 (40.8 ± 5.7% vs. 47.2 ± 5.5%, p = 0.005) and C3 (39.2 ± 5.5% vs. 47.3 ± 5.7%, p = 0.004), respectively. The LA conduit strain in D2 was significantly lower than that in C2 (28.9 ± 5.8% vs. 35.0 ± 5.0%, p = 0.006). The LA pump strain and the three phases of LA SR were not significantly different among the age groups. The LA stiffness index in D3 increased significantly compared to that in N3 (0.18 ± 0.05 vs. 0.13 ± 0.01, p CONCLUSIONS The LA reservoir strain might be as useful as LV-LS as an early marker of cardiac dysfunction in patients with T1DM. The correlation coefficient between the LA reservoir strain and LV-LS was not strong. Therefore, although LV-LS might affect the LA reservoir strain, it might represent other aspects of cardiac dysfunction. The increase of LA stiffness might represent the changes in LA wall properties and could be another useful indicator of cardiac dysfunction during long-term follow-ups, which is independent of LV-LS. Overall, these findings provide new insights into cardiac dysfunction in patients with T1DM.

Background The number of patients with diabetes is increasing worldwide. Macrovascular disease, chronic kidney disease, chronic respiratory disease, cancer and smoking frequently accompany type 2 diabetes. Few data are available related to mortality of Asians with diabetes associated with these serious comorbidities. The present study aimed to quantify the excess mortality risks of type 2 diabetic patients with comorbidities. Methods We analysed the available records of 30,834 Japanese patients with type 2 diabetes from the BioBank Japan Project between 2003 and 2007. Men and women were followed up for median 8.03 and 8.30 years, respectively. We applied Cox proportional hazard model and Kaplan–Meier estimates for survival curves to evaluate mortality in diabetic patients with or without macrovascular disease, chronic respiratory disease, chronic kidney disease, cancer and smoking. Results Adjusted hazard ratios (HRs) for mortality were 1.39 (95% CI, 1.09–1.78) for male sex, 2.01 (95% CI, 1.78–2.26) per 10-year increment of age. Adjusted HRs of primary interest were 1.77 (95% CI, 1.42–2.22), macrovascular disease; 1.58 (95% CI, 1.08–2.31), chronic respiratory disease; 2.03 (95% CI, 1.67–2.47), chronic kidney disease; 1.16 (95% CI, 0.86–1.56), cancer; and 1.74 (95% CI, 1.30–2.31), current smoking. Conclusions Diabetic patients with a past or current history of chronic kidney, macrovascular or respiratory diseases or smoking habit have exhibited the highest risk of mortality. Data were limited to those of survivors of comorbidities but we propose the need to improve comorbidities and terminate cigarette smoking for better prognosis in patients with diabetes., Highlights • Fatal diseases frequently accompany diabetes. • Data for survival of Asian patients with diabetes with comorbidities are scarce. • Comorbid chronic kidney disease was associated with the most fatalities. • Current smoking was as fatal as 10 years of ageing in diabetic patients. • Values of 1% HbA1c and 10 mmHg blood pressure confer 11% excess mortality risk.

Background Although the subclinical left ventricular (LV) dysfunction caused by diabetes mellitus (DM) results in a high risk of death and heart failure, the details of cardiac dysfunction across a wide age range remain unclear. The aim of this study was to assess LV dysfunction in patients with type 1 DM (T1DM) using layer-specific strain analysis by echocardiography.Methods and Results:The 52 patients (median age: 23 [range: 5-40] years) with T1DM were divided into 3 age groups (D1: 5-14 years, D2: 15-24 years, D3: 25-40 years); 78 age- and sex-similar controls were divided into 3 corresponding groups (C1, C2, and C3). Layer-specific longitudinal strain (LS) and circumferential strain (CS) of the 3 myocardial layers (endocardium, midmyocardium, and epicardium) were determined using echocardiography. Strains did not decrease in D1. Epicardial and midmyocardial CS at the basal level and LS in all layers were decreased in D2 compared with C2. CS at the basal level and LS in all layers were lower in D3 than in C3. The strains correlated with the duration of T1DM and LV wall thickness. Conclusions In patients with T1DM, longitudinal deformation in all layers and epicardial and midmyocardial circumferential deformation at the basal level decreased from the late teens, which correlated with the duration of the disease and LV hypertrophy.

To review the surgical treatment of hypospadias (HP) associated with disorders of sex development (DSD). HP cases were assessed for DSD by gross examination for atypical external genitalia, and assessment of hormone levels and karyotype. There were 58 HP cases with concomitant DSD treated between 1999 and 2017. DSD classification, type of HP, sex assignment, hormonal abnormality, surgical strategy, and post-urethroplasty complications (post-UPC) were reviewed. DSD were sex chromosome abnormalities (n = 4), 46,XY (n = 51), 46,XX (n = 1), and 47,XY + 21 (n = 2). HP was perineal: (n = 26), scrotal: (n = 16), penoscrotal: (n = 15), and midshaft: (n = 1); repair was primary (n = 6) or staged (n = 52). Mean age at final urethroplasty (UP) was 4.12 ± 0.21 years; all cases had soft tissue interposition at UP. At mean follow-up 5.16 ± 0.56 years after final UP, observed post-UPC (n = 8; 13.8%) were urethral stenosis (n = 3), urethral diverticulum (n = 2), urethrocutaneous fistula (n = 2), and curvature (n = 1). Mean onset of post-UPC was 1.24 ± 0.77 years (range 0.1–6.3). The second half of our cases (n = 29; treated 2015 ~) had significantly less post-UPC (0/29; 0%) than the first half (8/29; 27.6%) (p = 0.0075). Although UP for HP + DSD was formidably challenging, we achieved a significant decrease in post-UPC through a combination of surgical techniques and experience.

Background In Japan, prophylactic thyroidectomy involves out-of-pocket expense. The American Thyroid Association (ATA) recommends prophylactic thyroidectomy for medullary thyroid carcinoma (MTC) during early childhood in patients with multiple endocrine neoplasia type 2 (MEN2). The ATA reports a high frequency of postoperative complications in childhood, which also influenced the delay of prophylactic thyroidectomy in Japan. Methods This retrospective study of multiple medical centers in Japan included individuals aged RET mutations between 1997 and 2017. The onset and onset possibility were defined based on confirmed lesions or calcitonin levels. The definition of risk and prophylactic thyroidectomy were based on the ATA 2015 revised guideline. Results Twenty-one patients with MEN2 were enrolled (highest risk, n = 5; high risk, n = 5; and moderate risk, n = 11). The cumulative incidence of the onset/onset possibility reached 50% at 5 and 8 years and 100% at 9 years and 17 years in high- and moderate-risk patients, respectively. Of 7 patients with MEN2A, 71% underwent prophylactic thyroidectomy. Only one 5-year-old patient (C634Y) had increased serum calcitonin level after prophylactic thyroidectomy in the MEN2A group. The only permanent complication, which did not occur in patients who underwent total thyroidectomy alone, was hypoparathyroidism (33% of patients). This permanent complication occurred with clinically developed MTC. No permanent postoperative complications occurred in patients aged 5–6 years. Conclusions Prophylactic thyroidectomy reduces recurrence and postoperative complications in pediatric patients with MEN2. Early thyroidectomy based on only calcitonin level could possibly reduce thyroidectomy delay.

Isovaleric acidemia (IVA) is an autosomal recessive inborn error affecting leucine metabolism. It is caused by a deficiency in isovaleryl-CoA dehydrogenase (IVD), a mitochondrial matrix enzyme that catalyzes the oxidation of isovaleryl-CoA to 3-methylcrotonyl-CoA. IVD is a FAD-containing enzyme, consisting of four identical subunits. Clinical features of IVA include poor feeding, vomiting, lethargy, developmental delay, metabolic acidosis, and a characteristic "sweaty foot" odor. IVA is one of the target disorders for newborn screening by tandem mass spectrometry (MS/MS). The human IVD gene is located on chromosome 15q. To date, over 50 disease-causing mutations have been reported worldwide. In this study, we searched for IVD mutations in five Japanese patients with IVA (neonatal type, two patients; chronic intermittent type, two patients; and mild biochemical type, one patient). The diagnosis of IVA was confirmed by urinary organic acid analysis using gas chromatography and mass spectrometry. All coding exons and the flanking introns in the IVD gene were amplified by PCR and were directly sequenced. We thus identified six hitherto unknown mutations (p.G94D, p.E116K, p.M167T, p.L243P, p.L246P, and c.696+1G>T) and four previously reported (p.R53P, p.R395C, p.Y403C, and p.E411K) pathogenic mutations. All patients were compound heterozygotes, and each mutation was identified in a single patient. Pathogenicity of newly identified mutations was validated using computational programs. Among them, the p.M167T is believed to influence FAD binding, as the position 167 is present in one of the FAD-binding sites. Our results have illustrated the heterogeneous mutation spectrum and clinical presentation of IVA in the Japanese patients.

Context Mutations in the GH1 gene have been identified in patients with isolated growth hormone deficiency (IGHD). Mutations causing aberrant splicing of exon 3 of GH1 that have been identified in IGHD are inherited in an autosomal dominant manner, whereas other mutations in GH1 that have been identified in IGHD are inherited in an autosomal recessive manner. Objective Two siblings born from nonconsanguineous healthy parents exhibited IGHD. To elucidate the cause, GH1 in all family members was analysed. Results Two novel mutations in GH1, a point mutation in intron 3 and a 16-bp deletion in exon 3, were identified by sequence analyses. The intronic mutation was present in both siblings and was predicted to cause aberrant splicing. The deletion was present in one of the siblings as well as the mother with normal stature and was predicted to cause rapid degradation of mRNA through nonsense-mediated mRNA decay. The point mutation was not identified in the parents' peripheral blood DNA; however, it was detected in the DNA extracted from the father's sperms. As a trace of the mutant allele was detected in the peripheral blood of the father using PCR-RFLP, the mutation is likely to have occurred de novo at an early developmental stage before differentiation of somatic cells and germline cells. Conclusions This is the first report of mosaicism for a mutation in GH1 in a family with IGHD. It is clear that the intronic mutation plays a dominant role in the pathogenesis of IGHD in this family, as one of the siblings who had only the point mutation was affected. On the other hand, the other sibling was a compound heterozygote for the point mutation and the 16-bp deletion and it may be arguable whether IGHD in this patient should be regarded as autosomal dominant or recessive.

Background: Corticosteroids therapy, classically the first-line treatment for ulcerative colitis (UC), often causes serious side-effects. Theoretically, pulse steroid therapy where high doses are given for a shorter period may have maximal beneficial effects and minimal side-effects as induction therapy for UC. We have therefore retrospectively compared induction therapy using pulse steroids with conventional steroid treatment for children and adolescents with moderate-to-severe UC. Methods: We utilized conventional steroid treatment (prednisolone 1–1.5 mg/kg/day) as an induction treatment in 17 UC patients between 1985 and 2006. Alternatively we used a 3-day megadose pulse steroid therapy (methylprednisolone intravenously 20–30 mg/kg/day, max. 1000mg/day) in 20 UC patients from 1993 to 2006. Results: Pulse steroid therapy successfully induced rapid remission in UC patients with moderate-to-severe disease compared with conventional treatment (13.2 days vs 25.1 days; P < 0.05). The amelioration of Pediatric Ulcerative Colitis Activity Index score between before and 1 week after pulse steroid therapy was significantly more than that of conventional treatment (P < 0.01). No serious adverse effects were observed in the patients treated with pulse steroid therapy. However, the rate of the relapse episodes during the next 12 months after pulse steroid therapy was not significantly different from that after conventional treatment. Conclusion: These findings suggest that pulse steroid therapy is an option to be considered in children with moderate-to-severe UC.

We experienced a case of a Japanese boy who developed intractable idiopathic intracranial hypertension (IIH) during growth hormone (GH) treatment. At the age of 4 yr, the boy was diagnosed with idiopathic growth hormone deficiency, and recombinant human GH replacement was initiated. Nine months after initiation of the GH therapy, he began to complain of headache, but papilledema was not observed. His headache persisted thereafter, and right esotropia occurred 10 mo after the initiation of GH therapy, at which time papilledema was detected. No other neurological abnormalities were detected, and the findings of computed tomography and magnetic resonance imaging were normal. In a cerebrospinal fluid (CSF) examination, the pressure was markedly elevated to 450 mmH2O, but no other abnormality was recognized. Impaired CSF absorption was detected using the pressure-volume index technique. The CSF levels of GH and insulin-like growth factor I were not increased. GH therapy was withdrawn after it was suggested that the IIH was associated with the GH therapy, but the headache persisted. The intracranial hypertension did not respond to diuretics, and prednisolone was only transiently effective. Although the funduscopic findings were normalized, increased CSF pressure was still observed. For over 2 yr, repeated lumbar puncture was necessary to protect against visual defect. IIH is an uncommon adverse event during GH therapy, but it must be considered carefully.

Cytochrome P450 oxidoreductase (POR) deficiency is a rare autosomal recessive disorder characterized by skeletal dysplasia, adrenal dysfunction, disorders of sex development (DSD), and maternal virilization during pregnancy. Although multiple studies have been performed for this condition, several matters remain to be clarified, including the presence of manifesting heterozygosity and the underlying factors for clinical variability.The objective of the study was to examine such unresolved matters by detailed molecular studies and genotype-phenotype correlations.Thirty-five Japanese patients with POR deficiency participated in the study.Mutation analysis revealed homozygosity for R457H in cases 1-14 (group A), compound heterozygosity for R457H and one apparently null mutation in cases 15-28 (group B), and other combinations of mutations in cases 29-35 (group C). In particular, FISH and RT-PCR sequencing analyses revealed an intragenic microdeletion in one apparent R457H homozygote, transcription failure of apparently normal alleles in three R457H heterozygotes, and nonsense mediated mRNA decay in two frameshift mutation-positive cases examined. Genotype-phenotype correlations indicated that skeletal features were definitely more severe, and adrenal dysfunction, 46,XY DSD, and pubertal failure were somewhat more severe in group B than group A, whereas 46,XX DSD and maternal virilization during pregnancy were similar between two groups. Notable findings also included the contrast between infrequent occurrence of 46,XY DSD and invariable occurrence of 46,XX DSD and pubertal growth pattern in group A mimicking that of aromatase deficiency.The results argue against the heterozygote manifestation and suggest that the residual POR activity reflected by the R457H dosage constitutes the underlying factor for clinical variability in some features but not other features, probably due to the simplicity and complexity of POR-dependent metabolic pathways relevant to each phenotype.

BACKGROUND Gastric inflammation in patients with Helicobacter pylori infection is considered to be regulated by many kinds of inflammatory and cytoprotective factors. The present study examined the effects of cyclo-oxygenase (COX)-1, -2, and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) on gastric mucosal injury in children with H. pylori infection. METHODS The subjects were 24 children who underwent endoscopy for the evaluation of anemia or gastrointestinal symptoms, and they were divided into two groups: a H. pylori-positive group and -negative group. The numbers of neutrophils in the gastric mucosa of children with and without H. pylori infection and expression of COX-1, -2, and PPAR-gamma were examined, using reverse transcription-polymerase chain reaction. RESULTS The numbers of neutrophils were significantly higher in the H. pylori-positive group than in the H. pylori-negative group. The ratio of COX-1 mRNA to COX-2 mRNA in the H. pylori-positive group was significantly lower than that in the H. pylori-negative group. The ratio of PPAR-gamma m-RNA to beta-actin mRNA was significantly higher in the H. pylori-positive group than the H. pylori-negative group. CONCLUSIONS Enhanced production of COX-2 and PPAR-gamma in the gastric mucosa has cytoprotective and anti-inflammatory effects, although the relationship to the carcinogenic activity of COX-2 and PPAR-gamma should be clarified.

Lower gastrointestinal bleeding (LGB), particularly in newborns, is of serious concern and requires urgent investigation and hospital care. Whereas allergic proctocolitis caused by food protein is a significant cause of LGB in infants with eosinophilia, there are several cases of diseases with symptoms similar to those of allergic proctocolitis but without an apparent allergic reaction influence.We examined 2 neonates using rectosigmoidoscopy who showed eosinophilia and experienced fresh LGB soon after birth and before their first feedings. Serum eosinic cationic protein (ECP) and platelet activating factor (PAF) levels were also examined in the second case to confirm the involvement of eosinophils for its pathogenesis.Both patients were in a clinically stable condition, and their abdomens were soft. The results of their blood analyses, abdominal radiographs, and stool cultures were normal, but they had gross eosinophilia: the eosinophil counts were 9014/mm3 (patient 1) and 1955/mm3 (patient 2). Rectosigmoidoscopy with colonic mucosal biopsy revealed nodular lymphoid hyperplasia with a pale mucosal surface and massive oozing with diffuse eosinophilic infiltration in the lamina propria. In patient 2 the serum ECP and PAF levels were elevated to 123 microg/L (normal,14.7) and 13.1 micromol/L/min (normal,6). A few days after intravenous hydration therapy, LGB was no longer detected, and the serum ECP and PAF levels returned to normal.Inasmuch as these infants had LGB similar to allergic proctocolitis without any allergic reactions, we suggest that infiltrated eosinophils in the colonic mucosa could be involved in the pathogenesis of LGB in early infancy.

We report the efficacy of staged segmental urethroplasty (SSUP) versus non-staged urethroplasty (NSUP) for treating scrotal/perineal hypospadias (SPH). Between 1997 and 2015, 29 SPH patients underwent UP (SSUP: n = 15; NSUP: n = 14). Incidences of urethrocutaneous fistula (UF), stenosis of the neourethra (SNU), diverticula formation, and residual chordee (RC) were compared. Differences were statistically significant if p

Isovaleric acidemia (IVA) is an autosomal recessive inborn error affecting leucine metabolism. It is caused by a deficiency in isovaleryl-CoA dehydrogenase (IVD), a mitochondrial matrix enzyme that catalyzes the oxidation of isovaleryl-CoA to 3-methylcrotonyl-CoA. IVD is a FAD-containing enzyme, consisting of four identical subunits. Clinical features of IVA include poor feeding, vomiting, lethargy, developmental delay, metabolic acidosis, and a characteristic "sweaty foot" odor. IVA is one of the target disorders for newborn screening by tandem mass spectrometry (MS/MS). The human IVD gene is located on chromosome 15q. To date, over 50 disease-causing mutations have been reported worldwide. In this study, we searched for IVD mutations in five Japanese patients with IVA (neonatal type, two patients; chronic intermittent type, two patients; and mild biochemical type, one patient). The diagnosis of IVA was confirmed by urinary organic acid analysis using gas chromatography and mass spectrometry. All coding exons and the flanking introns in the IVD gene were amplified by PCR and were directly sequenced. We thus identified six hitherto unknown mutations (p.G94D, p.E116K, p.M167T, p.L243P, p.L246P, and c.696+1GT) and four previously reported (p.R53P, p.R395C, p.Y403C, and p.E411K) pathogenic mutations. All patients were compound heterozygotes, and each mutation was identified in a single patient. Pathogenicity of newly identified mutations was validated using computational programs. Among them, the p.M167T is believed to influence FAD binding, as the position 167 is present in one of the FAD-binding sites. Our results have illustrated the heterogeneous mutation spectrum and clinical presentation of IVA in the Japanese patients.

Objective: To examine the effects of stool dilution on the results of the faecal Helicobacter pylori antigen test. Methods: Stool samples from nine children with H. pylori infection were diluted with normal saline at dilutions of 1, 5, 10, 50, 100 and 500 times, and optical density (OD) was measured. Results: The faecal H. pylori antigen test yielded positive results for all samples at dilutions of 1:10 and less, although the sample from the case whose original stool showed the lowest OD value changed from positive to negative at a dilution of 1:50. Conclusion: Our results suggest that stools at dilutions of less than 1:10 usually do not yield false-negative results in the faecal H. pylori antigen test, even in patients with low faecal antigen levels.

SummaryObjective NR5A1 or steroidogenic factor 1 is a nuclear receptor that plays important roles in the hypothalamus–pituitary–steroidogenic axis. The clinical phenotype of most 46,XY mutation carriers includes disorders of sex development (DSD) without adrenal insufficiency, whereas 46,XX mutation carriers have phenotypes ranging from no symptoms to ovarian insufficiency. Although genetically engineered ventromedial hypothalamus-specific Nr5a1 knockout mice show anxiety behaviour, no psychiatric symptoms have been reported in human NR5A1 mutation carriers. We report clinical and molecular findings for individuals (from two families) with NR5A1 mutations, showing psychiatric symptoms. Design and methods We screened for NR5A1 mutations in a cohort of 34 patients with 46,XY DSD using PCR-based sequencing. Psychiatric symptoms were assessed using mental health assessment tools and structured clinical interviews. Functional properties of detected mutant NR5A1s were studied in silico and in vitro, including three-dimensional (3D) mutation models, subcellular NR5A1 protein localization and transactivation assays. Results We found 2 (46,XY) patients with NR5A1 heterozygous novel mutations (p.D257fs and p.V424del), which were transmitted from their respective mothers. The patients' clinical findings indicated DSD without adrenal insufficiency. Both mothers showed psychiatric symptoms, including excessive anxiety and/or depression. The mother and grandmother of one patient had premature ovarian insufficiency. Functional studies showed altered 3D models of p.V424del and normal subcellular NR5A1 localization and impaired transcriptional activation without dominant-negative effects in both mutations. Conclusions We found 2 (46,XX) NR5A1 mutation carriers with excessive anxiety and/or depression. These results suggest that excessive anxiety and/or depression are possible clinical phenotypes of 46,XX NR5A1 mutations.

Mutations in the GH1 gene have been identified in patients with isolated growth hormone deficiency (IGHD). Mutations causing aberrant splicing of exon 3 of GH1 that have been identified in IGHD are inherited in an autosomal dominant manner, whereas other mutations in GH1 that have been identified in IGHD are inherited in an autosomal recessive manner. Two siblings born from nonconsanguineous healthy parents exhibited IGHD. To elucidate the cause, GH1 in all family members was analysed. Two novel mutations in GH1, a point mutation in intron 3 and a 16-bp deletion in exon 3, were identified by sequence analyses. The intronic mutation was present in both siblings and was predicted to cause aberrant splicing. The deletion was present in one of the siblings as well as the mother with normal stature and was predicted to cause rapid degradation of mRNA through nonsense-mediated mRNA decay. The point mutation was not identified in the parents' peripheral blood DNA; however, it was detected in the DNA extracted from the father's sperms. As a trace of the mutant allele was detected in the peripheral blood of the father using PCR-RFLP, the mutation is likely to have occurred de novo at an early developmental stage before differentiation of somatic cells and germline cells. This is the first report of mosaicism for a mutation in GH1 in a family with IGHD. It is clear that the intronic mutation plays a dominant role in the pathogenesis of IGHD in this family, as one of the siblings who had only the point mutation was affected. On the other hand, the other sibling was a compound heterozygote for the point mutation and the 16-bp deletion and it may be arguable whether IGHD in this patient should be regarded as autosomal dominant or recessive.

In Japan, there is as yet no report on growth retardation in children with IBD. We therefore investigated the cause of growth retardation in Japanese children with IBD. We investigated the height, body weight, serum levels of albumin, IGF-I, CRP, and cytokines, and the amount of corticosteroid administered in children with Crohn's disease (CD, 𝑛 = 1 5 ) and ulcerative colitis (UC, 𝑛 = 1 8 ). Our results suggest that growth retardation is already present before the initial visit in children with CD, and chronic inflammation may be responsible this growth disturbance. Moreover, the amount of PSL used may contribute to growth retardation by decreasing the serum levels of IGF-I in children with IBD.

Background: Rooibos tea is known to be caffeine free with abundant flavonoids. Aspalathin and nothofagin, the main flavonoids contained in Rooibos tea, have stronger anti-oxidative activity than other flavonoids.As oxidative stress can induce inflammation, the anti-inflammatory effects of Rooibos tea were investigated using a rat colitis model. Methods: Seven-week-old Wister rats were divided into two groups: one group given Rooibos tea, and one given water. After four weeks of breeding, serum superoxide dismutase (SOD) levels were determined using the Electron Spin Resonance analysis. Urine 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentrations were also determined as reflections of DNA damage using enzyme-linked immunosorbent assay. Furthermore, rats were administrated dextran sodium sulfate (DSS), which is known to induce colitis in rodents, with or without Rooibos tea to evaluate its anti-inflammatory activity. Clinical symptoms, hemoglobin, serum iron and SOD levels were compared between the groups. Results: There were no significant differences in bodyweight gain or laboratory data between the groups. The serum SOD levels were significantly increased, and urine 8-hydroxy-2'-deoxyguanosine levels were significantly decreased in the Rooibos group compared with the controls (P < 0.05 in each).After DSS administration, the serum SOD levels were significantly higher in the Rooibos group compared to the controls (P < 0.05).As a result, a decreased hemoglobin level, observed in the control group, was prevented in the Rooibos group after the DSS challenge. Conclusion: Rooibos tea may prevent DNAdamage and inflammation by its anti-oxidative activity in vivo.As Rooibos tea is free from caffeine, routine intake may be safe and useful in reducing oxidative stress in children.

Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors [tissue inhibitors of metalloproteinases (TIMPs)] have been implicated in tissue injury and remodeling in many organs. The objective of this study was to evaluate the expression of MMP-3 and -9, and TIMP-1, -2, and -3 and their relationship to liver fibrosis in infants with biliary atresia. The expression of MMP-3 and-9 and TIMP-1, -2 and -3 was investigated in liver tissue samples of nine patients with biliary atresia. In addition, the expression of CCR-4 and CCR-5 was analyzed to investigate the activation of Th1 and Th2 cells. The mRNA levels were measured by semiquantitative reverse transcriptase polymerase chain reaction. The expression of MMP-3 was higher than that of MMP-9 in all samples (P

Background: The rates of acquisition and spontaneous eradication of Helicobacter pylori infection in children has yet to be established. To determine these rates in children living in an urban region of Japan, the levels of urine H. pylori antibodies in children of three different age groups were measured. Methods: A urine-based enzyme-linked immunosorbent assay (ELISA) was used to detect H. pylori antibodies twice within a 12 month interval over 2 years in 452 healthy children living in Tokyo. The subjects were divided into three groups: ages 4, 7, and 10 years. Results: The prevalence of H. pylori infection was not different among the groups, being between 4.0% and 6.7%. The rate of turn to positivity for H. pylori infection was 1.5% per year and the rate of turn to negativity was 1.1%, but in the 10 year age group the rates were markedly lower than in the younger children. Conclusion: The prevalence of H. pylori infection in Tokyo was 4.0–6.7% and was not different among 4, 7, and 10 year age groups.

Objective: Recent studies of urine-based enzyme-linked immunosorbent assays (ELISA) for detection of antibody to Helicobacter pylori (H. pylori) have already shown high sensitivity and specificity in adults. The diagnostic accuracy of these assays in children was investigated. Methods: The results of serum and urine-based ELISAs were compared with those of 13C-urea breath tests (13C-UBT) and/or detection of faecal H. pylori antigen in 68 children. The effect of urine total immunoglobulin G (IgG) levels on the ELISA results for anti-H. pylori antibodies in urine was also examined. Results: The sensitivity, specificity and accuracy of the serum ELISA were 72.7%, 96.3%, and 92.3% respectively, while those of the urine-based ELISA were 92.3%, 76.4%, and 79.4% respectively. The level of urine total IgG in children with false-positive results in the urine-based ELISA, was significantly higher than that in children who showed negative results in both the urine-based ELISA and the 13C-UBT and/or faecal H. pylori antigen tests. Human γ-globulin affected the urine-based ELISA results at final concentrations of 2.0 mg/dL, 3.0 mg/dL, and 4.0 mg/dL; the anti-H. pylori antibody values were significantly higher than the ELISA values without the addition of human γ-globulin. Conclusion: The findings suggested that the specificity of urine-based ELISA for detection of H. pylori antibodies is low in children, since high urinary levels of total IgG increase the likelihood of false-positive results.

Background The role of cytomegalovirus infection in triggering systemic lupus erythematosus remains a subject of debate. Here, we present a case of childhood systemic lupus erythematosus with concomitant cytomegalovirus infection, which sheds light on the relationship between these conditions and their treatment in pediatric patients. Case presentation A 12-year-old Japanese girl with no history of systemic illness was diagnosed with systemic lupus erythematosus and concomitant primary cytomegalovirus infection. Her anti-cytomegalovirus immunoglobulin G antibodies were elevated during diagnosis and treatment. Further, the patient’s cytomegalovirus pp65 antigenemia level was slightly elevated (1 cell per 5 × 104 cells). Treatment included the administration of ganciclovir, prednisolone, methylprednisolone, and cyclophosphamide, none of which prompted adverse effects. The patient was in good condition at the most recent follow-up. Conclusion Ganciclovir treatment is not completely safe, and there are no clinical guidelines regarding its use in patients with systemic lupus erythematosus triggered by cytomegalovirus infection. Our experience with this case suggests that the decision to administer ganciclovir treatment in pediatric cases should be guided by a variety of factors in addition to the cytomegalovirus antigenemia level. These factors include lymphopenia, renal biopsy results, and cytomegalovirus DNA levels detected by polymerase chain reaction. The details of our patient’s presentation and treatment should prove illustrative to other clinicians who face similar cases.