Citrin-deficient patient-derived induced pluripotent stem cells as a pathological liver model for congenital urea cycle disorders.

Citrin deficiency is a congenital secondary urea cycle disorder lacking useful disease models for effective treatment development. In this study, human induced pluripotent stem cells (iPSCs) were generated from two patients with citrin deficiency and differentiated into hepatocyte-like cells (HLCs). Citrin-deficient HLCs produced albumin and liver-specific markers but completely lacked citrin protein and expressed argininosuccinate synthase only weakly. In addition, ammonia concentrations in a medium cultured with citrin-deficient HLCs were higher than with control HLCs. Sodium pyruvate administration significantly reduced ammonia concentrations in the medium of citrin-deficient HLCs and slightly reduced ammonia in HLCs differentiated from control iPSCs, though this change was not significant. Our results suggest that sodium pyruvate may be an efficient treatment for patients with citrin deficiency. Citrin-deficient iPSCs are a pathological liver model for congenital urea cycle disorders to clarify pathogenesis and develop novel therapies.

Efficient protocol for the differentiation of kidney podocytes from induced pluripotent stem cells, involving the inhibition of mTOR.

The mechanistic/mammalian target of rapamycin (mTOR) is involved in a wide range of cellular processes. However, the role of mTOR in podocytes remains unclear. In this study, we aimed to clarify the role of mTOR in podocyte differentiation from human induced pluripotent stem cells (hiPSCs) and to establish an efficient differentiation protocol for human podocytes. We generated podocytes from hiPSCs by modifying protocol. The expression of the podocyte-specific slit membrane components nephrin and podocin was measured using PCR, western blotting, flow cytometry, and immunostaining; and the role of mTOR was evaluated using inhibitors of the mTOR pathway. Nephrin and podocin were found to be expressed in cells differentiated from hiPSCs, and their expression was increased by mTOR inhibitor treatment. S6, a downstream component of the mTOR pathway, was also found to be involved in podocyte differentiation. we evaluated its permeability to albumin, urea, and electrolytes. The induced podocytes were permeable to the small molecules, but only poorly permeable to albumin. We have shown that the mTOR pathway is involved in podocyte differentiation. Our monolayer podocyte differential protocol, using an mTOR inhibitor, provides a novel in vitro model for studies of kidney physiology and pathology.

Dysbiosis of the gut microbiota as a susceptibility factor for Kawasaki disease.

Introduction: Gut microbial imbalance (dysbiosis) has been reported in patients with acute Kawasaki disease (KD). However, no studies have analyzed the gut microbiota while focusing on susceptibility to KD. This study aimed to evaluate whether dysbiosis elevates susceptibility to KD by assessing children with a history of KD. Methods: Fecal DNA was extracted from 26 children with a history of KD approximately 1 year prior (KD group, 12 boys; median age, 32.5 months; median time from onset, 11.5 months) and 57 age-matched healthy controls (HC group, 35 boys; median age, 36.0 months). 16S rRNA gene analysis was conducted with the Illumina Miseq instrument. Sequence reads were analyzed using QIIME2. Results: For alpha diversity, Faith's phylogenetic diversity was significantly higher in the KD group. Regarding beta diversity, the two groups formed significantly different clusters based on Bray-Curtis dissimilarity. Comparing microbial composition at the genus level, the KD and HC groups were significantly different in the abundance of two genera with abundance over 1% after Benjamini-Hochberg false discovery rate correction for multiple comparisons. Compared with the HC group, the KD group had higher relative abundance of Ruminococcus gnavus group and lower relative abundance of Blautia. Discussion and conclusion: Ruminococcus gnavus group reportedly includes pro-inflammatory bacteria. In contrast, Blautia suppresses inflammation via butyrate production. In the predictive functional analysis, the proportion of gut microbiota involved in several pathways was lower in the KD group. Therefore, dysbiosis characterized by distinct microbial diversity and decreased abundance of Blautia in parallel with increased abundance of Ruminococcus gnavus group might be a susceptibility factor for KD.

Growth Hormone Therapy for Small for Gestational Age Short Stature Develops Type 2 Diabetes.

Growth Hormone therapy has been shown to induce transient insulin resistance in children, and there is concern regarding the diabetogenic potential of GH therapy in children born small for gestational age (SGA). In this case, female patient born SGA with a weight of 2,750 g (-1.73 standard deviation (SD)) and length of 45.5 cm (-2.6 SD). The patient's father and paternal grandfather were diagnosed with type 2 diabetes mellitus. At 3 years of age, the patient presented with short stature; height and weight were 85 cm (-2.5 SD) and 13 kg (-0.19 SD), respectively. She was placed on GH therapy. At 11 years of age, her fasting blood glucose and hemoglobin A1c levels were 116 mg/dL and 7.4%, respectively. Blood test results were negative for anti-glutamic acid decarboxylase and anti-islet antigen-2 antibodies. The patient discontinued GH therapy and started diet therapy and oral metformin (500 mg/day) administration. Five months later, the hemoglobin A1c level was 5.3% and glycemic control further improved. To our knowledge, family history may be an important risk factor for GH-induced diabetes. So, the GH dosage for patients born SGA with family history of diabetes should be adjusted so as not to be too excessive, and long-term follow-up studies will be required to evaluate fully the effects of GH therapy for them.

Abnormal Development of Microbiota May Be a Risk Factor for Febrile Urinary Tract Infection in Infancy.

Febrile urinary tract infection (fUTI) is common in infants, but specific risk factors for developing it remain unclear. As most fUTIs are caused by ascending infections of intestinal bacteria, dysbiosis-an imbalance in gut microbial communities-may increase fUTI risk. This study was conducted to test the hypothesis that abnormal development of gut microbiota during infancy increases the risk of developing fUTI. Stool samples were collected from 28 infants aged 3-11 months with first-onset fUTI (fUTI group) and 51 healthy infants of the same age (HC group). After bacterial DNA extraction, 16S rRNA expression was measured and the diversity of gut microbiota and constituent bacteria were compared between the two groups. The alpha diversity of gut microbiota (median Shannon index and Chao index) was significantly lower in the fUTI group (3.0 and 42.5) than in the HC group (3.7 and 97.0; p < 0.001). The beta diversity also formed different clusters between the two groups (p < 0.001), suggesting differences in their microbial composition. The linear discriminant analysis effect size showed that the fUTI group proportionally featured significantly more Escherichia-Shigella in the gut microbiota (9.5%) than the HC group (3.1%; p < 0.001). In summary, abnormal gut microbiota development during infancy may increase the risk of fUTI.

Diagnostic Significance of Absence of Post-Feeding Contraction of the Gallbladder in Biliary Atresia: Two Case Reports.

Ultrasonography is an essential part of the diagnostic process of biliary atresia (BA). The characteristic findings of BA include a hilar hyperechoic zone, the triangular cord sign (TCS), an absence of gallbladder contraction after feeding, and gallbladder atrophy. However, approximately 10% of patients with BA have a normal gallbladder. We herein present two cases of BA with normal morphology of the gallbladder as shown by ultrasonography. In the first case, the patient was positive for the TCS, negative for gallbladder atrophy, and positive for contraction of the gallbladder after feeding; the final diagnosis was hilar obstructive BA complicated by pancreaticobiliary maljunction. In the second case, the patient was positive for the TCS, negative for gallbladder atrophy, and negative for contraction of the gallbladder after feeding; the patient also had common bile duct obstruction and stenosis of the hepatic duct in the hilar region. Based on these two cases, we conclude that gallbladder findings are not diagnostic for BA because in some types, the gallbladder may be normal in morphology and even have the capacity for contraction after feeding.

Management of treatment-resistant nocturnal enuresis.

Nocturnal enuresis is defined as intermittent urinary incontinence during sleep in children 5 years of age and older, occurring at least once a month for at least 3 months. In Japan, pediatricians who do not specialize in nocturnal enuresis have become more proactive in treating the condition since 2016, when the guidelines for treating it were revised for the first time in 12 years. For monosymptomatic nocturnal enuresis, the first step is lifestyle guidance, with a focus on the restriction of fluid intake at night; however, if lifestyle guidance does not decrease the frequency of nocturnal enuresis, aggressive treatment should be added. The first choice of aggressive treatment is oral desmopressin, an antidiuretic hormone preparation, or alarm therapy. However, there remain patients whose wet nights do not decrease with oral desmopressin or alarm therapy. In such cases, it is necessary to reconfirm the method of desmopressin administration and check for factors that may decrease the efficacy of desmopressin. If alarm therapy does not increase the number of dry nights, it is possible that the patient is fundamentally unsuitable for alarm therapy. If dry nights do not increase with oral desmopressin or alarm therapy, the next treatment strategy should be considered immediately to keep the patient motivated for treatment.

Anti-Ku antibody-positive systemic sclerosis and idiopathic inflammatory myopathies overlap syndrome in children: a report of two cases and a review of the literature.

The occurrence of anti-Ku antibody-positive idiopathic inflammatory myopathy (IIM) in pediatric patients is rare, and therefore, the clinical phenotypes of this disease in such patients remain obscure. We herein report two cases of Japanese female pediatric patients with anti-Ku antibody-positive IIM. One case was unique in that it was complicated by pericardial effusion. Another patient had severe and refractory myositis with immune-mediated necrotizing myopathy. In addition, we reviewed literatures involving a total of 11 pediatric patients with anti-Ku antibody-positive IIM. The median age of the patients was 11 years, and most of them were girls. Skin rash, including erythematous nodules, malar rash, multiple brownish plaques, butterfly rash, heliotrope rash, periorbital edema, and Gottron's papules, was observed in 54.5% of the patients, scleroderma in 81.8%, and skin ulcer in 18.2%. Their serum creatine kinase level ranged from 504 to 10,840 IU/L. Furthermore, joint involvement was observed in 91% of the patients, interstitial lung disease in 18.2%, and esophageal involvement in 9.1%. All patients were treated with corticosteroids in combination with immunosuppressants. Pediatric patients with anti-Ku antibody-positive IIM had unique characteristics compared to adult patients. Skin manifestations, joint involvement and elevation of serum CK levels were more common in children than in adults. In contrast, ILD and esophageal involvement were less common in children than in adults. Although pediatric cases of anti-Ku antibody-positive IIM are rare, patients with IIM need to be tested for the presence of anti-Ku antibodies.

Comparison of the characteristics and factors influencing hospital visits among children with nocturnal enuresis in Japan: The Hirakata-Urayasu population-based cohort study.

OBJECTIVES: The aim of this study was to compare the demographic characteristics of school-aged children with nocturnal enuresis and factors influencing hospital visits between two regions in Japan. METHODS: A cross-sectional survey was conducted in Hirakata City, Osaka Prefecture, and Urayasu City, Chiba Prefecture. An anonymous online questionnaire was administered to all public elementary and junior high school students (aged 6-16 years) or their guardians. Questions included age, gender, perinatal history, frequency of nocturnal enuresis, frequency of bowel movements, comorbidities, and hospital visits for nocturnal enuresis. RESULTS: The survey response rates were 15.4% in Hirakata City and 37.0% in Urayasu City. In total, 426 children with nocturnal enuresis in Hirakata City and 270 in Urayasu City were included in the final analysis. In both cities, the boy-girl ratio was approximately 2:1, and the prevalence of nocturnal enuresis gradually decreased with age. Multivariate analysis revealed that children aged ≥11 years had a significantly higher proportion of hospital visits (OR, 2.61; 95% CI: 1.49-4.56; p = 0.001; OR, 2.72; 95% CI: 1.12-6.64; p = 0.027, respectively). However, the frequency of nocturnal enuresis did not affect hospital visits. CONCLUSIONS: The findings of this study suggest that parents with school-aged children have low awareness that nocturnal enuresis is a health problem and therefore subject to medical consultation. Although the proportion of hospital visits increases for children aged ≥11 years, children and families suffering from nocturnal enuresis should be encouraged to see a doctor instead of adopting a "wait and see attitude," even at a young age.

Sonic hedgehog N-terminal level correlates with adiponectin level and insulin resistance in adolescents.

OBJECTIVES: Extracellular vesicles (EVs) are small vesicles released by nearly all types of cells. They deliver different types of substances, including proteins and nucleic acids, to nearby or distant cells and play a role in the mediation of cell-to-cell communication. The aim of this study was to explore the association between EVs and insulin resistance in adolescents with obesity or type 2 diabetes mellitus (DM2). METHODS: The subjects were eight adolescents with DM2 (DM2 group; four males and four females; age: 18.1 ± 2.3 years), 18 adolescents with simple obesity (obesity group; 12 males and six females; age: 12.2 ± 3.4 years), and 20 controls (control group; 10 males and 10 females; age: 13.0 ± 1.4 years). As markers of EVs, serum CD9/CD63 and sonic hedgehog N-terminal (Shh-N) levels were measured using enzyme-linked immunosorbent assay. RESULTS: The CD9/CD63 level in the control group was similar to that in the DM2 group, whereas the obesity group had a significantly higher CD9/CD63 level. In the entire study group, correlations were observed between serum Shh-N level and Homeostasis Model Assessment of insulin resistance (HOMA-IR) score (r=0.371, p=0.0143), Homeostasis Model Assessment-ß cell function score (r=0.382, p=0.0115), serum insulin level (r=0.350, p=0.0171), and serum adiponectin level (r=0.367, p=0.0122). Multiple regression analysis revealed that serum Shh-N level was the most significant risk factor for HOMA-IR score and serum insulin level. CONCLUSIONS: Shh is correlated with insulin resistance via its association with adiponectin in adolescents.

Dysbiosis of the gut microbiota in children with severe motor and intellectual disabilities receiving enteral nutrition: A pilot study.

BACKGROUND: Children with severe motor and intellectual disabilities (SMIDs) frequently and continuously receive enteral nutrition and medications and lack adequate exercise, which may lead to dysbiosis, an imbalance in the composition of the gut microbiota. However, studies on the composition of gut microbiota in children with SMIDs are limited. Therefore, we aimed to examine the characteristics of the gut microbiota in children with SMIDs. METHODS: 16S rRNA gene sequencing was performed using fecal samples of 10 children with SMIDs, who received enteral nutrition through a gastric fistula or gastric tube (SMID group: median age, 10.0 years), and 19 healthy children (healthy control [HC] group: median age, 9.0 years). Microbial diversity, microbial composition, and abundance of butyric acid-producing bacteria were compared between the groups. Daily dietary fiber intake in the SMID group was evaluated using questionnaires. RESULTS: The Shannon and Simpson indices (alpha diversity indices) were significantly lower in the SMID group than those in the HC group. Beta diversity analysis identified different clusters. Compared with the HC group, Clostridiales and butyric acid-producing bacteria were less abundant and Bacteroidales were more abundant in the SMID group. Dietary fiber intake in the SMID group was approximately two-thirds of the estimated average requirement for healthy Japanese children. CONCLUSION: Children with SMIDs showed dysbiosis with alteration in the microbial diversity, which could partly be attributed to their low dietary fiber intake. Further studies, with the intervention of prebiotics, probiotics, and synbiotics, are warranted to improve dysbiosis in children with SMIDs.

Dysbiosis in Gut Microbiota in Children Born Preterm Who Developed Autism Spectrum Disorder: A Pilot Study.

The gut microbiota was reported to differ between children with autism spectrum disorder (ASD) and typically developing (TD) children, and dysbiosis of the gut microbiota in preterm infants is common. Here, we explored the characteristics of gut microbiota in children born preterm with ASD. We performed 16S rRNA gene sequencing using stool samples from ASD children born preterm and TD children born preterm. Alpha diversity was significantly greater in the ASD group. A comparison of beta diversity showed different clusters. Linear discriminant analysis effect size analysis revealed significantly more Firmicutes in the ASD group compared with the TD group. In conclusion, the gut microbiota in children born preterm differs between children with ASD and TD.

Effect of a Bifidobacterium-Containing Acid-Resistant Microcapsule Formulation on Gut Microbiota: A Pilot Study.

Approximately 10 Bifidobacterium species are known to inhabit the human intestinal tract. Bifidobacteria have been reported to possess a variety of probiotic benefits. However, when bifidobacteria are consumed internally as probiotics, the bacteria are killed by gastric acid. Therefore, we developed acid-resistant microcapsules containing Bifidobacterium breve M-16V and B. longum BB536, which are unaffected by gastric acid, and evaluated whether the microcapsule formulation increased the amount of bifidobacteria in the stool after administration compared with the powder formulation. The results revealed no significant difference in the percentage or number of B. longum between before and after administration of the powder or microcapsule formulation in children. By contrast, the bacterial count of B. breve was significantly increased after microcapsule formulation administration (1.5 × 105 copies/g after administration versus 2.8 × 104 copies/g before administration, p = 0.013). In addition, the increase in the bacterial count of B. breve in stools after administration of microcapsule formulation was approximately 1000-fold higher than that after powder formulation administration (p = 0.018). In conclusion, the results indicate that the microcapsule formulation is efficiently transferred to the large intestine without the adverse effects of gastric acidity in children.

Transition from undergraduates to residents: A SWOT analysis of the expectations and concerns of Japanese medical graduates during the COVID-19 pandemic.

INTRODUCTION: Interruptions in undergraduate clinical clerkship during the COVID-19 pandemic have reduced the confidence and preparedness of residents beginning their postgraduate training. We explore the thoughts of new residents about this transition and reflect on the support needed. METHODS: An exploratory qualitative case study was conducted with 51 residents. All had experienced interruptions in clinical training due to the pandemic and had just started their postgraduate training. Qualitative data were collected through 6 focus groups and 12 individual follow-up interviews. A thematic analysis was undertaken, and the data were categorised using a Strengths, Weaknesses, Opportunities, and Threats (SWOT) framework. RESULTS: Graduates beginning their residency were aware of their professionalism and independence during the transition. They also faced the predicament of needing close supervision while their supervisors managed pandemic conditions. Residents emphasised the importance of developing relationships with colleagues and supervisors during the transition to residency and wanted direct observation and detailed feedback from their supervisors during procedures. CONCLUSIONS: The experiences of residents were not uniformly negative. In fact, some had developed a positive mindset when entering the clinical field. Medical faculty members reflecting on interactions with new residents and planning future clinical internships could benefit from placing a high value on building relationships among residents, who may expect direct observation and detailed feedback from their supervisors.

Accuracy of diagnosing acute kidney injury by assessing urine output within the first week of life in extremely preterm infants.

BACKGROUND: Neonatal acute kidney injury (AKI) is associated with increased mortality and is often assessed with the neonatal modified Kidney Disease: improving Global Outcomes (KDIGO) classification, which uses changes in serum creatinine levels. However, because this classification has many drawbacks, a novel method, the neonatal Risk, Injury, Failure, Loss, and End-Stage Kidney Disease (nRIFLE) classification for diagnosing neonatal AKI according to urine output (UO), was recently proposed. To date, no data on the incidence of AKI according to nRIFLE are available for extremely preterm infants (born at gestational age less than 28 weeks). This study was conducted to clarify the association between incidence of AKI and in-hospital mortality in extremely preterm infants. METHODS: Of 171 extremely preterm infants hospitalized from 2006 to 2020, 84 in whom indwelling bladder catheters were placed for UO measurements within 24 h of life were included. The incidence of AKI was assessed using the nRIFLE classification. In-hospital mortality was compared between patients with AKI and those without it. RESULTS: The incidence of AKI during the first week of life was 56% and that of in-hospital mortality was significantly higher in patients with AKI (25.5%) than in those without it (2.8%). The odds ratio was 12.3 with 95% confidence interval ranging from 1.5 to 100.0. CONCLUSION: The incidence of AKI according to nRIFLE was higher than reported in most previous studies using the neonatal modified KDIGO classification, suggesting that assessment by nRIFLE criteria using UO may improve diagnostic accuracy of AKI in extremely preterm infants.

Psychogenic fever and postural tachycardia syndrome among school-aged children and adolescents with fever of unknown origin.

BACKGROUND: Although fever is a common symptom in pediatric practice, its origin is often unknown in pediatric patients. Psychogenic fever is a stress-related, psychosomatic disease observed especially in young women. This study aimed to estimate the prevalence of psychogenic fever in pediatric patients with fever of unknown origin by surveying the medical records of school-aged children and adolescents. METHODS: The study subjects included 47 patients aged 6-15 years who visited the Department of Pediatrics in Kansai Medical University Medical Center between January 2006 and December 2020 with fever of unknown origin. Data on age, sex, final estimated diagnosis, and comorbid psychosocial issues were collected from the medical records. RESULTS: The study was composed of 47 patients, including 22 male and 25 female patients (male/female ratio, 1:1.36). The mean age was 10.1 (standard deviation, 2.4) years for boys and 11.6 (standard deviation, 2.7) years for girls (p = .047). The final estimated diagnoses were psychogenic fever, physical disorder, infection of unknown origin, and miscellaneous in 18, 12, 12, and 5 patients, respectively. The most common comorbidity in these pediatric patients with psychogenic fever was postural tachycardia syndrome. CONCLUSION: Psychogenic fever was a common cause of fever of unknown origin in pediatric patients, and postural tachycardia was prevalent among children with psychogenic fever. Enhanced sympathetic response to stress might play an important role in both psychogenic fever and postural tachycardia.

Gut microbiota and allergic diseases in children.

The gut microbiota resides in the human gastrointestinal tract, where it plays an important role in maintaining host health. The human gut microbiota is established by the age of 3 years. Studies have revealed that an imbalance in the gut microbiota, termed dysbiosis, occurs due to factors such as cesarean delivery and antibiotic use before the age of 3 years and that dysbiosis is associated with a higher risk of future onset of allergic diseases. Recent advancements in next-generation sequencing methods have revealed the presence of dysbiosis in patients with allergic diseases, which increases attention on the relationship between dysbiosis and the development of allergic diseases. However, there is no unified perspective on the characteristics on dysbiosis or the mechanistic link between dysbiosis and the onset of allergic diseases. Here, we introduce the latest studies on the gut microbiota in children with allergic diseases and present the hypothesis that dysbiosis characterized by fewer butyric acid-producing bacteria leads to fewer regulatory T cells, resulting in allergic disease. Further studies on correcting dysbiosis for the prevention and treatment of allergic diseases are warranted.

Research Publication Experience as a Requirement for Board Examination Acceptance to Promote Scholarly Activities of Pediatric Residents.

INTRODUCTION: Scholarship is an essential component of postgraduate education. This study's objective was to investigate the effect of a new reform requiring research publication experience before taking the pediatric board examination to promote scholarly activities among pediatric residents in Japan. METHODS: We conducted an experimental study from 2015 to 2018 to investigate the effectiveness of this reform for promoting scholarly activities among Japanese pediatric residents. RESULTS: Of all 2524 participants, the number of examinees before and after the reform was 1580 and 944, respectively. The yearly number of the residents' presentations and publications during their residency was 1.2 (SD 0.9) and 0.06 (SD 0.16), respectively, before the reform and 1.3 (SD 1.0) and 0.21 (SD 0.18), respectively, after the reform. Multiple regression showed the post-reform examinees (ß = 0.37, p < 0.01) and the number of research presentations (ß = 0.28, p < 0.01) were significantly associated with the number of research publications during the residency. While no contributive variables were found in the institution types, residents in the Kyushu and Okinawa area (i.e., southern island area in Japan) published fewer articles than those in the Tokyo area (ß = -0.05, p = 0.03). CONCLUSIONS: The newly implemented policy requiring residents to publish research articles as a board examination prerequisite effectively promotes research activities among pediatric residents.