Association of hyaluronan and proteoglycan link protein 1 gene with the need of home oxygen therapy in premature Japanese infants with bronchopulmonary dysplasia.

BACKGROUND: Bronchopulmonary dysplasia (BPD) has a lasting effect on the respiratory function of infants, imposing chronic health burdens. BPD is influenced by various prenatal, postnatal, and genetic factors. This study explored the connection between BPD and home oxygen therapy (HOT), and then we examined the association between HOT and a specific single-nucleotide polymorphism (SNP) in the hyaluronan and proteoglycan link protein 1 (HAPLN1) gene among premature Japanese infants. MATERIALS AND METHODS: Prenatal and postnatal data from 212 premature infants were collected and analyzed by four SNPs (rs975563, rs10942332, rs179851, and rs4703570) around HAPLN1 using the TaqMan polymerase chain reaction method. The clinical characteristics and genotype frequencies of HAPLN1 were assessed and compared between HOT and non-HOT groups. RESULTS: Individuals with AA/AC genotypes in the rs4703570 SNP exhibited significantly higher HOT rates at discharge than those with CC homozygotes (odds ratio, 1.20, 95% confidence interval, 1.07-1.35, p = .038). A logistic regression analysis determined that CC homozygotes in the rs4703570 SNP did not show a statistically significant independent association with HOT at discharge. CONCLUSIONS: Although our study did not reveal a correlation between HAPLN1 and the onset of BPD, we observed that individuals with CC homozygosity at the rs4703570 SNP exhibit a reduced risk of HOT.

Diphenyl-tetrazol-propanamide Derivatives Act as Dual-Specific Antagonists of Platelet CLEC-2 and Glycoprotein VI.

BACKGROUND: Platelet C-type lectin-like receptor 2 (CLEC-2) induces platelet activation and aggregation after clustering by its ligand podoplanin (PDPN). PDPN, which is not normally expressed in cells in contact with blood flow, is induced in inflammatory immune cells and some malignant tumor cells, thereby increasing the risk of venous thromboembolism (VTE) and tumor metastasis. Therefore, small-molecule compounds that can interfere with the PDPN-CLEC-2 axis have the potential to become selective antiplatelet agents. METHODS AND RESULTS: Using molecular docking analysis of CLEC-2 and a PDPN-CLEC-2 binding-inhibition assay, we identified a group of diphenyl-tetrazol-propanamide derivatives as novel CLEC-2 inhibitors. A total of 12 hit compounds also inhibited PDPN-induced platelet aggregation in humans and mice. Unexpectedly, these compounds also fit the collagen-binding pocket of the glycoprotein VI molecule, thereby inhibiting collagen interaction. These compounds also inhibited collagen-induced platelet aggregation, and one compound ameliorated collagen-induced thrombocytopenia in mice. For clinical use, these compounds will require a degree of chemical modification to decrease albumin binding. CONCLUSION: Nonetheless, as dual activation of platelets by collagen and PDPN-positive cells is expected to occur after the rupture of atherosclerotic plaques, these dual antagonists could represent a promising pharmacophore, particularly for arterial thrombosis, in addition to VTE and metastasis.

Diagnosis and prevention of the vasodepressor type of neurally mediated syncope in Japanese patients.

We investigated circulatory dynamics in patients with vasodepressor type neurally mediated syncope (VT-NMS) by performing high-resolution Holter electrocardiography and a correlation analysis of changes in adenylate cyclase activity, blood pressure, and pulse during the head-up tilt test. Holter electrocardiography was performed for 30 patients. Adenylate cyclase activity was evaluated in lymphocytes from blood samples taken at rest and during the head-up tilt test. There was no change in autonomic nerve fluctuation during electrocardiography in VT-NMS patients, but our results showed a significant difference in blood pressure and adenylate cyclase activity between VT-NMS patients and healthy volunteers; the systolic blood pressure of VT-NMS patients decreased after 5 min, while at 10 min, the adenylate cyclase activity was the highest (0.53%) and the systolic blood pressure was the lowest (111.8 mm Hg). Pulse rates increased after 10 min. VT-NMS patients showed higher blood pressure, pulse rate, and adenylate cyclase activity during the tilt test than did healthy volunteers. In patients with syncope, standing for longer than 10 minutes may increase the risk of VT-NMS. From our results, we consider it likely that high systolic blood pressure and adenylate cyclase activity at rest cause fainting in VT-NMS patients. Our findings may be helpful for identifying individuals with a high risk of developing NMS in the healthy population.

Functionally confirmed compound heterozygous ADAM17 missense loss-of-function variants cause neonatal inflammatory skin and bowel disease 1.

A disintegrin and metalloprotease 17 (ADAM17) is the major sheddase that processes more than 80 substrates, including tumour necrosis factor-α (TNFα). The homozygous genetic deficiency of ADAM17 causing a complete loss of ADAM17 expression was reported to be linked to neonatal inflammatory skin and bowel disease 1 (NISBD1). Here we report for the first time, a family with NISBD1 caused by functionally confirmed compound heterozygous missense variants of ADAM17, namely c.1699T>C (p.Cys567Arg) and c.1799G>A (p.Cys600Tyr). Both variants were detected in two siblings with clinical features of NISBD1, such as erythroderma with exudate in whole body, recurrent skin infection and sepsis and prolonged diarrhoea. In a cell-based assay using Adam10/17 double-knockout mouse embryonic fibroblasts (Adam10/17-/- mEFs) exogenously expressing each of these mutants, phorbol 12-myristate 13-acetate-stimulated shedding was strongly reduced compared with wild-type ADAM17. Thus, in vitro functional assays demonstrated that both missense variants cause the loss-of-function of ADAM17, resulting in the development of NISBD1. Our study further expands the spectrum of genetic pathology underlying ADAM17 in NISBD1 and establishes functional assay systems for its missense variants.

Monitoring the autophagy-endolysosomal system using monomeric Keima-fused MAP1LC3B.

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes. Dysfunction of this system is associated with many pathological conditions such as cancer, inflammation and neurodegenerative diseases. Therefore, monitoring the cellular autophagy-endolysosomal activity is crucial for studies on the pathogenesis as well as therapeutics of such disorders. To this end, we here sought to create a novel means exploiting Keima, an acid-stable fluorescent protein possessing pH-dependent fluorescence excitation spectra, for precisely monitoring the autophagy-endolysosomal system. First, we generated three lines of transgenic (tg) mouse expressing monomeric Keima-fused MAP1LC3B (mKeima-LC3B). Then, these tg mice were subjected to starvation by food-restriction, and also challenged to neurodegeneration by genetically crossing with a mouse model of amyotrophic lateral sclerosis; i.e., SOD1H46R transgenic mouse. Unexpectedly, despite that a lipidated-form of endogenous LC3 (LC3-II) was significantly increased, those of mKeima-LC3B (mKeima-LC3B-II) were not changed under both stressed conditions. It was also noted that mKeima-LC3B-positive aggregates were progressively accumulated in the spinal cord of SOD1H46R;mKeima-LC3B double-tg mice, suggestive of acid-resistance and aggregate-prone natures of long-term overexpressed mKeima-LC3B in vivo. Next, we characterized mouse embryonic fibroblasts (MEFs) derived from mKeima-LC3B-tg mice. In contrast with in vivo, levels of mKeima-LC3B-I were decreased under starved conditions. Furthermore, when starved MEFs were treated with chloroquine (CQ), the abundance of mKeima-LC3B-II was significantly increased. Remarkably, when cultured medium was repeatedly changed between DMEM (nutrient-rich) and EBSS (starvation), acidic/neutral signal ratios of mKeima-LC3B-positive compartments were rapidly and reversibly shifted, which were suppressed by the CQ treatment, indicating that intraluminal pH of mKeima-LC3B-positive vesicles was changeable upon nutritional conditions of culture media. Taken together, although mKeima-LC3B-tg mice may not be an appropriate tool to monitor the autophagy-endolysosomal system in vivo, mKeima-LC3B must be one of the most sensitive reporter molecules for monitoring this system under in vitro cultured conditions.

Efficacy of an elimination diet in a patient with eosinophilic gastroenteritis : a pediatric case with multiple food allergies.

Eosinophilic gastrointestinal disorders are chronic inflammatory diseases in which eosinophils highly infiltrate into gastrointestinal tissue, resulting in gastrointestinal dysfunction. Here, we report a case of pediatric eosinophilic gastroenteritis (EGE). A 7-year-old boy with multiple food allergies (cow milk, hen's egg, fish,shellfish, and chicken) was admitted to our hospital because of continuous abdominal pain and vomiting. His soy allergy had been diagnosed to have oral tolerance based on an oral food challenge at the age of 6 years. He was diagnosed with EGE based on biopsy findings showing eosinophilic infiltration ( 20 eosinophils per high-power field) into the gastrointestinal mucosa. A diet eliminating soy, wheat, beef, pork, rice, and sesame in addition to the food that had already been eliminated and oral corticosteroids improved his symptoms and peripheral eosinophilia. A relapse of both abdominal pain and peripheral eosinophilia after the reintroduction of soy or pork identified them as foods causative of EGE. This report highlights the utility of elimination diets in improving EGE symptoms and the subsequent reintroduction of offending foods in identifying causative foods. Furthermore,EGE onset should be considered when introducing potentially allergic food in the management of food allergy. J. Med. Invest. 66 : 201-204, February, 2019.

Neurally mediated syncope diagnosis based on adenylate cyclase activity in Japanese patients.

The study aims to clarify the mechanism in patients with neurally mediated syncope (NMS), focusing on the adenylate cyclase (AC) activity level in lymphocytes. This study included 40 subjects: 22 healthy volunteers and 18 NMS patients. We investigated the changes in AC activity that occur during of syncope at rest and during the head-up tilt (HUT) test. We obtained 8 mL of blood at rest time and four times during the HUT test. Then, we measured the AC activity and the test reagent was added to the lymphocytes (10,000) and reacted for 30 min at room temperature. We were able to determine the standard value of AC activity when adrenaline (AD) and isoproterenol (IP) were added to lymphocytes. The results of our study showed one of the causes of NMS has a difference in AC activity level and classification of the patients into two different types of NMS was possible: either the vasodepressor type (VT) or mixed type (MT). At rest time, VT patients showed significantly higher AC activity (AD; 100 µM: p = 0.005, IP; 50 µM: p = 0.02) and MT patients showed significantly lower AC activity (AD; 10 µM: p = 0.02, IP; 50 µM: p = 0.004) than the average AC activity in healthy volunteers. Moreover, VT patients had significantly higher AC activity than healthy volunteers at the four points of the HUT test. MT patients had significantly lower AC activity (AD: p = 0.04 and IP: p = 0.04) than healthy volunteers at the rest time of HUT. Our study showed a significant difference in AC activities between NMS patients and healthy volunteers at rest. Therefore, a detailed NMS diagnosis can be made by examining AC activity levels in blood taken at rest time.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the quantitative analysis of mammalian-derived inositol poly/pyrophosphates.

Although myo-inositol pyrophosphates such as diphosphoinositol pentakisphosphate (InsP7) are important in biology, little quantitative information is available regarding their presence in mammalian organisms owing to the technical difficulties associated with accurately detecting these materials in biological samples. We have developed an analytical method whereby InsP7 and its precursor inositol hexakisphosphate (InsP6) are determined directly and sensitively using tandem mass spectrometry coupled with hydrophilic interaction liquid chromatography (HILIC). InsP6 and InsP7 peak symmetry is influenced greatly by the buffer salt composition and pH of the mobile phase used in HILIC analysis. The use of 300 mM ammonium carbonate (pH 10.5) as an aqueous mobile phase resolves InsP6 and InsP7 on a polymer-based amino HILIC column with minimal peak tailing. Method validation shows that InsP6 and InsP7 can be quantitated from 20-500 pmol with minimal intra-day/inter-day variance in peak area and retention time. The concentration of InsP6 in C57BL/6J mouse brain (40.68 ± 3.84 pmol/mg wet weight) is successfully determined. HILIC‒MS/MS analysis using HEK293 culture cells confirms previous observations that InsP7 is induced by NaF treatment and ectopic expression of InsP6K2, a primary kinase for InsP7 synthesis. Furthermore, this analysis reveals the abundance of InsP6 (50.46 ± 18.57 pmol/106 cells) and scarcity of InsP7 in human blood cells. The results demonstrate that HILIC‒MS/MS analysis can quantitate endogenous InsP6 and InsP7 in mouse and human samples, and we expect that the method will contribute to further understanding of InsP7 functions in mammalian pathobiology.

Ultrasound follow-up in a patient with intestinal obstruction due to post-traumatic intramural duodenal hematoma.

We report the case of a 7-year-old girl with intestinal obstruction due to post-traumatic intramural duodenal hematoma. She had fallen from the monkey bars the day before presenting to our hospital, and was admitted with signs of abdominal pain, vomiting, and nausea. Abdominal ultrasonography, computed tomography (CT), and magnetic resonance imaging (MRI) demonstrated a heterogeneous solid mass located within the duodenal wall, compressing the descending part of the duodenum. The inferior vena cava was also compressed by the mass lesion, although no associated symptoms were evident. Based on these findings, the mass lesion was considered to represent intramural hematoma causing intestinal obstruction. She was managed conservatively with total parenteral nutrition. Although CT and MRI are useful for differentiating hematoma from other intestinal tumors, ultrasonography is minimally invasive and easier to perform repeatedly. In case of duodenal hematoma, ultrasonography may be quite helpful for diagnosis and follow-up by monitoring tumor size and characteristics, and the degree of duodenal compression during conservative treatment.