Kazunori Miki

The Rab8 GTPase regulates apical protein localization in intestinal cells

A number of proteins are known to be involved in apical/basolateral transport of proteins in polarized epithelial cells. The small GTP-binding protein Rab8 was thought to regulate basolateral transport in polarized kidney epithelial cells through the AP1B-complex-mediated pathway. However, the role of Rab8 (Rab8A) in cell polarity in vivo remains unknown. Here we show that Rab8 is responsible for the localization of apical proteins in intestinal epithelial cells. We found that apical peptidases and transporters localized to lysosomes in the small intestine of Rab8-deficient mice. Their mislocalization and degradation in lysosomes led to a marked reduction in the absorption rate of nutrients in the small intestine, and ultimately to death. Ultrastructurally, a shortening of apical microvilli, an increased number of enlarged lysosomes, and microvillus inclusions in the enterocytes were also observed. One microvillus inclusion disease patient who shows an identical phenotype to Rab8-deficient mice expresses a reduced amount of RAB8 (RAB8A; NM_005370). Our results demonstrate that Rab8 is necessary for the proper localization of apical proteins and the absorption and digestion of various nutrients in the small intestine.

The sugar permeability test reflects disease activity in children and adolescents with inflammatory bowel disease

OBJECTIVES To investigate the relationship of intestinal permeability in children and adolescents with inflammatory bowel disease (IBD) to disease activity, disease extent, and response to therapy. STUDY DESIGN Patients with new and established diagnoses of IBD (12 Crohns disease [CD] and 18 ulcerative colitis [UC]) were studied. Intestinal permeability was evaluated by measuring with high-performance liquid chromatography 5-hour urinary excretion ratio of lactulose/L-rhamnose (L/Rh). RESULTS In 8 of 9 patients with active CD, the L/Rh ratio was higher than the reference range (0.006 to 0.074, n = 36). In inactive CD (n = 3) the L/Rh ratio was within the reference range. In 6 of 7 patients with active extensive UC, the L/Rh ratio was elevated. In inactive extensive UC (n = 6) the normal permeability ratio was shown. In both active CD and active extensive UC, the frequency of elevated intestinal permeability was significantly greater than values in both inactive forms. The permeability ratio was normal in 4 of 5 patients with active left-sided colitis. In 5 of 7 patients (3 CD, 4 UC), repeat permeability values entered the reference range after acute phase therapy. Two patients with persistently elevated intestinal permeability (1 CD, 1 UC) had a disease flare-up within 6 months. CONCLUSIONS Intestinal permeability is a marker of disease activity in CD and extensive UC. Serial permeability test may be useful in monitoring disease activity.

Three paediatric cases of primary sclerosing cholangitis treated with ursodeoxycholic acid and sulphasalazine

We present here three paediatric patients with primary sclerosing cholangitis. In case 1, the serum γ‐glutamyl transpeptidase was decreased only temporarily by ursodeoxycholic acid (UDCA) treatment and 34 months later, sulphasalazine was added because of microscopic colitis. The enzyme level decreased with dual therapy. Similarly, in case 3, first diagnosed as autoimmune hepatitis, the transpeptidase levels remained elevated for 18 months during treatment with UDCA, prednisolone and mizoribin. The enzyme decreased only after a diagnosis of primary sclerosing cholangitis complicated with ulcerative colitis was established and sulphasalazine was introduced. Case 2 also had Crohns colitis and was put on UDCA and sulphasalazine from the start. The enzyme level was normalized within 1 month and has remained normal for the following 5 years. Liver biopsies were analysed repeatedly in these three patients. In case 1, periductal fibrosis remained unchanged while being treated by UDCA. There appeared to be no progression in liver cirrhosis in case 3 while being treated by UDCA, prednisolone, and mizoribin. In case 2, who has been treated with both UDCA and sulphasalazine from the start, periductal fibrosis and portal fibrosis were remarkably improved 45 months later. We suggest that sulphasalazine in addition to UDCA might be a viable treatment for children with primary sclerosing cholangitis.

Two novel mutations in the ED1 gene in Japanese families with X-linked hypohidrotic ectodermal dysplasia.

X-linked hypohidrotic ectodermal dysplasia (XLHED), which is characterized by hypodontia, hypotrichosis, and hypohidrosis, is caused by mutations in ED1, the gene encoding ectodysplasin-A (EDA). This protein belongs to the tumor necrosis factor ligand superfamily. We analyzed ED1 in two Japanese patients with XLHED. In patient 1, we identified a 4-nucleotide insertion, c.119-120insTGTG, in exon 1, which led to a frameshift mutation starting from that point (p.L40fsX100). The patients mother was heterozygous for this mutation. In patient 2, we identified a novel missense mutation, c.1141G>C, in exon 9, which led to a substitution of glycine with arginine in the TNFL domain of EDA (p.G381R). This patients mother and siblings showed neither symptoms nor ED1 mutations, so this mutation was believed to be a de novo mutation in maternal germline cells. According to molecular simulation analysis of protein structure and electrostatic surface, p.G381R increases the distance between K375 in monomer A and K327 in monomer B, which suggests an alteration of overall structure of EDA. Thus, we identified two novel mutations, p.L40fsX100 and p.G381R, in ED1 of two XLHED patients. Simulation analysis suggested that the p.G381R mutation hampers binding of EDA to its receptor via alteration of overall EDA structure.

Hypophosphatemic Rickets Accompanying Congenital Microvillous Atrophy

This report concerns an 11‐year‐old boy who manifested hypophosphatemic rickets associated with congenital microvillous atrophy (CMA). He had been suffering from vomiting and severe diarrhea from the first day of life and had been treated with total parenteral nutrition (TPN) since he was 67 days old. At 4 years of age, intestinal biopsy resulted in a diagnosis of CMA. He was admitted to our hospital complaining of leg pain at the age of 11. Laboratory data revealed hypophosphatemia, elevated serum 1,25‐dihydroxyvitamin D (1,25(OH)2D) levels, and hypercalciuria. A roentgenogram showed rickets in the extremities. A balance study of phosphate in urine and stool indicated that the amount of phosphate leaking into the stool was greater than that into the urine. Moreover, the total amount of phosphate leaking from both the intestine and kidney exceeded the amount of phosphate intake from TPN. The rickets was healed by increasing the phosphate concentration in TPN. This case is different from X‐linked hypophosphatemic rickets but similar to hereditary hypophosphatemic rickets with hypercalciuria (HHRH) in terms of hypercalciuria and elevated serum 1,25(OH)2D levels. The effectiveness of phosphate treatments used here is also similar to that used for HHRH. However, this type of hypophosphatemic rickets is unique in that phosphate leaking into the intestine plays an important role in its pathogenesis.

Urinary sulfated bile acid concentrations in infants with biliary atresia and breast-feeding jaundice.

A selective screening for an early identification of biliary atresia (BA) and hepatobiliary disease is advocated 1 using tests for urinary bilirubin and serum direct bilirubin in the third week of life. However, venepuncture is not desirable in small infants for a primary screening purpose at a clinic. The urinary excretion of sulfated bile acid (USBA) increases with cholestasis through an activated alternate metabolic pathway of bile acid. 2 A direct enzymatic assay of USBA has been reported as a sensitive and rapid method of detecting cholestatic jaundice, replacing measurements of serum-direct bilirubin for selective screening for BA and neonatal hepatitis syndrome. 3 We have measured USBA in BA before surgery and other infantile cholestatic conditions since 1996. The present report summarizes the feasibility of this urinary test as a screening modality.

Utility of Duplex Doppler Ultrasound in Evaluating Portal Hypertension in Children

We measured the maximal velocity of the blood flow in the main portal trunk by duplex Doppler ultrasound in children suffering from a variety of liver diseases. The maximal velocity of the main portal vein in children with chronic active hepatitis and liver cirrhosis was decreased significantly as compared to those in control children (p < 0.01). The maximal velocity of the main portal trunk and K indocyanine green (ICG) had no significant correlation (r = 0.25, n = 27). The patient with the lowest portal velocity had esophageal and gastric varices with red color sign. We conclude that the measurement of the maximal velocity of the main portal trunk with duplex Doppler ultrasound is useful in evaluating portal hypertension in children.

Prevention of Neonatal HBV Infection with the Combination of HBIG and HBV Vaccine and Its Long‐Term Efficacy in Infants Born to HBeAg Positive HBV Carrier Mothers

Seventy‐three infants born to HBeAg positive HBV carrier mothers were protected from neonatal HBV infection with our standard prevention schedule consisting of two doses of HBIC (0, 2 mo) and three doses of HBV vaccine (2, 3, 5 mo). In 62 infants who successfully responded to HBV vaccine with a titer of anti‐HBs greater than 23, anti‐HBs titer was monitored for as long as 48 months (25.6 ±11.0 mo) and found to decrease as follows: 5.1 ± 1.7 at 12 mo., 4.5 ± 1.8 at 18 mo., 4.2 ± 1.8 at 24 mo., 4.0± 1.6 at 30 mo., 3.7 ± 1.7 at 36 mo., 32 ± 2.0 at 48 mo. During the follow‐up period, eight HBV events (11.9%) were demonstrated: one case showed an increase of anti‐HBs, three showed a reappearance of anti‐HBc alone, three showed a reappearance of anti‐HBc with increase of anti‐HBs, and one became a chronic HBV carrier. All infants were further divided into three groups by their maximal response of anti‐HBs to HBV vaccine: Group I (26), Group II (23‐25), and Group III (22). Group I sustained a higher titer from 12 to 30 months of age and had less HBV events than (3‐II and G‐III. Our study suggests that acquisition of a high titer of anti‐HBs is important in long‐term prevention of HBV infection as well as in the neonatal period in infants born to HBeAg positive HBV carrier mothers.

Mass Protection Program of Perinatal Hepatitis B Virus Infection in Japan and Impact of an Optional Booster Vaccination on Its Efficacy

We assessed the efficacy of a government‐sponsored mass protection program in Osaka, Japan, for perinatal HBV infection in infants born to HBeAg positive HBV carrier mothers. We also evaluated the impact of optional follow‐up procedures in such infants, including an evaluation of anti‐HBs response and a booster dose of HBV vaccine for poor responders. The results demonstrated that this mass protection program protected 94.4% of the infants from perinatal HBV infection in the Osaka area. However, the proportion of infants with an unprotective level of anti‐HBs was higher in the standard group than in the follow‐up group both at 1.0 and 1.5 years of age, which was also the case for HBV events. Furthermore, the present study showed that a booster dose of vaccine in poor responders was very effective in promoting an anti‐HBs response. In conclusion, we recommend that a follow‐up blood test to confirm a response of anti‐HBs to HBV vaccine should be performed at 4–8 weeks after the third injection of HBV vaccine in infants born to HBeAg positive HBV carrier mothers. We also recommend that a booster injection of HBV vaccine should be immediately given to poor responding infants who otherwise are at a considerable risk of developing HBV infection in late infancy.

Relationship between dose of antithyroid drugs and adverse events in pediatric patients with Graves’ disease

Abstract. Graves’ disease (GD) accounts for a large proportion of pediatric hyperthyroidism, and the first-line treatment is antithyroid drug (ATD) therapy. Methimazole (MMI) is effective in most patients but is associated with significant adverse events (AEs). We reviewed the medical records of GD patients (n = 56) with onset age of <15 yr and investigated the relationship between MMI dose and AEs. The study population comprised 11 male and 45 female patients and the median age at diagnosis was 11 yr. All patients were initially treated with ATDs. Among the 52 patients initially treated with MMI, 20 received a low dose, and 32 received a high dose of MMI (< 0.7 vs ≥ 0.7 mg/kg/day, respectively). AEs occurred in 20% of the patients in the low-dose MMI group, and in 50% patients in the high-dose MMI group (p = 0.031). A greater variety of AEs was observed in the high-dose group. Neutropenia and rash were observed in both groups. With treatment transition to low-dose MMI according to the Japanese Society for Pediatric Endocrinology guidelines, we expect a decrease in the incidence of AEs in future. However, we should be careful as neutropenia and rash can occur independently of the MMI dose.