Structural variation of the 3-acetamido-4,5,6-trihydroxyazepane iminosugar through epimerization and C-alkylation leads to low micromolar HexAB and NagZ inhibitors.

We report the synthesis of seven-membered iminosugars derived from a 3S-acetamido-4R,5R,6S-trihydroxyazepane scaffold and their evaluation as inhibitors of functionally related exo-N-acetylhexosaminidases including human O-GlcNAcase (OGA), human lysosomal ß-hexosaminidase (HexAB), and Escherichia coli NagZ. Capitalizing on the flexibility of azepanes and the active site tolerances of hexosaminidases, we explore the effects of epimerization of stereocenters at C-3, C-5 and C-6 and C-alkylation at the C-2 or C-7 positions. Accordingly, epimerization at C-6 (L-ido) and at C-5 (D-galacto) led to selective HexAB inhibitors whereas introduction of a propyl group at C-7 on the C-3 epimer furnished a potent NagZ inhibitor.

Selective trihydroxylated azepane inhibitors of NagZ, a glycosidase involved in Pseudomonas aeruginosa resistance to ß-lactam antibiotics.

The synthesis of a series of d-gluco-like configured 4,5,6-trihydroxyazepanes bearing a triazole, a sulfonamide or a fluorinated acetamide moiety at C-3 is described. These synthetic derivatives have been tested for their ability to selectively inhibit the muropeptide recycling glucosaminidase NagZ and to thereby increase sensitivity of Pseudomonas aeruginosa to ß-lactams, a pathway with substantial therapeutic potential. While introduction of triazole and sulfamide groups failed to lead to glucosaminidase inhibitors, the NHCOCF3 analog proved to be a selective inhibitor of NagZ over other glucosaminidases including human O-GlcNAcase and lysosomal hexosaminidases HexA and B.

Mutations in SRD5B1 (AKR1D1), the gene encoding delta(4)-3-oxosteroid 5beta-reductase, in hepatitis and liver failure in infancy.

BACKGROUND: A substantial group of patients with cholestatic liver disease in infancy excrete, as the major urinary bile acids, the glycine and taurine conjugates of 7alpha-hydroxy-3-oxo-4-cholenoic acid and 7alpha,12alpha-dihydroxy-3-oxo-4-cholenoic acid. It has been proposed that some (but not all) of these have mutations in the gene encoding delta(4)-3-oxosteroid 5beta-reductase (SRD5B1; AKR1D1, OMIM 604741). AIMS: Our aim was to identify mutations in the SRD5B1 gene in patients in whom chenodeoxycholic acid and cholic acid were absent or present at low concentrations in plasma and urine, as these seemed strong candidates for genetic 5beta-reductase deficiency. PATIENTS AND SUBJECTS: We studied three patients with neonatal onset cholestatic liver disease and normal gamma-glutamyl transpeptidase in whom 3-oxo-delta(4) bile acids were the major bile acids in urine and plasma and saturated bile acids were at low concentration or undetectable. Any base changes detected in SRD5B1 were sought in the parents and siblings and in 50 ethnically matched control subjects. METHODS: DNA was extracted from blood and the nine exons of SRD5B1 were amplified and sequenced. Restriction enzymes were used to screen the DNA of parents, siblings, and controls. RESULTS: Mutations in the SRD5B1 gene were identified in all three children. Patient MS was homozygous for a missense mutation (662 C>T) causing a Pro198Leu amino acid substitution; patient BH was homozygous for a single base deletion (511 delT) causing a frame shift and a premature stop codon in exon 5; and patient RM was homozygous for a missense mutation (385 C>T) causing a Leu106Phe amino acid substitution. All had liver biopsies showing a giant cell hepatitis; in two, prominent extramedullary haemopoiesis was noted. MS was cured by treatment with chenodeoxycholic acid and cholic acid; BH showed initial improvement but then deteriorated and required liver transplantation; RM had advanced liver disease when treatment was started and also progressed to liver failure. CONCLUSIONS: Analysis of blood samples for SRD5B1 mutations can be used to diagnose genetic 5beta-reductase deficiency and distinguish these patients from those who have another cause of 3-oxo-delta(4) bile aciduria, for example, severe liver damage. Patients with genetic 5beta-reductase deficiency may respond well to treatment with chenodeoxycholic acid and cholic acid if liver disease is not too advanced.

Measuring quality of life in children with inflammatory bowel disease: the impact-II (NL).

Inflammatory bowel disease (IBD) is a chronic debilitating disorder. Measures of quality of life are only available for adult patient populations. We developed a new disease-specific health-related quality of life instrument in Dutch for pediatric patients with IBD, called Impact-II (NL). We translated and strongly modified the original (Canadian) Impact questionnaire. It comprises 35 items in six domains. Eighty-three children (66%) completed the questionnaire, 39 children were assessed twice. Disease symptoms were recorded and disease course severity assessed through chart review. Summated disease activity scores and disease course severity scores were dichotomized into two categories. Reliability coefficients were good for five out of six domains (Cronbach's alpha ranged from 0.57 to 0.86) and measures of test-retest stability in clinically stable patients were good for all domains (intra-class correlation coefficients ranged from 0.67 to 0.91). The instrument showed good discriminant validity between symptom groups and disease course severity on all domains. Convergent validity with a validated generic instrument [TNO-AZL Children's Quality of life questionnaire (Tacqol)] showed satisfactory coefficients. In conclusion, the developed questionnaire shows good psychometric properties. Test-retest stability and responsiveness to change should be further assessed in larger patient samples. Cross-cultural translation and validation procedures into other languages are being conducted to enable international use of Impact-II.

Normal ranges of 24-hour pH-metry established in corrected esophageal atresia.

BACKGROUND: This study was undertaken to determine baseline values of 24-hour pH-metry in children who had undergone correction for esophageal atresia. METHODS: A 24-hour pH-metry without medication was conducted in 13 patients with an uncomplicated postoperative course after correction of esophageal atresia. The pH-metry was performed in the hospital with a flexible glass electrode. RESULTS: The mean reflux index was 4.08%, the mean total number of reflux periods with a pH less than 4 was 21, and the mean number of periods of pH less than 4 lasting longer than 5 minutes was 2.5. CONCLUSIONS: Twenty-four-hour pH-metry values in asymptomatic esophageal atresia patients are the same as in children from the same age group with normal anatomy.

Bone mineral density and nutritional status in children with chronic inflammatory bowel disease.

BACKGROUND: Osteoporosis has been reported in adult patients with inflammatory bowel disease. AIMS: To evaluate bone mineral density (BMD), nutritional status, and determinants of BMD in children with inflammatory bowel disease. PATIENTS: Fifty five patients (34 boys and 21 girls, age range 4-18) were studied; 22 had Crohn's disease and 33 ulcerative colitis. METHODS: Lumbar spine and total body BMD, and body composition were assessed by dual energy x ray absorptiometry (DXA). Results were expressed as standard deviation scores (SDS). Lean body mass was also assessed by bioelectrical impedance analysis (BIA). Yearly measurements during two years were performed in 21 patients. RESULTS: The mean SDS of lumbar spine BMD and total body BMD were significantly lower than normal (-0.75 and -0.95, both p < 0.001). Height SDS and body mass index SDS were also decreased. The decrease in BMD SDS could not be explained by delay in bone maturation. The cumulative dose of prednisolone correlated negatively with lumbar spine BMD SDS (r = -0.32, p < 0.02). Body mass index SDS correlated positively with total body BMD SDS (r = 0.36, p < 0.02). Patients with Crohn's disease had significantly lower lumbar spine and total body BMD SDS than patients with ulcerative colitis, even after adjustment for cumulative dose of prednisolone. In the longitudinal data cumulative dose of prednisolone between the measurements correlated negatively with the change in lumbar spine and total body BMD SDS. Lean tissue mass measured by DXA had a strong correlation with lean body mass measured by BIA (r = 0.98). CONCLUSIONS: Children with inflammatory bowel disease have a decreased BMD. Children with Crohn's disease have a higher risk of developing osteopaenia than children with ulcerative colitis. Corticosteroid therapy and nutritional status are important determinants of BMD in these patients.

Determinants of body composition measured by dual-energy X-ray absorptiometry in Dutch children and adolescents.

Knowledge about body composition is important in metabolic and nutritional studies. In this cross-sectional study the body composition of 403 healthy white Dutch children and adolescents was evaluated by using dual-energy X-ray absorptiometry (DXA). Possible determinants of body composition were analyzed. In 85 subjects the results of bioelectrical impedance analysis (BIA) were compared with DXA. Fat mass, lean tissue mass, and bone mineral content were greater in older boys and girls. Percentage body fat was greater in older girls but not in boys and it was higher in girls than in boys at all ages. From the age of 14 y boys had higher lean tissue mass and bone mineral content than girls. Tanner stage had a significant relation with body composition in both sexes. Percentage body fat was lower in boys in stage 4 than in stage 3 and was higher in consecutive Tanner stages in girls. After adjustment for age, Tanner stage was significantly positively related to lean tissue mass and bone mineral content in boys and girls and to percentage body fat and fat mass in girls. The profession of the parents and the education of the father had a significant negative correlation with percentage body fat and fat mass in girls (P < 0.01). Physical activity was related to lean tissue mass (P = 0.001) but not to fat mass in boys after adjustment for age. A high correlation and a small difference was found between lean body mass by BIA and lean tissue mass by DXA. Body composition in healthy Dutch children and adolescents is related to age, sex, Tanner stage, socioeconomic status, and physical activity.

Clinical use of acid steatocrit.

Malabsorption of fat is an important gastrointestinal cause of malnutrition and growth retardation in childhood. The gold standard for the evaluation of fat malabsorption is the faecal fat balance method. The acid steatocrit method has recently been introduced as a simple method to evaluate faecal fat. The present study was aimed at evaluating the acid steatocrit in clinical practice. Faecal fat excretion and acid steatocrit results were determined in 42 children, half with and half without fat malabsorption. Acid steatocrit results correlated significantly with both faecal fat excretion (p < 0.01) and faecal fat concentration (p < 0.001). Sensitivity and specificity of the acid steatocrit for the diagnosis of malabsorption were 90% and 100%, respectively. We consider the acid steatocrit method useful for the screening and monitoring of patients with steatorrhoea.

Fecal polyamine concentration in children with and without nutrient malabsorption.

BACKGROUND: Fermentation products of malabsorbed nutrients are thought to be responsible for intestinal adaptation following small bowel resection in rats. It has been suggested that either short-chain fatty acids or polyamines (mainly putrescine and cadaverine) could be the fermentation products involved. There are no data available on fecal polyamine content in humans. The present study compared the fecal polyamine concentrations in children with and without malabsorption. METHODS: Sixteen (8 girls, 8 boys) malabsorption patients (cystic fibrosis: 13, short bowel syndrome: 2, biliary atresia: 1) with a mean age of 8 years were compared to 17 (9 girls, 8 boys) sick children without malabsorption (mean age 5.7 years). Three-day fecal collections were performed and analyzed for fat and polyamine concentrations. High-performance liquid chromatography (HPLC) was used for the measurement of polyamine concentrations. RESULTS: Mean and SEM for fecal fat excretion was 13.4 +/- 2.5 g/day and 1.5 +/- 0.3 g/day in the malabsorption and control group respectively. Median fecal cadaverine and putrescine concentrations were 3723 mumol.kg-1 feces and 4737 mumol.kg-1 feces for the malabsorption group and 114 mumol.kg-1 feces and 306 mumol.kg-1 feces for the control group (p < 0.007 and < 0.00001 respectively). No significant differences were found for fecal spermine and spermidine concentrations between the two groups. CONCLUSIONS: Children with malabsorption show very high fecal putrescine and cadaverine concentrations. Our results support the hypothesis that fecal polyamines could be important.

[Favorable results of percutaneous endoscopic gastrostomy in children in need of long-term tube feeding]. / Gunstige resultaten van percutane endoscopische gastrostomie bij kinderen die langdurig sondevoeding nodig hebben.

OBJECTIVE: Analysis of indications, weight and peroperative and postoperative complications in 32 consecutive children who underwent percutaneous endoscopic gastrostomy (PEG). DESIGN: Retrospective. SETTING: Sophia Children's Hospital, Rotterdam, The Netherlands. METHOD: Survey of medical records. RESULTS: In 26 children the indication for PEG was necessity of prolonged tube feeding on account of severe psychomotor retardation. The 6 others had no such retardation and had various indications. Mean hospital stay was 6.7 days, median 6 days. Three patients had immediate postoperative complications: two had leakage, one infection. In 22 patients there were no late complications. One patients died following status epilepticus. One patient had a late peritonitis, and two others gastroesophageal reflux severe enough to require surgical intervention. Six patients had mild long-term complications, mainly infectious. All children showed improvement of weight for length. CONCLUSION: The PEG procedure is well-tolerated, effective way of tube feeding in children, with a relatively low complication rate.

In vivo measurement of chloride and water secretion in the jejunum of cystic fibrosis patients.

In the present study, we have investigated the possible consequences of the chloride channel defect in the intestine of cystic fibrosis (CF) patients for electrolyte and water transport in the jejunum in vivo, using a multilumen, double occluding balloon catheter, and an Ag/AgCl intraluminal electrode. During a chloride-free perfusion, to optimize the sensitivity of our measurements, the transmural potential difference (PD) (lumen with reference to serosal side) was found to be significantly higher in the jejunum of CF patients (+8.0 +/- 2.1 mV; n = 5) than in healthy control subjects (-2.2 +/- 2.0 mV; n = 9). The chloride concentration measured in chloride-free jejunal perfusates of CF patients was significantly lower than in controls (10.9 +/- 2.3 and 41.4 +/- 8.2 mM, respectively). Possible differences in net chloride and water secretion did not reach statistical significance (chloride secretion controls: -2.1 +/- 0.9 mmol/10 cm/h; CF: -0.8 +/- 0.2 mmol/10 cm/h; water secretion controls: -0.8 +/- 2.5 mL/10 cm/h; CF: -11.7 +/- 8.9 mL/10 cm/h). In control subjects, intraluminally applied theophylline stimulated the secretion of water (delta 23.4 +/- 4.6 mL/10 cm/h) and chloride (delta 4.1 +/- 1.1 mmol/10 cm/h), but not in CF patients (respectively delta 3.6 +/- 3.3 mL/10 cm/h and delta 1.1 +/- 1.1 mmol/10 cm/h). In controls, theophylline caused a significant increase in lumen negativity (PD -10.2 +/- 2.6 mV), but no change could be seen in CF patient transmural PD. These observations provide in vivo evidence for a decreased chloride permeability in the jejunum in CF, resulting in a significant reduction in net electrolyte and water secretion in the presence, but not in the absence, of an intestinal secretagogue.

Screening for coeliac disease in Dutch children with associated diseases.

Our objective was to assess the frequency of coeliac disease in children with associated disorders in the province of "Zuid-Holland". The Netherlands. We therefore screened 115 children with Down's syndrome, 62 children with juvenile rheumatoid arthritis (JRA) and 46 children with diabetes mellitus for CD using the IgA-class of antigliadin, antiendomysium and antireticulin antibodies in serum, and a functional sugar absorption test. The antiendomysium antibody test was the screening test that performed the best. Every patient who has at least one positive test underwent a jejunal biopsy for the diagnosis of CD. No association could be demonstrated between CD and diabetes mellitus. The frequency of CD in Down's syndrome was 7.0%, which is much higher than that found from screening the general population. CD was found in one child with JRA (1.5%), who also had Down's syndrome. We recommend screening for CD in all persons with Down's syndrome using at least the antiendomysium antibody test.

High frequency of celiac disease in Down syndrome.

We screened 115 children with Down syndrome for celiac disease, using antigliadin, antiendomysium, and antireticulin serum antibodies and an intestinal permeability test. Celiac disease was diagnosed in eight children, giving a frequency of 7.0%. We recommend screening for celiac disease in all persons with Down syndrome, with the use of at least the antiendomysium antibody determination.

Efficacy of a recombinant fowl pox-based Newcastle disease virus vaccine candidate against velogenic and respiratory challenge.

A fowl pox-based recombinant virus TROVAC-NDV (vFP96.5) was developed expressing the fusion and hemagglutinin-neuraminidase glycoproteins from a velogenic strain of Newcastle disease virus (NDV). Studies in specific-pathogen-free birds indicated that inoculation of a single dose of the recombinant led to the induction of significant levels of hemagglutination-inhibiting antibody that were maintained to 8 wk postinoculation. Further, the recombinant induced protective immunity against a combined intramuscular velogenic NDV challenge and respiratory NDV challenge. In commercial broiler chickens that were inoculated in the presence of maternally derived NDV immunity, the level of the NDV-specific humoral response was dampened, but significant levels of protection against both a lethal intramuscular NDV challenge and a fowl poxvirus challenge were obtained.

Herpesvirus of turkey recombinant viruses expressing infectious bursal disease virus (IBDV) VP2 immunogen induce protection against an IBDV virulent challenge in chickens.

Two recombinant herpesviruses of turkey (HVT) expressing the VP2 protein of infectious bursal disease virus (IBDV or Gumboro disease virus) have been constructed: vHVT001 and vHVT002. The VP2 open reading frame was inserted at the locus of the small subunit of ribonucleotide reductase gene (HSV-1 UL40 homolog) without any exogenous promoter in vHVT001 and at the locus of gl gene (HSV-1 US7 homolog) under the control of the human cytomegalovirus immediate-early promoter in vHVT002. The isolation of these recombinant viruses indicated that the deleted genes were not required for replication of HVT in chicken embryo fibroblasts. Efficacy of these recombinant viruses against IBDV strain 52/70 and Marek's disease virus (MDV strain RB1B) virulent challenges was evaluated in chickens vaccinated at 1 day of age. In the IBDV challenge, a good protection against mortality and bursal gross lesion was observed in vHVT002-vaccinated chickens: 100% with 10(5) PFU dose and 60% with 10(4) PFU dose; in contrast, only a weak level of protection was achieved after vaccination with vHVT001. Protection levels against MDV challenge obtained with vHVT001 and vHVT002 were low (around 10%) compared to that induced by the parental HVT (84%). In spite of the low protection level against MDV, this is the first report which describes induction of full protection against IBDV with a single inoculation of a recombinant virus.

Low incidence of childhood celiac disease in The Netherlands.

The incidence and prevalence of celiac disease vary internationally. We studied the incidence of identified cases of childhood celiac disease in six Dutch provinces that cover 47.9% of the surface area and 67.6% of the total population of The Netherlands. Children with celiac disease aged 0-14 y, diagnosed from January 1975 to January 1991, were traced by 1) contacting all pediatricians in this area, 2) examining the date of the Dutch National Medical Registration, and 3) investigating the membership records of the Dutch Celiac Disease Society. These data were cross-checked by the Dutch Network and National Database of Pathology. Of the 97.9% of pediatricians who answered our inquiry, 46.1% were treating celiac patients. A total of 342 celiac patients were identified. Informed consent that permitted examination of their medical files was given by 97.3% of the parents of the celiac children. The mean crude incidence rate of diagnosed childhood celiac disease, calculated per 1000 live births per year, was 0.18. However, a significant increase in reported incidence was demonstrated from 0.10 in 1976 to 0.32 in 1990. The best estimate, for the years 1985-1990, is 0.22/1000 live births, which is much lower than in most European countries.

A proposition for the diagnosis and treatment of gastro-oesophageal reflux disease in children: a report from a working group on gastro-oesophageal reflux disease. Working Group of the European Society of Paediatric Gastro-enterology and Nutrition (ESPGAN).

In this paper, a Working Group on Gastro-Oesophageal Reflux discusses recommendations for the first line diagnostic and therapeutic approach of gastro-oesophageal reflux disease in infants and children. All members of the Working Group agreed that infants with uncomplicated gastro-oesophageal reflux can be safely treated before performing (expensive and often unnecessary) complementary investigations. However, the latter are mandatory if symptoms persist despite appropriate treatment. Oesophageal pH monitoring of long duration (18-24 h) is recommended as the investigation technique of choice in infants and children with atypical presentations of gastro-oesophageal reflux. Upper gastro-intestinal endoscopy in a specialised centre is the technique of choice in infants and children presenting with symptoms suggestive of peptic oesophagitis. Prokinetics, still a relatively new drug family, have already obtained a definitive place in the treatment of gastro-oesophageal reflux disease in infants and children, especially if "non-drug" treatment (positional therapy, dietary recommendations, etc.) was unsuccessful. It was the aim of the Working Group to help the paediatrician with this consensus statement and guide-lines to establish a standardised management of gastro-oesophageal reflux disease in infants and children.

Laparoscopic management of tubal ectopic pregnancy.

As with laparotomy, laparoscopic treatment of ectopic pregnancy (EP) can be either conservative or radical. After conservative laparoscopic treatment by salpingotomy the risk of failure (between 4 and 6%) is comparable with that observed after similar treatment by laparotomy. The fertility results after laparoscopic treatment of EP are comparable if not better than those observed after similar treatment by laparotomy. These two reasons, together with the considerable advantages of endoscopy over laparotomy, mean that today laparoscopic treatment is without question the best surgical treatment for EP. The prognosis for post-EP fertility is unrelated to the characteristics of the EP (size, rupture, location). The post-EP fertility depends mainly on the patient's previous history. We have established the Therapeutic Scoring System for EP by assessing the different factors affecting future fertility and multivariable analysis. This score allowed us to choose the most suitable treatment to preserve fertility and reduce the risk of recurrence between conservative laparoscopic treatment, laparoscopic salpingectomy with or without contralateral tubal sterilization.

Newcastle disease virus (NDV) vaccine based on immunization with avian cells expressing the NDV hemagglutinin-neuraminidase glycoprotein.

Newcastle disease virus (NDV) is a paramyxovirus that bears two envelope glycoproteins at the virion surface. These proteins, fusion and hemagglutinin-neuraminidase (HN), are involved in the immune response against NDV infection. Recombinant cells constitutively expressing at their surface the HN protein from the velogenic Texas strain were generated by introducing the HN gene with a helper-free AEV-based vector. These recombinant cells were used to immunize chickens by various protocols, and birds were subsequently challenged with a lethal NDV injection. Both NDV protection and serologic response were observed.