Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema.

Primary lymphoedema is a rare, autosomal dominant disorder that leads to a disabling and disfiguring swelling of the extremities and, when untreated, tends to worsen with time. Here we link primary human lymphoedema to the FLT4 locus, encoding vascular endothelial growth factor receptor-3 (VEGFR-3), in several families. All disease-associated alleles analysed had missense mutations and encoded proteins with an inactive tyrosine kinase, preventing downstream gene activation. Our study establishes that VEGFR-3 is important for normal lymphatic vascular function and that mutations interfering with VEGFR-3 signal transduction are a cause of primary lymphoedema.

The effects of testosterone on craniosynostotic calvarial cells: a test of the gene/environmental model of craniofacial anomalies.

INTRODUCTION: The gene-environmental interaction model for craniofacial development proposes that if a genetic predisposition for an anomaly is coupled with an environmental factor that can exacerbate this predisposition, more severe phenotypes will result. Here, we utilize cells derived from our non-syndromic rabbit model of craniosynostosis to test the hypothesis that an insult, testosterone (TP) administration (exogenous source) will alter the osteogenic activity of these cells. DESIGN: Calvarial cells from wild-type (WT) (N=13) or craniosynostotic (CS) rabbits (N=11) were stimulated with TP, an androgen receptor blocker, flutamide, and combined treatments. Proliferation and differentiation assays were conducted after 7 days. anova and t-tests were used to determine differences in stimulation and cell type. RESULTS: The CS cells had significantly greater proliferation after TP administration compared to WT. There were no appreciable changes in differentiation after TP stimulation. Flutamide administration or combined TP and flutamide administration decreased both proliferation and differentiation for both cell types similarly. CONCLUSIONS: Testosterone exposure caused an increase in cell proliferation for CS osteoblast cells. However, a therapy targeted to mitigate this response (flutamide therapy) similarly affected CS and WT cells, suggesting that the administration of flutamide or TP in the presence of flutamide decreases osteogenesis of these cells. Thus, although our data support a mechanism of gene-environmental interaction, these results would not support a therapeutic intervention based on this interaction.

A defect in sodium-dependent amino acid uptake in diabetic rabbit peripheral nerve. Correction by an aldose reductase inhibitor or myo-inositol administration.

A myo-inositol-related defect in nerve sodium-potassium ATPase activity in experimental diabetes has been suggested as a possible pathogenetic factor in diabetic neuropathy. Because the sodium-potassium ATPase is essential for other sodium-cotransport systems, and because myo-inositol-derived phosphoinositide metabolites regulate multiple membrane transport processes, sodium gradient-dependent amino acid uptake was examined in vitro in endoneurial preparations derived from nondiabetic and 14-d alloxan diabetic rabbits. Untreated alloxan diabetes reduced endoneurial sodium-gradient dependent uptake of the nonmetabolized amino acid 2-aminoisobutyric acid by greater than 50%. Administration of an aldose reductase inhibitor prevented reductions in both nerve myo-inositol content and endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Myo-inositol supplementation that produced a transient pharmacological elevation in plasma myo-inositol concentration, but did not raise nerve myo-inositol content, reproduced the effect of the aldose reductase inhibitor on endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Phorbol myristate acetate, which acutely normalizes sodium-potassium ATPase activity in diabetic nerve, did not acutely correct 2-aminoisobutyric uptake when added in vitro. These data suggest that depletion of a small myo-inositol pool may be implicated in the pathogenesis of defects in amino acid uptake in diabetic nerve and that rapid correction of sodium-potassium ATPase activity with protein kinase C agonists in vitro does not acutely normalize sodium-dependent 2-aminoisobutyric acid uptake.

Direct method for detecting small quantities of hepatitis B virus DNA in serum and plasma using the polymerase chain reaction.

Serum components inhibit DNA polymerase, thereby obviating direct detection of serum viral DNA sequences by the polymerase chain reaction (PCR). This has necessitated extraction of nucleic acid from sera before performing PCR and has resulted in loss of sensitivity. By adsorbing virus to a solid surface (microcentrifuge tubes or antibody coated microparticles) followed by proteinase K digestion, as little as three viruses per 200 microliters serum may be directly detected by PCR without nucleic acid extraction. The sensitivity is dependent on the surface area of the adsorptive surface and is increased by having antibodies on the adsorptive surface. The nucleic acid sequence of the amplified DNA fragments may be directly determined by the dideoxy method. Of 24 plasma samples from HBsAg+ volunteer blood donors, HBV DNA was detected in 7 by dot blot assay, 7 by liquid hybridization, and 9 by PCR. PCR detected DNA in every sample that was positive by another assay. Analysis of serial samples of two patients with acute self-limited hepatitis B found detectable HBsAg and pre-S2 antigenemia before HBV DNA by the PCR method. These results suggest that surface antigenemia may precede viremia during acute hepatitis.

Psychomotor slowing is associated with distal symmetrical polyneuropathy in adults with diabetes mellitus.

To test the hypothesis that diabetes mellitus is associated with cognitive dysfunction, a battery of neuropsychological tests was administered to 75 diabetic adults and an equal number of demographically similar nondiabetic control subjects. Compared with control subjects, diabetic subjects performed significantly more poorly on measures of psychomotor efficiency and spatial information processing. In contrast, no between-group differences appeared on measures of verbal intelligence, learning, memory, problem solving, or simple motor speed. Results from multiple regression analyses showed that clinically significant distal symmetrical polyneuropathy was strongly associated with psychomotor slowing, whereas, glycosylated hemoglobin values were weakly associated with both psychomotor slowing and spatial processing. No other biomedical variables predicted cognitive test performance. These neurobehavioral data are consistent with the hypothesis that a "central neuropathy" may be associated, at least in part, with chronic hyperglycemia.

Polyol pathway activity and myo-inositol metabolism. A suggested relationship in the pathogenesis of diabetic neuropathy.

Two major metabolic perturbations, increased polyol (sorbitol) pathway activity and reduced tissue myo-inositol content, are induced in peripheral nerve by hyperglycemia. Although they are commonly invoked as alternative biochemical pathogenetic mechanisms for diabetic neuropathy, their possible interrelationship has never been adequately explored. Therefore, we studied the effect of polyol pathway blockade with sorbinil, a specific inhibitor of aldose reductase, on nerve myo-inositol content in acutely streptozotocin-diabetic rats. Sorbinil administration completely prevented the fall in nerve myo-inositol, thereby implicating increased polyol pathway activity as a likely factor in the fall in nerve myo-inositol content in experimental diabetes.

The glucokinase glucose sensor in human pancreatic islet tissue.

The enzyme glucokinase controls glucose metabolism in islets and is proposed to be the glucose sensor in pancreatic beta-cells. This concept was developed from studies with rodents and it remained to be explored whether it also applies to man. Studies in man were hampered, however, by the difficulty in obtaining well-preserved pancreatic islet tissue and also because the high activity of hexokinase made it difficult to measure glucokinase. To overcome these obstacles, quantitative histochemical sampling techniques were developed allowing precise dissection of pure human islet tissue and a newly designed radiometric microassay was used, avoiding hexokinase interference, and providing the sensitivity necessary to measure the relatively low glucokinase activity in small samples of tissue obtained from brain-dead tissue donors. The present data indicate that glucokinase is present in human pancreatic islet tissue and is not found in the exocrine pancreas. The enzyme's Vmax with D-glucose as substrate was similar to the Vmax for glucose utilization reported previously for intact, isolated human islets and the enzyme's Km for D-glucose was about 5 mM. Since glucokinase was also present in islet tissue of hamster, mouse, and rat, it is suggested that the glucokinase-glucose sensor concept has general applicability and that it could explain many aspects of the physiology and pathology of glucose homeostasis. This well-defined pancreatic islet glucokinase-glucose sensor should, therefore, be incorporated in any comprehensive model of glucose homeostasis.

Molecular regulation of lymphangiogenesis and targets for tissue oedema.

New insight has recently been obtained into the molecular mechanisms regulating the function of lymphatic endothelial cells. Vascular endothelial growth factors-C and -D have been shown to stimulate lymphangiogenesis, and their receptor VEGFR-3 has been linked to human hereditary lymphoedema, although there is evidence that other genes are also involved. These data suggest that it may become possible to stimulate lymphatic growth and function and to treat tissue oedema involved in many diseases.

Subclinical eating disorders and glycemic control in adolescents with type I diabetes.

Several recent case reports have shown that anorexia nervosa and bulimia negatively affect glycemic control in diabetic patients. However, there have been no systematic studies to assess the prevalence of clinical or subclinical eating disorders among diabetic patients or to determine the impact of such disturbances on glycemic control. This study reports a survey of 202 adolescents, aged 12-18 yr, seen in the Diabetes Clinic, Children's Hospital of Pittsburgh, who were asked to complete the Binge Eating Scale (BES) and the EAT-26 questionnaire. Responses of diabetic patients to the EAT-26 questionnaire were compared with those of a nondiabetic control group and were related to measures of glycemic control. Diabetic subjects scored higher on the total EAT-26 than nondiabetic control subjects, ordinarily indicative of more eating pathology. However, diabetic subjects scored higher only on the dieting subscale of this questionnaire, probably reflecting adherence to the diabetes dietary regimen. Subjects with diabetes scored lower, or did not differ significantly, from nondiabetic control subjects on measures of oral control and bulimia. Among diabetic subjects, self-reported bulimic behaviors were related to poorer glycemic control. Patients with the highest scores on the BES had an average HbA1 of 13.1% compared with 11.8% for age- and sex-matched patients at the 50th percentile, and 10.8% for patients in the lowest 10th percentile. Further studies are needed to determine whether modification of these eating behaviors would improve glycemic control.

Effect of thyroid hormones on human mononuclear leukocyte lysosomal acid lipase activity.

Lysosomal acid lipase (LAL), the enzyme which hydrolyzes lipoprotein cholesteryl esters, is under thyroid hormone regulation in experimental animals. To evaluate whether human LAL is also under thyroid regulation, we measured mononuclear leukocyte LAL activity in 31 hypothyroid, hyperthyroid, and euthyroid children under treatment. LAL activity was positively correlated with serum T3 and serum T4 levels, and was significantly higher in hyperthyroid children than in those who were hypothyroid. In another study, 5 adult euthyroid males were given 50 micrograms L-T3 every 8 h for 4 days. Their LAL activities increased significantly after treatment. The data suggest that LAL activity in man, as in experimental animals, is influenced by thyroid hormone status. Hormonal control of LAL activity may be important in the regulation of human cholesterol metabolism.

Premature pubarche: etiological heterogeneity.

Premature pubarche is characterized by pubic hair, adult type body odor, acne, and axillary hair before 8 yr of age in girls and 9.5 yr of age in boys. Causes of this premature virilization include premature adrenarche, mild errors of steroidogenesis, precocious puberty, and adrenal and gonadal tumors. To determine whether any clinical parameters are helpful in distinguishing which children should undergo further evaluation for mild congenital adrenal hyperplasia, we performed ACTH stimulation tests in 69 children with premature pubarche and 8 pubertal controls. Patients were categorized as having typical (pubic hair with or without axillary hair and body odor) or atypical (pubic hair and genital enlargement) premature pubarche. Blood samples, before and 30 min after iv bolus administration of synthetic ACTH, were obtained for progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, 11-deoxycortisol, and cortisol measurements. The patients were divided into 4 groups based on their individual responses to ACTH stimulation: premature adrenarche (no apparent defect in steroidogenesis), possible decreased 21-hydroxylase activity, possible decreased 3 beta-hydroxysteroid dehydrogenase activity, and indeterminate responses. Five of 11 (45%) children with atypical premature pubarche and 7 of 58 (12%) children with typical premature pubarche were found to have evidence of mild defects in steroidogenesis. Similar to previous reports in postpubertal women, only responses to ACTH stimulation allowed accurate classification of these patients.

Pulsatile growth hormone secretion in children with acute lymphoblastic leukemia after 1800 cGy cranial radiation.

The relationship between intensity of central nervous system preventive therapy and the development of hypothalamic pituitary dysfunction is unclear in patients with acute lymphoblastic leukemia. In a previous report, we demonstrated uniform decreases in spontaneous secretion of growth hormone following 2400 cGy whole brain radiation. In this study, we measured basal growth hormone levels every 20 minutes over 24 hr in five survivors of childhood acute lymphoblastic leukemia treated with 1800 cGy cranial radiation. Four of the patients had been off therapy 2 9/12-4 3/12 years. Growth hormone secretion in these patients, as indicated by mean growth hormone concentration, pulse amplitude and frequency, was clearly greater than that seen following 2400 cGy and appeared to be normal compared with sex- and Tanner stage-matched literature controls. However, serial growth measurements showed significant decreases in height percentiles in two of these children. The fifth patient, who had already approached her adult height at the time of diagnosis, had been off therapy only 1 year and had a mean growth hormone level intermediate between those of normal controls and previously reported children treated with 2400 cGy. These data suggest (a) that the effect of radiation therapy on spontaneous pulsatile growth hormone secretion may be dose related, and (b) that short stature in a given patient may not be indicative of subnormal basal growth hormone levels. Further longitudinal investigation may clarify whether early transient changes in GH secretion occur that may normalize over time.

Acute profound dystonia in infants with glutaric acidemia.

Acute profound dystonia developed in three previously well infants who were found to have glutaryl-CoA dehydrogenase deficiency in cultured skin fibroblasts. Two patients had excessive urinary excretion of glutaric acid, but one did not. Neuroradiologic studies performed in all three patients at the onset of their illnesses revealed large CSF-containing spaces both within the sylvian fissures and anterior to the temporal lobes. Pathologic examination of the brain of one patient demonstrated cerebral and cerebellar atrophy, shrinkage of the putamen, and white matter vacuolation. Glutaric acidemia may be a common cause of acquired persistent dystonia or choreoathetosis in infancy.

Renal ouabain inhibitable Na-K ATPase activity and myoinositol supplementation in experimental diabetes mellitus.

Alterations in ouabain inhibitable Na-K ATPase activity, polyol pathway activity, and myoinositol metabolism are part of a unifying hypothesis proposed to explain the pathogenesis of the chronic complications of diabetes mellitus. Direct measurements of renal ouabain inhibitable Na-K ATPase activity in animals with streptozotocin-induced diabetes show increased or decreased activity, depending on the nephron segment examined and the duration of diabetes. While myoinositol feeding corrects depressed Na-K ATPase activity in peripheral nerve of streptozotocin diabetic rats, the effect of myoinositol feeding on altered renal Na-K ATPase activity is unknown. To assess the effect of experimental diabetes on renal ouabain inhibitable Na-K ATPase activity and test the involvement of the polyol/inositol pathway, we assayed kidneys from normal, streptozotocin diabetic, and myoinositol-supplemented diabetic rats for renal ouabain-inhibitable Na-K ATPase, alkaline phosphatase, and tau-glutamyltranspeptidase (tau-GT) activity. Ouabain inhibitable Na-K ATPase activity, expressed per milligram of protein, is increased in the inner medulla of the diabetic kidney compared with normal and, expressed per microgram DNA, is increased in both the inner medulla and cortex. Myoinositol supplementation did not affect the increase in renal enzyme activity seen with streptozotocin diabetes. These observations suggest that the regulation of renal ouabain inhibitable Na-K ATPase activity, in streptozotocin diabetes, does not depend on supplemental myoinositol. These findings do not exclude the possibility that changes in polyol or myoinositol concentrations in a specific nephron segment may have pathogenetic significance for diabetic nephropathy.

Effect of diabetes and insulin therapy on human mononuclear leukocyte lysosomal acid lipase activity.

Acid lipase activity was measured in mononuclear leukocytes obtained from 18 newly diagnosed type-I diabetics and from six newly diagnosed type-II diabetics before and five to ten days after the initiation of insulin therapy. Using either 4-methylumbelliferyl oleate or cholesteryl[1-14C]oleate as substrate for the enzyme assays, acid lipase activity in the diabetics was not found to be significantly different from that in the controls, significantly less (P less than 0.05) after insulin therapy in type-I diabetics, and unchanged following treatment in the type-II patients. These results differ from those found in previous studies of acid lipase activity in type-II diabetics, in whom insulin therapy evoked a doubling of enzyme activity. The effect of insulin treatment on the activity of lysosomal acid lipase requires further evaluation.

Hypoplasia of the pancreas in a patient with type I diabetes mellitus.

Pancreatic hypoplasia is an uncommon developmental defect that has not been well documented in association with type I diabetes mellitus. We report the case of a patient with an atypical clinical onset of type I diabetes mellitus who died following pancreas transplantation. Autopsy showed the surprising finding of hypoplasia of the native pancreas with other features indicating the concurrence of type I diabetes mellitus. These findings lead to speculation about the occurrence and interaction of these two diseases in our patient.

Otitis media and hearing loss in Turner syndrome.

Twenty-two phenotypic females with Turner syndrome underwent prospective otologic evaluation including a standard history, physical examination, audiogram, and tympanogram. Eight of these patients had computed tomography of the temporal bones. Eighty-two percent of the patients had a history of chronic or recurrent ear infections. Eleven patients (50%) had previous myringotomy and tube placement and 4 (16%) had undergone tympanoplasty or tympanomastoidectomy for sequelae of otitis media. Ten patients (45%) had middle ear effusions evident on examination. Sixteen patients (73%) had hearing loss in at least one ear at the time of examination. Sensorineural losses were evident in 37% of patients. No malformations of the otic capsule were noted on computed tomography. The high prevalence of both hearing loss and otitis media in Turner syndrome warrants otologic and audiologic assessment of patients with this chromosomal anomaly.

Orthotopic liver transplantation for ornithine transcarbamylase deficiency with hyperammonemic encephalopathy.

Ornithine transcarbamylase (OTC) deficiency is an X chromosome-linked disorder causing hyperammonemic encephalopathy with a very poor prognosis. We describe here two patients with OTC deficiency, one a late on-set female patient (case 1) and the other a neonatal-onset male patient (case 2), who were successfully treated with orthotopic liver transplantation (OLTx). The OTC activity in the excised liver was 10% and 0% of control, respectively. Hyperammonemic encephalopathy was controlled with medical therapy in case 1 until the of 5 years, but the complicated course in case 2 in which hyperammonemia required peritoneal dialysis and hemodialysis in the neonatal period necessitated OLTx with a reduced-size liver at the age of 80 days. Both patients had restoration of serum ammonia level to normal in 2 and 3 days after liver replacement, and both patients have normal neurological and developmental status after 2 and 0.5 years of postoperative follow-up. These cases illustrate not only the metabolic cure of this disorder, but also the need to preserve neurological integrity by aggressive medical management of the hyperammonemia preoperatively and early surgical intervention when indicated, even if this is required very early in life.

A new method for assessment of changes in retinal blood flow.

This study validates the use of residence time distribution (RTD) functions in human subjects to assess changes in retinal flow by using the widely recognized model of flow changes due to oxygen breathing. Changes in retinal blood flow may provide important information for clinical decision-making in several populations, including those with diabetic retinopathy, sickle cell disease and retinitis pigmentosa. Normal volunteer subjects were studied before and after oxygen breathing. After i.v. injection, relative fluorescence was obtained using scanning laser ophthalmoscopy/image processing in all vessel branches (average, 17). For each experiment, 64 frames (2/s) were digitized and were normalized using the RTD method. Vessel diameters were measured using densitometry techniques on fundus photos, where the diameter data made it possible to weight each vessel according to relative cross-sectional area to obtain a true mean circulation time (MCT). MCT increased for the group of subjects when breathing oxygen compared to normal air (P = 0.001), representing a decrease in retinal blood flow. Average MCT increased 2.82 +/- 2.51 s for all subjects, with an increase of 2.93 +/- 2.26 s in repeat trials for one subject. The proposed method uses information from all retinal vessels and allows the assessment of overall, as well as selected, regional retinal flow. It is more comprehensive than previous methods analysing single vessel flow. This method will be potentially useful for assessing hemodynamic changes in the retina associated with a wide range of eye disease.

Appraising boardroom performance.

Rare is the company that does not periodically review the performance of its staff, business units, and suppliers. But rare, as well, is the company that does such a review of one of its most important contributors--its board of directors. Reviewing a board's performance is not an easy proposition: it has to be done by the members themselves, people who generally have many other responsibilities and whose time is always at a premium. But done properly, appraisals can help boards become more effective by clarifying individual and collective responsibilities. They can help improve the working relationship between a company's board and its senior management. They can help ensure a healthy balance of power between the board and the CEO. And, once in place, an appraisal process is difficult to dismantle, making it harder for a new CEO to dominate a board or avoid being held accountable for poor performance. Done properly is the key here, though. Done incorrectly, board appraisals can degenerate into self-serving evaluations or unpleasant, time-wasting exercises. Worse, they can evolve into rigid mechanical processes that discourage innovation. In fact, all of the approaches the authors observed in two years of research were incomplete. The authors have therefore drawn on the strengths of several different approaches to synthesize a best-practice process that is both rigorous and comprehensive.