The severe short stature in two siblings with a heterozygous IGF1 mutation is not caused by a dominant negative effect of the putative truncated protein.

OBJECTIVE: While in previous studies heterozygosity for an Insulin-Like Growth Factor 1 (IGF1) defect only modestly decreased height and head circumference, we recently reported on two siblings with severe short stature with a maternally transmitted heterozygous duplication of 4 nucleotides, resulting in a frame shift and a premature termination codon in the IGF1 gene. In this paper we describe the structural and functional characteristics of the putative truncated IGF-I protein. DESIGN: Two children, their mother and maternal grandfather carried the mutation. In addition, two family members who were not affected were included in the study. Mutant (MT) IGF-I was synthesized in oxidized and reduced form using two methods. Neutral gel filtration studies were carried out with wild-type (WT) and synthetic MT IGF-I. Binding analysis of synthetic MT IGF-I to the IGF1R and insulin receptors were performed with EBNA-293 cells, stably transfected with the IGF-I receptor, and IM9 cells. L6 cells were used to examine the mitogenic potency and the potential antagonizing effect of synthetic MT IGF-I by [(3)H]-thymidine incorporation assays. RESULTS: In the sera of both the carriers and non-carriers the proportion of (125)I-IGF-I that was associated with the 150 kDa complex was somewhat less (varying between ~37 and ~52%) than in normal pooled serum (~53-~63%) and, instead, slightly increased amounts of radioactivity were eluted in the 40-50 kDa fraction (consisting of binary IGF-IGFBP complexes) or remained unbound. Synthetic MT IGF-I did not bind to the IGF-I receptor, nor antagonize the growth-promoting effect of IGF-I. It did bind to IGFBPs, but was barely incorporated into 150 kDa complexes. Because in all cases WT IGF-I immunoreactivity was recovered in one peak, corresponding to the MW of WT IGF-I, i.e. ~7.6 kDa, an interaction of circulating truncated mutant peptide with WT IGF-I is very unlikely. CONCLUSIONS: There is no evidence that the severe short stature associated with heterozygosity for this novel IGF1 mutation in children born from a mother with the same mutation is caused by a dominant negative effect of the truncated protein. We speculate that the growth failure is caused by a combination of partial IGF-I deficiency, placental IGF-I insufficiency, and other genetic factors.

Short stature associated with a novel heterozygous mutation in the insulin-like growth factor 1 gene.

CONTEXT: Homozygous IGF1 deletions or mutations lead to severe short stature, deafness, microcephaly, and mental retardation. Heterozygosity for an IGF-I defect may modestly decrease height and head circumference. OBJECTIVE: The objective of the study was to investigate the clinical features of heterozygous carriers of a novel mutation in the IGF1 gene in comparison with noncarriers in a short family and to establish the effect of human GH treatment. SUBJECTS: Two children, their mother, and their maternal grandfather carried the mutation and were compared with two relatives who were noncarriers. RESULTS: The two index cases had severe short stature (height sd score -4.1 and -4.6), microcephaly, and low IGF-I levels. Sequencing of IGF1 revealed a heterozygous duplication of four nucleotides, resulting in a frame shift and a premature termination codon. The mother and maternal grandfather had the same IGF1 mutation. Adult height (corrected for shrinking and secular trend) and head circumference sd score of carriers of the paternally transmitted mutation was -2.5 and -1.8, in comparison with -1.6 and 0.3 in noncarriers, respectively. After 2 yr of GH treatment, both index cases exhibited increased growth. CONCLUSIONS: Heterozygosity for this novel IGF1 mutation in children born from a mother with the same mutation, presumably in combination with other genetic factors for short stature, leads to severe short stature, which can be successfully treated with GH.

[Acute caffeine intoxication after intake of 'herbal energy capsules']. / Acute coffeïne-intoxicatie na het innemen van 'herbal energy'-capsules.

Two males, 15 and 17 years old respectively, presented at the Emergency Department complaining of cramping abdominal pain, nausea and vomiting after ingestion of energy capsules. Physical examination revealed sinus tachycardia and slight abdominal pain. Laboratory examination showed substantial hypokalaemia and mild hyperglycaemia. Questioning revealed that they had taken 5 and 3 'herbal energy capsules' respectively and that these capsules supposedly contained 200 mg of caffeine each. Toxicological analysis showed a greatly increased serum caffeine concentration in both patients. The peak concentrations calculated were in the highly toxic range and could have led to severe acute complications such as convulsions. Pharmaceutical analysis demonstrated that these 'Supercap Xtreme'-capsules contained 700 mg caffeine or more. All symptoms presented were compatible with caffeine intoxication. The content of these capsules is not reliable and could lead to life-threatening intoxication.

[Persistent neonatal hypoglycaemia caused by arterial positioning of the umbilical venous catheter]. / Persisterende onverklaarde neonatale hypoglykemie door arteriële positie van een navelvenelijn.

Two neonates, a girl born at 40 2/7 weeks weighing 4165 g and a boy born at 37 6/7 weeks weighing 4040 g, received umbilical venous catheters to help manage hypoglycaemia. The catheter was ineffective or only effective when high doses of glucose were used, due to what later appeared to be arterial positioning of the catheter. Both patients recovered without consequences. Persistent hypoglycaemia is a common problem in newborns and can cause severe neurological sequelae. A relatively uncommon cause is malpositioning of the umbilical catheter. Positioning in an artery leads to direct infusion of glucose into the pancreas, which causes hyperinsulinaemia and can lead to potentially dangerous nonketotic hypoglycaemia. Arterial positioning of the umbilical catheter should be ruled out at an early stage. Correct catheter positioning can be determined using careful inspection of the umbilical veins, radiological examination of the catheter position, blood gas analysis or vascular pulsation.

[Serious side effects of frequently used antibiotics in childhood: biliary sludge or stones induced by ceftriaxone and thrombocytopenia induced by co-trimoxazole]. / Ernstige bijwerkingen van veelgebruikte antibiotica bij kinderen: galgruis of-stenen door ceftriaxon en trombocytopenie door co-trimoxazol.

Two patients, a girl and a boy, both aged 8.5 years, presented with serious side effects caused by ceftriaxone and co-trimoxazole, respectively. The first patientwas treated with ceftriaxone (100 mg/kg/day with a body weight of 35.6 kg) on suspicion of a neuroborreliosis, but developed an acute cholecystitis with cholelithiasis 3 weeks after the antibiotic had been withdrawn. He underwent a laparoscopic cholecystectomy. Ceftriaxone binds calcium in the biliary tract, forming biliary sludge or stones. The second patient developed thrombocytopenia during treatment with co-trimoxazole (58 mg/kg/day with a body weight of 25.4 kg) because of a urinary-tract infection. After discontinuation of the co-trimoxazole the thrombocytopenia resolved spontaneously. The pathophysiological mechanism involved may be either a direct toxic effect of trimethoprim or an immune-mediated reaction to sulfamethoxazole. According to current guidelines, the dosage of the drug was too high in both cases. It is important to ensure a correct dosage in children, since side effects are potentially dose-related.

[Accurate assessment of heart murmurs in children: thorough medical history and physical examination required]. / Beoordeling van hartgeruisen bij kinderen: zorgvuldige anamnese en lichamelijk onderzoek vereist.

4 children, boys aged 12, 5, 1.5 and 11 years, had a heart murmur. The 12-year-old boy could also not finish a football match and appeared to have atrioseptal defects (ASD). The 1.5-year-old boy had pulmonary symptoms that were not responsive to asthma medication; he also had ASD. The 11-year-old boy had had chest pain and pressure following exertion for 2 years; he appeared to have an aortic stenosis. Symptoms disappeared in all 3 patients after surgical correction. In the 5-year-old asymptomatic boy the murmur was deemed to be innocent following medical history and physical examination. Children frequently have heart murmurs. Most heart murmurs are innocent but some are caused by heart defects. Careful evaluation of the medical history and physical examination are critical in the differentiation of innocent and pathological heart murmurs. Routine supplementary diagnostic tests in children with heart murmurs are of limited value and are often misleading. One should inquire about specific and nonspecific symptoms and also perform systematic inspection, palpation and auscultation to identify any characteristics that suggest a heart murmur caused by a heart defect.

[Diagnostic images (169). A toddler not feeling well and having a rash. Eczema herpeticum]. / Diagnose in beeld (169). Een peuter met algehele malaise en huiduitslag. Eczema herpeticum.

A 1-year-old boy with eczema presented with fever and a rash, which was diagnosed as eczema herpeticum.

Monocyte chemoattractant protein-1 and interleukin-8 levels in urine and serum of patents with hemolytic uremic syndrome.

The epidemic form of the hemolytic uremic syndrome (HUS) in children is hallmarked by endothelial cell damage, most predominantly displayed by the glomerular capillaries. The influx of mononuclear (MO) and polymorphonuclear cells (PMNs) into the glomeruli may be an important event in the initiation, prolongation, and progression of glomerular endothelial cell damage in HUS patients. The molecular mechanisms for the recruitment of these leukocytes into the kidney are unclear, but monocyte chemoattractant protein-1 (MCP-1) and IL-8 are suggested to be prime candidates. In this study, we analyzed the presence of both chemokines in 24-h urinary (n = 15) and serum (n = 14) samples of HUS children by specific ELISAs. Furthermore, kidney biopsies of three different HUS children were examined for MO and PMN cell infiltration by histochemical techniques and electron microscopy. Whereas the chemokines MCP-1 and IL-8 were present in only very limited amounts in urine of 17 normal control subjects, serial samples of HUS patients demonstrated significantly elevated levels of both chemokines. HUS children with anuria showed higher initial and maximum chemokine levels than their counterparts without anuria. A strong positive correlation was observed between urinary MCP-1 and IL-8 levels. Whereas initial serum IL-8 levels were significantly increased in HUS children, serum MCP-1 levels were only slightly elevated compared with serum MCP-1 in control children. No correlation was found between urinary and serum chemokine concentrations. Histologic and EM studies of HUS biopsy specimens clearly showed the presence of MOs and to a lesser extent of PMNs in the glomeruli. The present data suggest an important local role for MOs and PMNs in the process of glomerular endothelial-cell damage. The chemokines MCP-1 and IL-8 may possibly be implicated in the pathogenesis of HUS through the recruitment and activation of MOs and PMNs, respectively.

Verocytotoxin inhibits mitogenesis and protein synthesis in purified human glomerular mesangial cells without affecting cell viability: evidence for two distinct mechanisms.

Acute renal failure is one of the hallmarks of the hemolytic uremic syndrome (HUS). Infection with a verocytotoxin (VT)- or Shiga-like toxin (SLT)-producing Escherichia coli has been strongly implicated in the etiology of the epidemic form of HUS. The functional receptor for these closely related toxins appears to be a glycosphingolipid, globotriaosylceramide (Gb3). Endothelial damage in the glomeruli and arterioles of the kidney induced by VT is believed to play a crucial role in the pathogenesis of HUS. However, little information is available regarding the effects of VT on mesangial cells, which also play an important role in glomerular function. In this study, the effects of VT on human mesangial cells in vitro were investigated. Mesangial cells were enriched by collecting hillock-shaped outgrowths derived from adult human glomeruli and subsequently purified by elimination of contaminating epithelial cells by immunoseparation with ulex europaeus lectin-I (UEA-I)-coated dynabeads. The obtained and subcultured mesangial cell populations were >98% pure. Their mesangial nature was established by the presence of a-smooth muscle cell actin in highly confluent cultures and the absence of cytokeratin or platelet/endothelial cell adhesion molecule-1. Mesangial cells bound VT to bands of Gb3 and a closely related glycolipid, which is similar to a glycolipid involved in the VT-dependent cytokine production in monocytes. VT did not induce the release of cytokines or chemokines in mesangial cells. In VT-susceptible cells, binding of VT to Gb3 causes cell death by the inhibition of protein synthesis. Although protein synthesis was inhibited in mesangial cells, all cells remained viable, both under basal and tumor necrosis factor-alpha-stimulated conditions. However, the marked reduction in protein synthesis may impair a proper response of the cells in conditions of increased demand of newly synthesized proteins. Furthermore, VT markedly inhibited DNA synthesis and proliferation of mesangial cells. The inhibition of mitogenesis was also found with the B-subunit of VT-1 alone, albeit to a lesser extent, without a significant effect on protein synthesis. Because the inhibition of protein synthesis involves the A-subunit, this suggests that two distinct mechanisms contribute to the effects of VT on protein synthesis and mitogenesis. Intracellular routing of VT (A- and B-subunits) may vary between cell types and result in differential effects on human mesangial cells when compared with other cell types.

Effects of TNF alpha on verocytotoxin cytotoxicity in purified human glomerular microvascular endothelial cells.

In the pathogenesis of the hemolytic uremic syndrome (HUS), endothelial damage of glomeruli and arterioles of the kidney appears to play a central role. Previous studies have shown that verocytotoxin-1 (VT-1) cytotoxicity on human vein endothelial cells require additional stimuli, in particular the inflammatory mediator tumor necrosis factor alpha (TNF alpha). In this study the effects of VT on human glomerular microvascular endothelial cells (GMVEC) were examined. A reproducible method was developed for the isolation and purification of large numbers of highly purified GMVEC. The obtained GMVEC were over 99% pure; their endothelial origin was demonstrated by the expression of the endothelial antigens von Willebrand factor, EN-4, PECAM-1 and V,E-cadherin. Upon stimulation with TNF alpha the cells expressed the endothelial-specific adhesion molecule E-selectin. A limited number of fenestral structures was observed by scanning electron microscopy (SEM), suggesting glomerular origin of the endothelial cells. Cytotoxicity of VT-1 to GMVEC was evaluated by determination of the number of viable adherent cells and by assay of overall protein synthesis after exposure to varying concentrations of VT-1. In non-stimulated GMVEC, cytotoxicity of VT-1 was inversely related to the degree and duration of confluence, subconfluent cells being the most sensitive. In highly confluent GMVEC, VT cytotoxicity required pre-exposure of the cells to the inflammatory mediator TNF alpha, which induced an increase in the number of VT receptors on GMVEC. Thin layer chromatography of extracted glycolipids from the GMVEC showed binding of VT-1 to globotriaosylceramide (Gb3), known to be the functional receptor for VT. There were no major differences in protein synthesis inhibition with equal concentrations VT-1 and VT-2. In conclusion, in this study we provide a reproducible method to isolate, purify and culture well characterized human GMVEC on a routine basis. In vitro studies with these GMVEC demonstrate that VT cytotoxicity depends on the degree of confluence and the additional preexposure to the inflammatory mediator TNF alpha. These observations provide further insight into the complex events that may occur in glomeruli in the pathogenesis of HUS.

Effects of verocytotoxin-1 on nonadherent human monocytes: binding characteristics, protein synthesis, and induction of cytokine release.

The epidemic form of the hemolytic uremic syndrome (HUS) has been associated with a verocytotoxin producing Escherichia coli infection. Endothelial cell damage of glomeruli and arterioles of the kidney plays a central role in the pathogenesis of HUS. A number of observations in vivo and in vitro indicate that inflammatory mediators contribute to this process. In this study we investigated the binding of 125I-verocytotoxin-1 (VT-1) to freshly isolated human nonadherent monocytes as well as the nature of the ligand to which VT-1 binds on monocytes. On the average, freshly isolated monocytes have 0.07 x 10(5) specific binding sites for 125I-VT-1 per cell. Preincubation of nonadherent monocytes with bacterial lipopolysaccharide (LPS) caused a 23- to 30-fold increase of specific binding sites for VT-1 as shown by Scatchard plot analysis. Thin-layer chromatography of extracted neutral glycolipids of the cells and subsequent binding of 125I-VT-1 showed that human monocytes bind VT-1 to a globotriaosylceramide (Gb3) species that is different from that found on endothelial cells, probably a short-chain fatty acyl Gb3 or an alpha-OH-Gb3. In addition, we evaluated the functional consequences of VT-1 binding to human monocytes by investigating the effects of VT-1 on the total protein synthesis and, specifically, the production of the cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8. We observed that VT-1 did not inhibit overall protein synthesis, nor under basal conditions, neither after stimulation with LPS, in contrast to previous observations with endothelial cells. Furthermore, we found that VT-1 induces the synthesis of the cytokines IL-1 beta, TNF-alpha, IL-6, and IL-8 in nonstimulated monocytes by a LPS-independent cell activation. The increase in the production of cytokines was parallelled by an increase in mRNA, as was demonstrated for IL-6 by reverse transcription-polymerase chain reaction. These data suggest that inflammatory mediators locally produced by VT-1-stimulated monocytes may contribute to the pathogenic mechanism of the HUS.