Jens Klinge

Incidence and significance of 22q11.2 hemizygosity in patients with interrupted aortic arch.

Interruption of the aortic arch (IAA) is a severe malformation of the heart with known association to DiGeorge syndrome (DGS) and 22q11.2 hemizygosity. The aim of this study was to establish incidence and significance of 22q11.2 hemizygosity in an unbiased sample of patients with IAA. All 15 children with IAA who were referred to our hospital in a 3-year period were tested by chromosome and fluorescence in situ hybridization (FISH) analysis with the probes D22S75, Tuplel, and cHKAD26 and by a set of 10 simple tandem repeat polymorphic (STRP) markers. In nine of 11 children with IAA type B, 22q11.2 hemizygosity was demonstrated by FISH and STRP analysis, but in none of the four children with type A. In all but one child, deletion size was approximately 3 Mb. The girl with the smaller deletion of approximately 1.5 Mb differed because of an Ullrich-Turner syndrome-like phenotype and severe T-cell defect. Additionally, in one patient with phenotypic signs of DGS, a small deletion distal to the known DGS region containing the marker D22S308 was suspected by STRP analysis. One deletion was shown to be inherited from a healthy father and one IAA type A recurred in a sib. T-cell anomalies were evident in eight of the nine children with classical deletion, five of whom suffered also from hypoparathyroidism. With respect to cause and clinical course, IAA type A and B were shown to represent different entities. This study showed that variable symptoms of 22q11.2 hemizygosity may cluster.

Intermittent administration of furosemide versus continuous infusion in the postoperative management of children following open heart surgery.

Objective: To compare the amount of furosemide needed to fulfil defined criteria for renal output if given intermittently or as a continuous infusion and to compare the effect of these two regimens on hemodynamic variables and urine electrolyte concentrations. Design: Prospective randomized study of postoperative hemodynamically stable pediatric cardiac patients. The patients were given furosemide according to the urine output, either as an intermittent bolus injection or as a continuous infusion. Setting: Pediatric intensive care unit in a university hospital Patients: The patients were randomly assigned before admission to either the intermittent i. v. or the continuous furosemide i. v. infusion group. Measurements and results: Demographic and hemodynamic data were recorded for a maximum of 72 h, as were furosemide dose, urine output, and fluid and inotropic drug requirements. Forty-six patients completed the study. Maximal hourly urine output was significantly higher in the intermittent group. A significantly lower dose of furosemide in the intermittent group produced the same 24-h urine volume as in the continuous infusion group. Conclusions: Intermittent furosemide administration may be recommended in hemodynamically stable postoperative pediatric cardiac patients because of less drug requirement. However, the high maximal urine output may cause hemodynamic problems in patients who depend on high inotropic support.

Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display.

Clotting factor VIII (fVIII)-inhibitory antibodies represent a major problem in the treatment of haemophilia A. To understand the inactivation mechanisms and to pave the way towards modifications of recombinant clotting factors that reduce their immunogenicity, the exact localization of immunodominant epitopes is required. Here, a random peptide phage display library was employed to identify epitopes of polyclonal fVIII antibodies isolated from patients plasma by affinity chromatography. FVIII-binding specificity and inhibitory activity of the isolated fVIII antibodies were confirmed by ELISA and Bethesda assays. Phage selection on the individual samples yielded several phages which were displaced from binding to the respective antibody preparation by fVIII. Their homology with amino acid motifs of human fVIII and immunoprecipitation results with radioactively labelled fVIII fragments suggested putative epitopes in the A1, A2 and C1 domains of fVIII for one and in the C2 domain for another patient. Synthetic peptides corresponding to the A2, C1 and C2 domain epitopes blocked antibody binding to fVIII and partially neutralized the inhibitory activity of the respective plasma in Bethesda assays. These results provide the proof of principle that random peptide libraries can be used for the mapping of epitopes in a polyclonal antibody preparation.

AGE-RELATED REFERENCE VALUES FOR ACTIVATION MARKERS OF THE COAGULATION AND FIBRINOLYTIC SYSTEMS IN CHILDREN

The physiology of the hemostatic system in infancy and childhood is different from that in adulthood. Despite differences in several components of the coagulation and fibrinolytic systems, there is no evidence that children are at greater risk for hemorrhagic or thrombotic problems compared with adults (1,2). Advances in understanding of the biochemistry of the hemostatic mechanism have led to the development of sensitive immunochemical methods for measuring peptides or enzyme-inhibitor complexes that are liberated with the activation of the coagulation and fibrinolytic systems in vivo (3). Activation markers of coagulation and fibrinolysis have been measured in newborns, infants and children with a variety of underlying disorders (4-16). However, reference ranges for children of different age groups have hitherto not been established. The aim of the present study was to document thrombin-antithrombin III-complex (TAT), prothrombin fragment 1 + 2 (F1 + 2), plasmin-alpha 2-antiplasmin-complex (PAP) and D-dimer in healthy children and to determine whether age-related differences can be demonstrated.

Balloon dilatation of critical left heart stenoses in low birthweight infants

We describe the results of balloon angioplasty in 5 infants with body weights of 850‐2400 g. Three patients with severe aortic valve stenosis and two patients with isthmic coarctation of the aorta experienced relief of stenosis. Two patients with aortic valve stenosis developed thrombosis of the femoral artery; however, complete resolution of the compromised pulse occurred following thrombolytic therapy. In both patients with isthmic coarctation, pulses on the right leg remained diminished. All patients are doing well 0.28 to 3.32 y after the procedure; none has required additional therapy. Our results in a limited number of consecutive low birthweight infants show that balloon dilatation is feasible and can be performed successfully even in neonates with body weights <1500g. According to our experience, balloon dilatation in infants with body weights >2000 g does not differ significantly from standard procedures. In very small infants, however, balloon angioplasty requires special precautions to avoid temperature loss. Arterial access is the major problem in small children, and requires further improvement.

Endothelial cells play an important role in the antiaggregatory effect of nitric oxide.

ObjectiveTo investigate the role of endothelial cyclooxygenase in the antiaggregatory effect of nitric oxide, and to investigate the significance of the time span between contact of nitric oxide and platelets and laboratory evaluation by platelet aggregation. DesignProspective, controlled, in vitro study. SettingResearch laboratory of a university hospital. ParticipantsThree healthy volunteers. InterventionsIncubation of platelets with different concentrations (30 &mgr;M, 100 &mgr;M, 500 &mgr;M, 1000 &mgr;M) of the nitric oxide-donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) for varying incubation times (0 hrs, 1 hr, 2 hrs, 4 hrs) with and without endothelial cells. Induction of platelet aggregation with adenosine diphosphate. Inhibition of the effect of SNAP by 100 &mgr;M of the guanylate cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). Inhibition of prostacyclin production by endothelial cells with COX inhibitors acetyl salicylic acid (1 mM) and indomethacin (10 &mgr;M). Measurements and Main ResultsIncubation with endothelial cells (= controls) had no effect on platelet aggregation. Platelet aggregation was significantly inhibited by all concentrations of SNAP. Time course studies with 30 &mgr;M of SNAP showed an inhibitory effect only after 0, 1, and 2 hrs of incubation, whereas after 4 hrs of incubation the inhibition of platelet aggregation could not be detected any more. Endothelial cells significantly increased the inhibitory effect of SNAP after 1 and 2 hrs of incubation. Incubation with ODQ with and without endothelial cells reversed the SNAP-mediated inhibition of maximum platelet aggregation regardless of the incubation time. Pretreatment of the endothelial cells with the COX inhibitors acetyl salicylic acid and indomethacin blocked the increased inhibitory effect of the endothelial cells after 1 and 2 hrs of incubation. ConclusionsThe time span between nitric oxide contact with platelets and induction of platelet aggregation by adenosine 5′-diphosphate is important for correct estimation of the antiaggregatory effect of nitric oxide. Endothelial cyclooxygenase plays an important role in the nitric oxide-mediated inhibition of platelet aggregation.

The Role of α2-Antiplasmin in the Inhibition of Clot Lysis in Newborns and Adults

The fibrinolytic system is involved in a wide variety of biological phenomena and differs physiologically in newborns compared to older children or adults. Newborns plasminogen differs from adult plasminogen in carbohydrate composition, cell binding and activation kinetics. The fetal plasminogen has an increased concentration of sialic acid similar to fetal fibrinogen. In a previously reported study on plasminogen activation kinetics, we demonstrated differences in the reaction kinetics between fetal plasmin and plasmin inhibitors as compared to the reaction between adult plasmin and inhibitors. Hitherto, there are no investigations on the role of alpha 2-antiplasmin in the inhibition of clot lysis in newborns. We studied the contributions of purified alpha 2-antiplasmin to the regulation of fibrin clot lysis by use of a microtiter clot lysis assay. The lysis time of clots without adding purified alpha 2-antiplasmin correlated to the activator dose. When purified alpha 2-antiplasmin was incorporated at final concentrations ranging from 10 to 40 micrograms/ml, strong dose-dependent inhibition resulting in a prolongation of 50% lysis time was observed. The inhibition in newborns at all alpha 2-antiplasmin concentrations was less pronounced than that of the adults if 50% lysis time was < 55 min. If 50% lysis time was > 55 min. the prolongation of 50% lysis time was more pronounced in newborn than in adult plasma. The fact that we have less effects of alpha 2-antiplasmin in newborn infants at short reaction times despite the lower plasminogen levels, is consistent with slower reaction kinetics between plasmin and alpha 2-antiplasmin in newborns. These differences raise an explanation for the findings of Idell et al. [Am J Respir Crit Care Med 1994; 149:767-775] in premature baboons with neonatal respiratory distress syndrome (RDS). The knowledge of different reaction kinetics between plasmin and alpha 2-antiplasmin and the role of alpha 2-antiplasmin in the inhibition of clot lysis in newborns can be helpful to elucidate the significance of the fetal fibrinolytic system in neonatal RDS and to establish treatment strategies for fibrinolytic therapy of progressive RDS.

Postnatal respiratory distress in a dichorial twin with congenital thoracic neuroblastoma after assisted reproduction by intracytoplasmatic sperm injection

We report on the case of a female twin with congenital thoracic neuroblastoma after conception via intracytoplasmatic sperm injection (ICSI). Birth occurred at 37 + 1‐week gestation per primary sectio caesarea. Acute respiratory distress necessitated intubation and mechanical ventilation. Ultrasound and magnetic resonance imaging (MRI) showed a mass in the right upper thorax compressing the trachea. The tumor was subtotally excised and histological analysis revealed neuroblastoma. No further treatment was given. The residual primary tumor regressed spontaneously. Four years after diagnosis, both twins are healthy and normally developed. Pediatr Blood Cancer 2007;48:358–360.

A 2-year-old boy with recurrent severe bleeding: von Willebrand type 2B and ITP--or von Willebrand type 2B alone?

We report on a 2-year-old boy presenting in a peripheral clinic with epistaxis and hematemesis 2 weeks after onset of febrile bronchitis. He had pronounced anemia (Hb 7.3 g/dl) and a low thrombocyte count (Fig. 1). A blood smear showed non-speci®c thrombocytopenia. Antibiotic and prednisone administration was successful and platelets increased. Later the child needed ENT surgery after several severe bleeding episodes. Hemorrhage was due to thrombocytopenia as low as 33,000/ll. As the parents refused further steroids, the child was treated, though without bene®t, by a non-medical practitioner. Only after 11 months were bleeding time (677 s) and clotting factors analyzed: FVIII 36%, vWFantigen 47%, ristocetin cofactor 10%. vWD type 1 was diagnosed at a central laboratory. Bleeding episodes were then easily controlled by DDAVP. As thrombocytopenia is more frequently associated with vWD2B than with vWD1 we proceeded to veri®cation of the diagnosis elsewhere. In fact collagen binding activity was 3% and multimeric structure corresponded to vWD2. Ristocetin-induced platelet aggregation of normal together with patient plasma at 0.5 mg/ml indicated subtype 2B. Later DDAVP testing (0.4 lg/kg body weight) of the child without infection showed a decrease of platelets from 250,000/ll to 170,000/ll. Molecular analysis revealed a heterozygous mutation in the A1-domain (G4196 A) leading to a single amino acid substitution of valine to methionine at position 1316.

Endothelial cells influence the sodium nitroprusside mediated inhibition of platelet aggregation by an as yet unkown pathway

The clinical use of Sodium nitroprusside (SNP) may be associated with an alteration of platelet function. The main focus of this study was the effect of SNP on platelet aggregation in the absence or presence of endothelial cells.Methods: Platelets were incubated with different concentrations of SNP with and without endothelial cells. Platelet aggregation was induced by ADP.Results: Platelet aggregation was significantly inhibited by all concentrations of SNP. Endothelial cells significantly increased this inhibitory effect of SNP. Time course studies showed an inverse correlation of incubation time to platelet aggregation inhibition in the absence of endothelial cells, and a direct correlation in the presence of endothelial cells. Blocking platelet and endothelial cell guanylate cyclase with 1 H-(1,2,4)-oxadiazolo(4,3-a) quinoxalin-1-one (ODQ), or pretreatment of the endothelial cells with cyclooxygenase – inhibitors, had no influence on the increased inhibitory effect of the endothelial cells. Cyanide reversed the inhibitory effect of SNP completely.Conclusion: Endothelial cells play an important role in the SNP mediated inhibition of platelet aggregation. The effect is reversible only by cyanide, not by blocking classical NO signal transduction.