Acquired von Willebrand syndrome in congenital heart disease surgery: results from an observational case-series.

Essentials Bleeding complications during congenital heart disease surgery in neonatal age are very common. We report the perioperative incidence of acquired von Willebrand syndrome (aVWS) in 12 infants. aVWS was detected in 8 out of 12 neonates and infants intraoperatively after cardiopulmonary bypass. Ten patients received von Willebrand factor concentrate intraoperatively and tolerated it well. SUMMARY: Background Cardiac surgery of the newborn and infant with complex congenital heart disease (CHD) is associated with a high rate of intraoperative bleeding complications. CHD-related anatomic features such as valve stenoses or patent arterial ducts can lead to enhanced shear stress in the blood stream and thus cause acquired von Willebrand syndrome (aVWS). Objective To evaluate the intraoperative incidence and impact of aVWS after cardiopulmonary bypass (CPB) in neonates and infants with complex CHD. Patients/Methods We conducted a survey of patients aged < 12 months undergoing complex cardiac surgery in our tertiary referral center. Twelve patients, whose blood samples were analyzed for aVWS before CPB and immediately after discontinuation of CPB on a routine basis, were eligible for the analysis. von Willebrand factor antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo), collagen binding activity (VWF:CB), VWF:multimers and factor VIII activity (FVIII:C) were determined. Results aVWS was diagnosed by VWF multimer analysis in 10 out of 12 patients (83%) prior to surgery and intraoperatively at the end of CPB in 8 out of 12 patients (66%). Ten patients received VWF/FVIII concentrate intraoperatively as individual treatment attempts during uncontrolled bleeding. They tolerated it well without intraoperative thrombotic events. One patient suffered a transient postoperative cerebral sinuous vein thrombosis. Conclusions aVWS is of underestimated incidence in complex CHD surgery. These data may offer a new approach to reduce the risk of severe bleedings and to achieve hemostasis during high-risk pediatric cardiac surgery by tailoring the substitution with von Willebrand factor concentrate.

Pitfalls in Echocardiography: Coarctation of the Aorta Presenting as Dilated Cardiomyopathy (DCM).

Purpose: The morphologic spectrum of aortic coarctation extends from discrete isthmic obstruction to tubular hypoplasia of the entire aortic arch. Neonates with coarctation frequently present with congestive heart failure and critically reduced perfusion of the descending aorta following ductal closure. During the recent years we observed several infants with coarctation who presented beyond the neonatal period with dilated cardiomyopathy (DCM). We reviewed our patients with coarctation to determine whether this presentation represents an exception or is relevant for the differential diagnosis of children with DCM. Materials and Methods: From 1/2001 to 12/2013 74 babies with isolated coarctation were diagnosed in our institution. 50 patients presented in the neonatal period and 24 patients beyond the first month. Results: 5/74 infants presented after the neonatal period with poorly contractile, dilated left ventricles. Echocardiographic detection of the coarctation was facilitated by application of the ductal view and by Doppler interrogation of the celiac artery revealing a significantly diminished systolic flow velocity. All patients underwent resection of the coarctation and end-to-end anastomosis of the aorta. Postoperative normalization of left ventricular function was observed within a median interval of 2 months. Conclusion: Coarctation of the aorta presenting as DCM accounted for 21 % of our infants with coarctation who presented beyond the neonatal period and 7 % of those in the first year of life. The stenosis was difficult to detect because of its distal location and normal configuration of the aortic arch. Isthmic coarctation should be included in the differential diagnosis of infants with DCM and requires careful examination of the isthmic region in these children.

Severe chest pain caused by Mycoplasma myocarditis in an adolescent patient.

Intermittent chest pain is a common symptom in adolescent patients, which can be attributed to musculoskeletal disorders in the majority of cases. While most patients have a benign course of their pain episodes, the differential diagnosis includes diseases which are associated with serious and even life-threatening complications. Therefore, careful examination and systematic diagnostic evaluation is important. We report on an adolescent, who presented with severe chest pain and distinct electrocardiographic findings caused by Mycoplasma pneumoniae myocarditis.

Factor X deficiency and intracranial bleeding: who is at risk?

Very few mutations of the gene encoding for coagulation factor X (FX) have been found associated with intracranial haemorrhage (ICH) due to FX deficiency (FXD). No guidelines exist as to when prophylaxis in FXD should be started and how patients at risk for ICH can be identified. We report on a novel mutation causative for ICH in a family of Iranian origin and provide a summary of all published mutations in the FX gene related to ICH. The index patient is an infant with umbilical bleeding requiring blood transfusion in the postnatal period. The international normalized ratio (6.01) and activated partial thromboplastin time (117 s) were prolonged. Coagulation factor analysis was normal except for FX activity (<1%). At 4 months, the child suffered a spontaneous severe intracranial haemorrhage. The child was the product of a consanguineous union. Four of five available family members from three generations displayed minor bleeding symptoms and mildly reduced FX. Sequencing of FX gene demonstrated homozygosity for a novel duplication A (c.1402_1403dupA)* in exon 8 and heterozygosity in four family members. We compare this case to all 15 patients with FXD and ICH and their 11 known mutations described so far. This case illustrates a pattern of FXD (a male neonate with umbilical or gastrointestinal bleeding, very low FX:C (<1%) and an underlying homozygous genotype) who may be at high risk for ICH. In these cases, we recommend to start early prophylactic substitution of FX to prevent a possible life-threatening haemorrhage.

Interictal and postictal performances on dichotic listening test in children with focal epilepsy.

Dichotic listening test (DL) is an important tool to disclose speech dominance in healthy subjects and in clinical cases. The aim of this study was to probe if focal epilepsy in children reveals a corresponding suppression of the ear reports contralateral to seizure onset site. Thus, 15 children and adolescents with clinically and electroencephalographically diagnosed focal epilepsy selected for left-hemisphere speech dominance without mental retardation were compared to matched controls according to age, gender, IQ and handedness. All children were assessed with DL for three times: Interictally (t(0)), postictally 5' (t(1)) and 1h (t(2)). At t(0), all groups revealed a right ear advantage (REA), indicating a left-hemisphere speech dominance. There was a continuous increase in right correct score (REC) over the trials for normal controls. Five minutes postictally, there was an abrupt decrease in REC with a sustained left ear correct score (LEC) for children with epilepsy, independent of which side suffered from seizures. This effect was maintained even after 1h. Thus, in children with left-hemisphere speech dominance the epileptic discharges caused a suppression of REC regardless of origin. The seizures may have a prolonged impact on attention and auditory perception for a considerable time after consciousness has been regained.

Neonatal Marfan syndrome: unusually large deletion of exons 24-26 of FBN1 associated with poor prognosis.

Neonatal Marfan syndrome is a very rare subset of the classical Marfan syndrome with pronounced phenotypic expression especially of the cardiovascular manifestations. It is associated with a very poor prognosis, with approximately 50% of affected infants dying from cardiac failure during the first year of life. We present a newborn with the classical phenotype of neonatal Marfan syndrome. Within few hours after birth, progressive and refractory heart failure developed. Postmortal molecular study revealed an unusually large deletion of exons 24-26 within the so-called neonatal region of the gene FBN1, which might explain the unfavourable course of the disease in our patient.

[Salmonella enteritidis infection presenting with septic shock, renal failure and cutaneous manifestations]. / Septischer Schock mit Niereninsuffizienz und Hautmanifestationen als Komplikation einer Salmonella-enteritidis-Infektion.

Infections by Salmonella enteritidis commonly present with diarrhoea, vomiting and fever and complications such as septicaemia, pleural effusion and acute renal failure are usually rare. There are only few reports of cutaneous manifestations and especially septic shock in patients with Salmonella enteritidis infection. We report on a previously healthy seven-year-old boy suffering from Salmonella enteritidis septicaemia presenting with septic shock, pleural effusion, renal failure and an unusual maculopapular skin eruption on both wrists and ankles. The boy had no underlying immunodeficiency.

Transarterial and transvenous approach for transcatheter closure of a large coronary artery fistula with the Amplatzer vascular plug.

Interventional occlusion of coronary artery fistulas has become a well-accepted alternative to surgical therapy. However, occlusion of high-flow lesions in children, necessitating implantation of large occluding devices, may be limited by the requirement of large delivery catheters. This report describes the interventional occlusion of a large coronary artery fistula in an 8-year-old girl. Complete occlusion was achieved by subsequent transvenous and transarterial implantation of two Amplatzer vascular plugs (AVP). The AVP is an interesting alternative for interventional occlusion of large coronary artery fistulas in children that can be deployed safely by rather small guiding catheters.

The Wada test in Austrian, Dutch, German, and Swiss epilepsy centers from 2000 to 2005: a review of 1421 procedures.

Twenty-six Austrian, Dutch, German, and Swiss epilepsy centers were asked to report on use of the Wada test (intracarotid amobarbital procedure, IAP) from 2000 to 2005 and to give their opinion regarding its role in the presurgical diagnosis of epilepsy. Sixteen of the 23 centers providing information had performed 1421 Wada tests, predominantly the classic bilateral procedure (73%). A slight nonsignificant decrease over time in Wada test frequency, despite slightly increasing numbers of resective procedures, could be observed. Complication rates were relatively low (1.09%; 0.36% with permanent deficit). Test protocols were similar even though no universal standard protocol exists. Clinicians rated the Wada test as having good reliability and validity for language determination, whereas they questioned its reliability and validity for memory lateralization. Several noninvasive functional imaging techniques are already in use. However, clinicians currently do not want to rely solely on noninvasive functional imaging in all patients.

[A life-threatening cardiomyopathy following port-a-cath infection under immune tolerance therapy]. / Lebensbedrohliche Kardiomyopathie als Folge einer Portkatheterinfektion bei Immuntoleranztherapie.

The development of inhibitors in patients with severe haemophilia A is a serious complication requiring long term immune tolerance therapy (ITT). ITT frequently requires implantable central venous access, mostly port catheters. Their use may be complicated by thrombosis and infection. We report on an 18 year old patient with severe haemophilia A who had developed a high-titre factor VIII inhibitor in the age of five years. ITT required the implantation of a port system. The postoperative course was complicated by severe septicaemia with congestive cardiac failure. The port catheter was removed due to recurrent fever after 26 days. Our patient developed dilative cardiomyopathy. ITT had to be stopped and was replaced by on demand therapy with an activated prothrombin complex concentrate. Cardiomyopathy resulted in congestive heart failure, severe ventricular arrhythmias and the death of the young man. In patients with haemophilia, dilative cardiomyopathy and development of inhibitors the possibility of cardiac transplantation should be evaluated before increasing inhibitors and the development of pulmonary hypertension exclude this therapeutical option.

Monitoring of technical oils in supercritical CO(2) under continuous flow conditions by NIR spectroscopy and multivariate calibration.

Metal parts and residues from machining processes are usually polluted with cutting or grinding oil and have to be cleaned before further use. Supercritical carbon dioxide can be used for extraction processes and precision cleaning of metal parts, as developed at Forschungszentrum Karlsruhe. For optimizing and efficiently conducting the extraction process, in-line analysis of oil concentration is desirable. Therefore, a monitoring method using fiber-optic NIR spectroscopy in combination with PLS calibration has been developed. In an earlier paper we have described the instrumental set-up and a calibration model using the model compound squalane in the spectral range of the CH combination bands from 4900 to 4200cm(-1). With this model only poor prediction results were obtained if applied to technical oil samples in supercritical CO(2). In this paper we describe a new calibration model, which was set up for the squalane/carbon dioxide system covering the 323-353K temperature and the 16-35.6MPa pressure range. Here, calibration data in the spectral range from 6100 to 5030cm(-1) have been used. This range includes the 5100cm(-1) CO(2) band of the Fermi triad as well as the hydrocarbon 1st overtone CH stretching bands, where spectral features of oil compounds and squalane are more similar to each other. The root mean-squared error of prediction obtained with this model is 4mgcm(-3) for carbon dioxide and 0.4mgcm(-3) for squalane, respectively. The utilizability of the newly developed PLS calibration model for predicting the oil concentration and CO(2) density of solutions of technical oils in supercritical carbon dioxide has been tested. Three types of "real world" cutting and grinding oil formulations were used in these experiments. The calibration proved to be suitable for determining the technical oil concentration with an error of 1.1mgcm(-3) and the CO(2) density with an error of 6mgcm(-3). Therefore, it seems possible to apply this in-line analytical approach on the basis of a cost-effective and time-saving model compound calibration for the surveillance of real world de-oiling and other extraction process based on supercritical carbon dioxide, and furthermore to establish an automated process termination criterion based on this technique.

Leukoencephalopathy with ataxia, hypodontia, and hypomyelination.

The authors describe four unrelated girls with a distinctive neurologic disorder with early-onset progressive ataxia and hypodontia with a characteristic pattern of delayed dentition. Cerebral MRI shows hypomyelinated white matter and cerebellar atrophy; 1H-MRS of white matter reveals a marked elevation of myo-inositol.

The restricted expression of granzyme M in human lymphocytes.

We have analyzed the expression of human granzyme M (Gzm M) in various human leukocyte subsets using the specific mAb 4H10. Using FACS and Western blotting analysis we compared the expression of Gzm M with that of other granzymes (Gzm A and Gzm B) and the lytic protein perforin. Human Gzm M was constitutively highly expressed in NK cells as was perforin and Gzm A. Surprisingly, freshly isolated NK cells had very low (sometimes undetectable) levels of Gzm B. In contrast to Gzm B and perforin, Gzm M was not detected in highly purified CD4(+) and CD8(+) T cells either constitutively or after short term activation in vitro. However, low levels of Gzm M were observed in some T cell clones on prolonged passage in vitro. Gzm M was not detected in highly purified neutrophils, monocytes, or tumor cells of the myelomonocytic lineage. Examination of minor T cell subsets from human peripheral blood showed detectable Gzm M in CD3(+), CD56(+) T cells and gammadelta T cells. A histological staining procedure was developed that demonstrated a granular staining pattern for Gzm M and a cellular distribution similar to that observed by Western blotting. These data indicate that the expression of Gzm M does not always correlate with the lytic activity of cytotoxic cells. However, expression of Gzm M in NK cells, CD3(+), CD56(+) T cells, and gammadelta T cells suggests that this enzyme may play some role in innate immune responses.

Activating Ly-49D NK receptors: expression and function in relation to ontogeny and Ly-49 inhibitor receptors.

Developmental changes in the repertoire of activating Ly-49 family members have not been examined previously. In the present study, we have examined the expression and function of the activating Ly-49s (D and H) from birth through 8 weeks of age. We demonstrate that 1) activating Ly-49s are expressed early, 2) their expression intensity is not different from adult NK cells, and 3) activating receptors are functional. Examination of the inhibitory Ly-49s also demonstrated functional capacity immediately upon expression. To examine the kinetics of expression of the repertoire of activating Ly-49 members, we utilized five- and six-color flow cytometric analyses of NK cells from birth through adulthood. Previous studies examining the inhibitory Ly-49 repertoire have proposed that expression is regulated by the product rule. Our results indicated that Ly-49D, which recognizes H-2Dd, had a discordantly high coexpression of the inhibitory Ly-49s that recognized H-2Dd (Ly-49A and Ly-49G2). The product rule of Ly-49 expression does not explain the coexpression of selected activating and inhibitory receptors. This high level of coexpression of H-2Dd recognizing activating and inhibitory Ly-49s suggests an in vivo selection or regulated coexpression.

Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase.

Bacillus licheniformis alpha-amylase (BLA) is a starch-degrading enzyme that is highly thermostable although it is produced by a rather mesophilic organism. Over the last decade, the origin of BLA thermal properties has been extensively investigated in both academic and industrial laboratories, yet it is poorly understood. Here, we have used structure-based mutagenesis in order to probe the role of amino acid residues previously proposed as being important for BLA thermostability. Residues involved in salt-bridges, calcium binding or potential deamidation processes have been selected and replaced with various amino acids using a site-directed mutagenesis method, based on informational suppression. A total of 175 amylase variants were created and analysed in vitro. Active amylase variants were tested for thermostability by measuring residual activities after incubation at high temperature. Out of the 15 target residues, seven (Asp121, Asn126, Asp164, Asn192, Asp200, Asp204 and Ala269) were found to be particularly intolerant to any amino acid substitutions, some of which lead to very unstable mutant enzymes. By contrast, three asparagine residues (Asn172, Asn188 and Asn190) could be replaced with amino acid residues that significantly increase the thermostability compared to the wild-type enzyme. The highest stabilization event resulted from the substitution of phenylalanine in place of asparagine at position 190, leading to a sixfold increase of the enzyme's half-life at 80 degrees C (pH 5.6, 0.1 mM CaCl(2)). These results, combined with those of previous mutational analyses, show that the structural determinants contributing to the overall thermostability of BLA concentrate in domain B and at its interface with the central A domain. This region contains a triadic Ca-Na-Ca metal-binding site that appears extremely sensitive to any modification that may alter or reinforce the network of electrostatic interactions entrapping the metal ions. In particular, a loop spanning from residue 178 to 199, which undergoes pronounced conformational changes upon removal of calcium, appears to be the key feature for maintaining the enzyme structural integrity. Outside this region, most salt-bridges that were destroyed by mutations were found to be dispensable, except for an Asp121-Arg127 salt-bridge that contributes to the enhanced thermostability of BLA compared to other homologous bacterial alpha-amylases. Finally, our studies demonstrate that the natural resistance of BLA against high temperature is not optimized and can be enhanced further through various means, including the removal of possibly deamidating residues.

Heme oxygenase-1 inhibits TNF-alpha-induced apoptosis in cultured fibroblasts.

Heme oxygenase (HO)-1 catalyzes the oxidative cleavage of heme to yield equimolar amounts of biliverdin, iron, and carbon monoxide. HO-1 is a stress response protein, the induction of which is associated with protection against oxidative stress. The mechanism(s) of protection is not completely elucidated, although it is suggested that one or more of the catalytic by-products provide antioxidant functions either directly or indirectly. The involvement of reactive oxygen species in apoptosis raised the question of a possible role for HO-1 in programmed cell death. Using the tetracycline-regulated expression system, we show here that conditional overexpression of HO-1 prevents tumor necrosis factor-alpha-induced apoptosis in murine L929 fibroblasts. Inhibition of apoptosis was not observed in the presence of tin protoporphyrin, a specific inhibitor of HO activity, and in cells overexpressing antisense HO-1. Interestingly, exogenous administration of a low concentration of carbon monoxide also prevented tumor necrosis factor-alpha-induced apoptosis in L929 fibroblasts. Inhibition of tumor necrosis factor-alpha-induced apoptosis by HO-1 overexpression was reversed by 1H-(1,2, 4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of guanylate cyclase, which is a target enzyme for carbon monoxide. Taken together, our data suggest that the antiapoptotic effect of HO-1 may be mediated via carbon monoxide.

Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization.

Huntington disease (HD) is a genetically dominant condition caused by expanded CAG repeats coding for glutamine in the HD gene product huntingtin. Although HD symptoms reflect preferential neuronal death in specific brain regions, huntingtin is expressed in almost all tissues, so abnormalities outside the brain might be expected. Although involvement of nuclei and mitochondria in HD pathophysiology has been suggested, specific intracellular defects that might elicit cell death have been unclear. Mitochondria dysfunction is reported in HD brains; mitochondria are organelles that regulates apoptotic cell death. We now report that lymphoblasts derived from HD patients showed increased stress-induced apoptotic cell death associated with caspase-3 activation. When subjected to stress, HD lymphoblasts also manifested a considerable increase in mitochondrial depolarization correlated with increased glutamine repeats.

Amino acid sequence, crystallization and structure determination of reduced and oxidized cytochrome c6 from the green alga Scenedesmus obliquus.

Cytochrome c6from the unicellular green alga Scenedesmus obliquus was sequenced, crystallized in its reduced and oxidized state and the three-dimensional structure of the protein in both redox states was determined by X-ray crystallography. Reduced cytochrome c6crystallized as a monomer in the space group P 21212, whereas the oxidized protein crystallized as a dimer in the space group P 3121. The structures were solved by molecular replacement and refined to 1. 9 and 2.0 A, respectively. Comparison of the structures of both redox states revealed only slight differences on the protein surface, whereas a distortion along the axis between the heme iron and its coordinating Met61 residue was observed. No redox-dependent movement of internal water molecules could be detected. The high degree of similarity of the surfaces and charge distributions of both redox states, as well as the dimerization of cytochrome c6as observed in the oxidized crystal, is discussed with respect to its biological relevance and its implications for the reaction mechanisms between cytochrome c6and its redox partners. The dimer of oxidized cytochrome c6may represent a molecular structure occurring in a binary complex with cytochrome b6f. This assembly might be required for the correct orientation of cytochrome c6with respect to its redox partner cytochrome b6f, facilitating the electron transfer within the complex. If the dimerization is not redox-dependent in vivo, the almost identical surfaces of both redox states do not support a long range differentiation between reduced and oxidized cyt c6, i.e. a random collision model for the formation of an electron transfer complex must be assumed.