A novel mutation in BCS1L associated with deafness, tubulopathy, growth retardation and microcephaly.

UNLABELLED: We report a novel homozygous missense mutation in the ubiquinol-cytochrome c reductase synthesis-like (BCS1L) gene in two consanguineous Turkish families associated with deafness, Fanconi syndrome (tubulopathy), microcephaly, mental and growth retardation. All three patients presented with transitory metabolic acidosis in the neonatal period and development of persistent renal de Toni-Debré-Fanconi-type tubulopathy, with subsequent rachitis, short stature, microcephaly, sensorineural hearing impairment, mild mental retardation and liver dysfunction. The novel missense mutation c.142A>G (p.M48V) in BCS1L is located at a highly conserved region associated with sorting to the mitochondria. Biochemical analysis revealed an isolated complex III deficiency in skeletal muscle not detected in fibroblasts. Native polyacrylamide gel electrophoresis (PAGE) revealed normal super complex formation, but a shift in mobility of complex III most likely caused by the absence of the BCS1L-mediated insertion of Rieske Fe/S protein into complex III. These findings expand the phenotypic spectrum of BCS1L mutations, highlight the importance of biochemical analysis of different primary affected tissue and underline that neonatal lactic acidosis with multi-organ involvement may resolve after the newborn period with a relatively spared neurological outcome and survival into adulthood. CONCLUSION: Mutation screening for BCS1L should be considered in the differential diagnosis of severe (proximal) tubulopathy in the newborn period. WHAT IS KNOWN: • Mutations in BCS1L cause mitochondrial complex III deficiencies. • Phenotypic presentations of defective BCS1L range from Bjornstad to neonatal GRACILE syndrome. What is New: • Description of a novel homozygous mutation in BCS1L with transient neonatal acidosis and persistent de Toni-Debré-Fanconi-type tubulopathy. • The long survival of patients with phenotypic presentation of severe complex III deficiency is uncommon.

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody.

BACKGROUND: beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial alpha(V)beta(3). Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. OBJECTIVES: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on beta(3)-integrins. METHODS: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC(50) were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb-GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. RESULTS: We observed specific and high-affinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIb-GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with alpha(V)beta(3), and also demonstrated proangiogenic effects in tube formation and CAM assays. CONCLUSIONS: These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.

Coagulation markers predict survival in cancer patients.

In order to prospectively evaluate the predictive value of coagulation markers such as the fibrin Ddimer for survival of cancer patients, we analyzed their role in a prospective study at a University Hospital Institute of Medical Oncology. 268 consecutive outpatients with cancer were included, 72 in remission and 196 with active disease. All cause mortality in relation to the marker levels was measured. 99/268 patients died during the observation period of 4,484 patient months (mean: 17 months). Patients with active disease had a significant, 1.5-5-fold increased marker concentration compared to patients in remission. When analyzed in quartiles, the data showed a lower than predicted death rate in the first quartile and a significantly elevated mortality in the fourth marker quartile. The odds ratio for death predicted by the fibrin monomer (FM) in the fourth vs. the first quartile was 4.1 (95% C.I.: 1.7-9.7) and p = 0.005 for the multivariate analysis of the markers. We conclude that a single determination of coagulation markers, particularly of TAT, FM, and Ddimer is sufficient to strongly predict survival in cancer patients over the following 1-3 years.

Silicon-based biosensors for rapid detection of protein or nucleic acid targets.

BACKGROUND: We developed a silicon-based biosensor that generates visual, qualitative results or quantitative results for the detection of protein or nucleic acid targets in a multiplex format. METHODS: Capture probes were immobilized either passively or covalently on the optically coated surface of the biosensor. Intermolecular interactions of the immobilized capture probe with specific target molecules were transduced into a molecular thin film. Thin films were generated by enzyme-catalyzed deposition in the vicinity of the surface-bound target. The increased thickness on the surface changed the apparent color of the biosensor by altering the interference pattern of reflected light. RESULTS: Cytokine detection was achieved in a 40-min multiplex assay. Detection limits were 4 ng/L for interleukin (IL)-6, 31 ng/L for IL1-beta, and 437 ng/L for interferon-gamma. In multianalyte experiments, cytokines were specifically detected with signal-to-noise ratios ranging from 15 to 80. With a modified optical surface, specificity was also demonstrated in a nucleic acid array with unambiguous discrimination of single-base changes in a 15-min assay. For homozygous wild-type and homozygous mutant samples, signal-to-noise ratios of approximately 100 were observed. Heterozygous samples yielded approximately equivalent signals for wild-type and mutant capture probes. CONCLUSIONS: The thin-film biosensor allows rapid, sensitive, and specific detection of protein or nucleic acid targets in an array format with results read visually or quantified with a charge-coupled device camera. This biosensor is suited for multianalyte detection in clinical diagnostic assays.

Low molecular weight dextran sulfate prevents complement activation and delays hyperacute rejection in pig-to-human xenotransplantation models.

Dextran sulfate of 5000 molecular weight (DXS 5000) is known to block complement activation as well as the intrinsic coagulation cascade by potentiation of C inhibitor. The effect of DXS 5000 on hyperacute rejection (HAR) was tested in pig-to-human xenotransplantation models. For in vitro testing, a cytotoxicity assay was used with the pig kidney cell line PK15 as target cells and fresh, undiluted human serum as antibody and complement source. Ex vivo pig lung perfusion was chosen to assess DXS 5000 in a physiologic model. Pig lungs were perfused with fresh, citrate-anticoagulated whole human blood to which 1 or 2 mg/ml DXS 5000 were added; the lungs were ventilated and the blood de-oxygenated. Pulmonary vascular resistance (PVR) and blood oxygenation (deltapO2) were monitored throughout the experiment. Autologous pig blood and human blood without DXS 5000 served as controls. In the PK 15 assay DXS 5000 led to a complete, dose-dependent inhibition of human serum cytotoxicity with an average IC50 of 43 +/- 18 microg/ml (n=8). Pig lungs perfused with untreated human blood (n=2) underwent HAR within 105 +/- 64 min, characterized by increased PVR, decrease of deltapO2, and generalized edema. Microscopically, capillary bleeding as well as deposition of human antibodies, complement and fibrin could be observed. Addition of DXS 5000 (n=4) prolonged lung survival to 170 +/- 14 min for 1 mg/ml and 250 +/- 42 min for 2 mg/ml. and PVR values as well as edema formation were comparable to control lungs that were perfused with autologous pig blood (n=2). Activation of complement (activation products in serum, deposition on lung tissue) and the coagulation system (fibrin monomers) were significantly diminished as compared to human blood without DXS 5000. Binding of anti-Gal antibodies was not influenced, and in vitro experiments showed no evidence of complement depletion by DXS 5000. In conclusion, DXS 5000 is an efficient complement inhibitor in pig-to-human xenotransplantation models and therefore a candidate for complement-inhibitory/anti-inflammatory therapy either alone or in combination with other substances and warrants further investigation.

Interference-based detection of nucleic acid targets on optically coated silicon.

Sequence-specific detection of polynucleotides typically requires modified reporter probes that are labeled with radioactive, fluorescent, or luminescent moieties. Although these detection methods are capable of high sensitivity, they require instrumentation for signal detection. In certain settings, such as clinical point of care, instrumentation might be impractical or unavailable. Here we describe a detection approach in which formation of a nucleic acid hybrid is enzymatically transduced into a molecular thin film that can be visually detected in white light. The system exploits a flat, optically coated silicon-based surface to which capture oligonucleotides are covalently attached. The optimized system is capable of detection of nucleic acid targets present at sub-attomole levels. To supplement visual detection, signals can be quantitated by a charge-coupled device. The design and composition of the optical surface, optimization of immobilization chemistry for attachment of capture probes, and characterization of the efficiency of the hybridization process are presented. We describe the application of this system to detection of a clinically relevant target, the mecA gene present in methicillin-resistant Staphylococcus aureus.

Antiplatelet effects of clopidogrel compared with aspirin after myocardial infarction: enhanced inhibitory effects of combination therapy.

OBJECTIVES: We sought to compare the inhibitory effects of the combination of two doses of aspirin plus clopidogrel with either drug alone on platelet aggregation and activation. BACKGROUND: Enhanced platelet inhibitory effects of clopidogrel by aspirin on platelet aggregation and activation are suggested by experimental studies but have not been shown in humans. METHODS: The effects of clopidogrel 75 mg or aspirin 100 (300) mg on platelet aggregation and activation by flow cytometry after stimulation with various agonists were determined in 30 patients with a past history of myocardial infarction. RESULTS: Clopidogrel alone or in combination with aspirin markedly inhibited adenosine diphosphate (ADP)-mediated platelet aggregation compared with monotherapy with aspirin (24.6 +/- 3.3% or 26.6 +/- 2.7% vs. 44.7 +/- 2.9%; p < 0.001). Combined treatment significantly inhibited collagen-induced aggregation compared with aspirin and clopidogrel (16.4 +/- 2.4%, 36.5 +/- 4.2% and 59.3 +/- 5.1%, respectively;, p < 0.001) and resulted in considerable inhibition of aggregation induced by thrombin receptor agonist peptide (TRAP, p < 0.03). Clopidogrel with or without aspirin significantly suppressed expression of platelet activation markers CD 62p, CD 63 and PAC-1 after stimulation with ADP or thrombin (p < 0.001). In addition, the combined treatment was more effective than either agent alone after activation with low dose thrombin (p < 0.05). Both doses of aspirin equally potentiated the platelet inhibitory effects of clopidogrel. CONCLUSIONS In this prospective clinical ex vivo platelet study, clopidogrel was more effective than aspirin in inhibiting ADP-mediated platelet aggregation and activation. Clopidogrel in combination with aspirin showed synergistic inhibitory effects after stimulation with collagen and thrombin compared with monotherapies. Thus, this dual antiplatelet treatment strategy deserves further evaluation in clinical trials for secondary prevention of acute myocardial infarction or unstable angina.

Preparation and characterization of 'heparinocytes': erythrocytes with covalently bound low molecular weight heparin.

In an attempt to create the possibility of stable, long acting, intravascular anticoagulation, low molecular weight heparin was modified by introducing a sulfhydryl group into the molecule (LMWH-SH). Human erythrocytes were covalently grafted with LMWH-SH by the use of a heterobifunctional coupling reagent which reacts with the SH group of LMWH-SH and surface exposed amino groups of erythrocytes now called 'heparinocytes' (HC). HC were morphologically indistinguishable from untreated erythrocytes and displayed identical osmotic resistance. The functionality of HC was analyzed by classical coagulation tests in which they dose dependently inhibited clot formation. HC were also functional in recalcified whole blood inhibiting thrombin formation as assessed by the cleavage of the chromogenic substrate S-2238. The system appears applicable as a potential autologous, long-term anticoagulant treatment or prophylaxis.

Hemostatic activation under anticoagulant treatment: a comparison of unfractionated heparin vs. nadroparin in the treatment of proximal deep vein thrombosis.

BACKGROUND: Multiple clinical trials have been performed to compare standard heparin with low molecular weight heparin in the therapy of deep vein thrombosis, but little is known about the time course of the markers of hemostatic system during the treatment with these two heparin regimens. METHODS: Twenty patients with proximal deep vein thrombosis confirmed by duplex ultrasound and phlebography were randomly assigned to either unfractionated heparin (UH) given as an intravenous bolus of 80 U/kg followed by a constant infusion of 18 U/kg/h, or nadroparin 185 AXa IU/kg once daily subcutaneously. Oral anticoagulants were started at day 4. Markers of hemostatic activation (F1+2, FPA, TAT, D-dimer) were measured daily for 4 days. Primary endpoints were the time course of these markers; secondary endpoints consisted in the evaluation of thromboembolic and hemorrhagic complications by clinical outcome and Marder score. RESULTS: Treatment with UH resulted in a rapid achievement of therapeutic heparin levels. UH reduced markers of fibrin formation and fibrinolysis more rapidly than nadroparin (p < 0.05). Within the nadroparin group activation of prothrombotic markers four hours after the subcutaneous injection (peak level) was significantly lower when compared with the time prior to injection (trough level). Secondary endpoints showed no significant difference between the two groups. CONCLUSION: Continuous intravenous perfusion of UH administered on a basis of a weight-adjusted nomogram controlled markers of the hemostatic system more rapidly than once-daily subcutaneously administered weight-adjusted nadroparin.

Thin film biosensor for rapid visual detection of nucleic acid targets.

BACKGROUND: We have developed a silicon-based biosensor that generates a visual signal in response to nucleic acid targets. METHODS: In this system, capture oligonucleotide probes are immobilized on the surface of the biosensor. Interaction of the capture probes with a complementary target and a biotinylated detector oligonucleotide allows initiation of formation of an organic thin film on the biosensor. Thin film formation is completed by enzymatic activity of peroxidase conjugated to an anti-biotin antibody. Peroxidase catalyzes deposition of an insoluble product onto the silicon surface, generating a uniform thin film. The increased thickness on the surface alters the perceived color of the biosensor through changes in the interference patterns of reflected light from the surface, causing a color change from gold to purple. RESULTS: The biosensor results may be evaluated by direct visual inspection or quantified by ellipsometry. Results are obtained in 25 min with a detection limit of 5 pmol/L (150 amol/sample). Selectivity of the biosensor is demonstrated by discrimination of single nucleotide mismatches. Multitarget arrays are also analyzed with the thin film biosensor, and the system is capable of detecting targets from human serum and urine. CONCLUSIONS: The biosensor surface is inexpensive to produce, and the assay format is simple and rapid. The thin film biosensor is adaptable to a wide variety of nucleic acid detection applications, including rapid diagnostic testing for infectious disease panels, antibiotic resistance panels, or allelic discrimination of specific genetic markers.

Fibrinogen St. Gallen I (gamma 292 Gly--> Val): evidence for structural alterations causing defective polymerization and fibrinogenolysis.

Fibrinogen St. Gallen I was detected in an asymptomatic Swiss woman. Routine coagulation tests revealed a prolonged thrombin and reptilase time. Functionally measured fibrinogen levels were considerably lower than those determined immunologically. Polymerization of fibrin monomers derived from purified fibrinogen was delayed in the presence of either calcium or EDTA. Normal fibrinopeptide A and B release by thrombin was established. An abnormal degradation of fibrinogen St. Gallen I by plasmin was observed. Fragment D1 of normal fibrinogen was fully protected against further proteolysis in the presence of 10 mM calcium, whereas fibrinogen St. Gallen I was partially further degraded to fragments D2 and D3. In the presence of 10 mM EDTA, the conversion of variant fragment D1 to D2 was accelerated whereas the degradation of fragment D2 to D3 was delayed in comparison to degradation of fragments D1 and D2 of normal fibrinogen. Three high-affinity calcium binding sites were found in both normal and variant fibrinogen. Mutation screening with SSCP analysis suggested a mutation in exon VIII of the gamma-chain gene. Cycle sequencing of this gene portion revealed a single base substitution from G to T of the base 7527, leading to replacement of gamma 292 glycine by valine. The same mutation has already been described for the fibrinogen variant Baltimore I. Molecular modeling was performed of a part of the gamma-chain containing the mutation site, based on recently published X-ray crystal structures of human fibrinogen fragment D and of a 30 kD C-terminal part of the gamma-chain. Significant structural alterations due to the substitution of glycine by valine at gamma 292 were observed, e.g. spreading of the protein backbone, probably leading to a modified accessibility of the plasmic cleavage sites in the gamma-chain at 356 Lys and 302 Lys. A shift of gamma 297 Asp that is involved in interactions of fragment D with the Gly-Pro-Arg-Pro-peptide was noted by molecular modeling. The latter observation is compatible with delayed polymerization of fibrin monomers.

Effect of intravenous heparin infusion on thrombin-antithrombin complex and fibrinopeptide A in unstable angina.

BACKGROUND: In unstable angina, the clinical efficacy of heparin is limited in time, and recurrence of adverse events has been reported after discontinuation of the anticoagulant. METHODS: In 21 episodes of unstable angina, we used the plasma level of fibrinopeptide A (FPA) and of thrombin-antithrombin complex (TAT) to evaluate the pattern of thrombin inhibition by heparin and the effect of stopping heparin and initiating aspirin. RESULTS: At admission, the plasma level of FPA was increased: median value 3.7 ng/mL compared with 5.5 ng/mL in a control group of 20 patients with early myocardial infarction (not significant). The following findings were observed during a 4-day course of intravenous heparin infusion: (1) FPA decreased significantly 6 hours after the start of therapy; (2) FPA was lower when activated partial thromboplastic time (aPTT) was >1.5 times baseline; (3) there was a significant negative correlation between aPTT and FPA. Twenty-four hours after heparin was discontinued and aspirin initiated, a significant increase in TAT and FPA in plasma was observed. CONCLUSIONS: The results confirm ongoing fibrin formation in the active phase of unstable angina, indicate incomplete and variable inhibition of thrombin by heparin during continuous infusion, and suggest a risk of re-emergence of thrombosis (in spite of initiating aspirin) 24 hours after withdrawal of heparin. Data demonstrate a better control of thrombin activity when heparin is infused at rates that maintain aPTT at >1.5 times baseline, as currently recommended in unstable angina.

Mechanisms of cerebrovascular events as assessed by procoagulant activity, cerebral microemboli, and platelet microparticles in patients with prosthetic heart valves.

BACKGROUND AND PURPOSE: Cerebrovascular events (CVE) in patients with prosthetic heart valves (PHV) have remained a severe and frequent complication despite oral anticoagulation with or without aspirin. We studied the possible pathophysiological involvement of platelet-derived microparticles (PMP) as a contributing factor for the increased incidence of CVE in patients with PHV. METHODS: We compared in a retrospective, case-control study the clinical outcome after the implantation of the PHV with several different independent morphological and functional methods, including simultaneous transcranial Doppler monitoring of both middle cerebral arteries, PMP detection by flow cytometry with use of platelet-specific antibodies, coagulation markers, and determination of the procoagulant activity by Russell's viper venom time, a phospholipid-dependent coagulation assay. RESULTS: Eight of 26 patients with PHV had 9 CVE during 136 person-years of observation. Transcranial Doppler monitoring revealed an increased frequency of microembolic signals recorded over a 30-minute period in patients with CVE (75+/-25; median, 55; range, 27 to 248) compared with those without CVE (23+/-12; median, 7; range, 0 to 153; P<0.05) or with control subjects (0; P<0.001). Flow cytometry analysis showed increased levels of PMP in patients with compared to those without CVE (4.1+/-0.6% versus 2.4+/-0.4% of all fluorescence-positive events gated; P<0.05). Increased procoagulant activity was documented by the shortened Russell's viper venom time expressed as an increased level of platelet equivalents per microliter of plasma in patients compared with control subjects (+24.7+/-14.9%; P<0.01). Subgroup analysis revealed that patients with CVE had a higher excess of platelet equivalents per microliter of plasma than patients without CVE in relation to the controls (+68.7+/-36.7%; P<0.05). Mildly elevated thrombin-antithrombin III complexes (2.9+/-0.7; median, 2.3; normal, <2.0 microg/L) suggested incompletely suppressed thrombin formation, and fibrin generation (fibrinopeptide A) was in the upper normal range (2.1+/-0.2; median, 1.8; normal, <2.0 ng/mL), despite adequate anticoagulation (INR=3.6+/-0.1). CONCLUSIONS: Our data show increased microembolic signals, platelet microparticles, and procoagulant activity in symptomatic patients with PHV and provide a potential pathophysiological explanation of CVE.

Cyclosporine-induced detachment of vascular endothelial cells initiates the intrinsic coagulation system in plasma and whole blood.

Cyclosporine A (CsA) is supposed to alter the metabolism of vascular endothelial cells, leading to a prothrombotic state. We examined by which mechanism human umbilical vein endothelial cells (HUVECs) treated with CsA would promote coagulation in human plasma and in whole blood. Treatment of HUVECs with CsA at concentrations clinically used led to dose-dependent cell detachment, with the subsequent exposure of the highly procoagulant connective tissue. As determined by scanning electron microscopy, cell counting of detached and adherent cells, and antigenic measurement of collagen exposure, HUVECs treated with 0.4 micrograms/ml CsA (or more) for 4 days exhibited significant amounts of subendothelial areas. On CsA-treated HUVEC monolayers, the clotting time of recalcified citrated platelet-rich plasma (PRP), but not platelet-poor plasma (PPP), was dose-dependently shortened. Likewise, the onset of thrombin generation was significantly earlier. Except at a high concentration of 8.0 micrograms/ml CsA, there was no procoagulant effect when PPP was used. To investigate CsA-treated HUVECs in whole blood, cells were cultivated on globular microcarriers and were incubated with nonanticoagulated whole blood. When untreated cells were used, generation of factor Xa, thrombin, and kallikrein was completely suppressed for 30 minutes. HUVEC beads treated with 0.4 and 0.8 micrograms/ml CsA, however, led to a dose-dependent generation of all three coagulation factors, with peak values at 2.5 to 5 minutes. Extrinsic activation was excluded, since CsA treatment did not induce tissue factor activity in HUVECs. Furthermore, the thrombomodulin activity of HUVECs w as not altered by CsA. In conclusion, treatment of HUVECs with CsA for 4 days at concentrations clinically used leads to the exposure of subendothelial areas that induce activation of the intrinsic coagulation in recalcified PRP and nonanticoagulated whole blood.

Use of epoetin alfa in autologous blood donation programs for patients scheduled for elective cardiac surgery.

The optimum dosage of subcutaneous (s.c.) epoetin alfa was assessed in a double-blind study in 31 patients scheduled for cardiac surgery. Patients received a total of four doses of either epoetin alfa 150 IU/kg (n = 11), epoetin alfa 300 IU/kg (n = 10), or placebo (n = 10) administered as single s.c. injections at weekly intervals starting 23 days prior to surgery. AB was collected with isovolemic replacement prior to each of the first three doses of medication. During the AB donation period, Hb levels decreased significantly (P < .05) from baseline to surgery in the placebo group (16.5%), compared with no significant decrease in either of the epoetin alfa groups (8.1% and 9.7% in the 150 IU/kg and 300 IU/kg groups, respectively). In addition, the difference between groups with regard to the decrease in Hb level reached statistical significance (P < .05) for the 150 IU/kg group versus placebo. Epoetin alfa treatment was also associated with significantly higher reticulocyte counts and serum erythropoietin levels in the preoperative period compared with placebo.