Normal synthesis and expression of endothelial IIb/IIIa in Glanzmann's thrombasthenia

Glanzmann's thrombasthenia is a bleeding disorder, inherited in an autosomal recessive way and characterized by an absence or deficiency of the platelet glycoprotein (GP) IIb/IIIa complex. Recently, we and others demonstrated that cultured human umbilical vein endothelial cells synthesized a membrane protein complex similar to the platelet GP IIb/IIIa complex. In this article, we demonstrate that endothelial cells isolated from the umbilical vein of a newborn with Glanzmann's thrombasthenia, as compared with normal endothelial cells, show no difference in their ability to synthesize and express this GP IIb/IIIa complex. Our results indicate that Glanzmann's thrombasthenia is not accompanied by an "endotheliopathy."     

Site‐Dependent Reference Point Microindentation Complements Clinical Measures for Improved Fracture Risk Assessment at the Human Femoral Neck

In contrast to traditional approaches to fracture risk assessment using clinical risk factors and bone mineral density (BMD), a new technique, reference point microindentation (RPI), permits direct assessment of bone quality; in vivo tibial RPI measurements appear to discriminate patients with a fragility fracture from controls. However, it is unclear how this relates to the site of the most clinically devastating fracture, the femoral neck, and whether RPI provides information complementary to that from existing assessments. Femoral neck samples were collected at surgery after low‐trauma hip fracture (n = 46; 17 male; aged 83 [interquartile range 77–87] years) and compared, using RPI (Biodent Hfc), with 16 cadaveric control samples, free from bone disease (7 male; aged 65 [IQR 61–74] years). A subset of fracture patients returned for dual‐energy X‐ray absorptiometry (DXA) assessment (Hologic Discovery) and, for the controls, a micro‐computed tomography setup (HMX, Nikon) was used to replicate DXA scans. The indentation depth was greater in femoral neck samples from osteoporotic fracture patients than controls (p < 0.001), which persisted with adjustment for age, sex, body mass index (BMI), and height (p < 0.001) but was site‐dependent, being less pronounced in the inferomedial region. RPI demonstrated good discrimination between fracture and controls using receiver‐operating characteristic (ROC) analyses (area under the curve [AUC] = 0.79 to 0.89), and a model combining RPI to clinical risk factors or BMD performed better than the individual components (AUC = 0.88 to 0.99). In conclusion, RPI at the femoral neck discriminated fracture cases from controls independent of BMD and traditional risk factors but dependent on location. The clinical RPI device may, therefore, supplement risk assessment and requires testing in prospective cohorts and comparison between the clinically accessible tibia and the femoral neck. © 2015 American Society for Bone and Mineral Research.     

Positive remodeling at 3 year follow up is associated with plaque free coronary wall segment at baseline: A serial IVUS study

Aims

At present it is unknown what limits the arterial remodeling process during atherosclerotic plaque formation. In healthy arteries remodeling is regulated by the shear stress induced response by the endothelium. As endothelium at the plaque site is assumed to be dysfunctional, we tested the hypothesis that plaque free wall (PFW) determines vascular remodeling during atherosclerotic plaque build-up.

Methods & results

66 human coronary ROIs (38 patients) were studied at baseline and at 3 years follow up applying intravascular ultrasound (IVUS). From the IVUS images the lumen and external elastic membrane contours were delineated to assess wall thickness (WT), vessel area (VA), Plaque Area (PA) and plaque burden (PA/VA*100%). WT < 0.5 mm was defined as normal and determined the arc of the PFW (0–360°). Positive remodeling was defined as relative difference of VA over time >5%. At baseline, IVUS-PFW was inversely related to plaque burden (p < 0.05). Positive remodeling was most frequently observed in ROIs with IVUS-PFW > 180° (i.e. larger than half of the circumference) compared to PFW < 180° (55% vs. 12%, p < 0.05). Accordingly, plaques with IVUS-PFW > 180° at baseline had the largest change in VA (1.1 ± 2.1 vs. −0.4 ± 0.6 mm², p < 0.05) with an odds ratio of 9.2 to develop positive remodeling.

Conclusions

Our serial IVUS data show that IVUS-PFW is a determinant of vascular remodeling. ROIs with PFW > 180 at baseline had the highest probability to undergo positive remodeling.     

On the IVUS plaque volume error in coronary arteries when neglecting curvature

Plaque volume determined by common linear 3-D IVUS analysis systems will show under- or overestimation in curved vessel segments because these systems approximate the true 3-D transducer pull-back trajectory by a straight line. We developed a mathematical model that showed that the error is primarily dependent on the curvature of the pull-back trajectory and not on vessel tortuosity. Furthermore, we measured this error in vivo in the coronary arteries of 15 patients, comparing the plaque volume using a true 3-D reconstruction method with that of the linear approach. The in vivo plaque volume error ranged from 2.3% to −1.2% for 15 coronary segments with lengths ranging from 38.8 to 89.1 mm (62.2 ± 13 mm). The volume error introduced by linear 3-D IVUS analysis systems is dependent on the curvature of the pull-back trajectory. The error measured in vivo was small and inversely related to segment length.     

Fibrinopeptide A and the phosphate content of fibrinogen in venous thromboembolism and disseminated intravascular coagulation

Concentrations of plasma fibrinopeptide A (FPA) were measured by radioimmunoassay in 50 patients with venous thromboembolism or disseminated intravascular coagulation or both. A consistent discrepancy was observed in values obtained with two anti-FPA antisera. Analysis of extracts from plasma of these patients by high-performance liquid chromatography (HPLC) revealed the presence of a phosphorylated and an unphosphorylated form of the A peptide. Differences in concentrations of FPA measured with the two antisera could be accounted for by their different reactivity with phosphorylated FPA (FPA-P). The differences were abolished by treatment with alkaline phosphatase. A good correlation was observed between the FPA-P content of free A- peptide material and of fibrinogen in plasma as determined by HPLC (r = .88, P less than .001, n = 11). In patients with elevated FPA levels, the mean FPA-P content of fibrinogen was significantly higher (P less than .002, n = 13) than in patients with normal FPA levels (n = 8) and in healthy controls (n = 14). Phosphorus in fibrinogen did not correlate with fibrinogen degradation products or fibrinogen levels and became normal on adequate anticoagulation. Therefore, blood-clotting activation may lead to a high phosphate content of fibrinogen and of free FPA in plasma.     

Negative predictive value of a vision screening program aimed at children aged 3 to 4 years old

PURPOSE: Nova Scotia has a vision screening program which assesses children aged 4[1\2] to 5[1\2] years. However, its use in younger children proved impossible. This study will examine a modified screening protocol for the younger children (3 to 4 years old) and determine its negative predictive value and minimum age for reliable application. MATERIALS AND METHODS: Public health nurses administered the study protocol to 3- to 4-year-old children. One hundred seventy-eight children were screened over two summers. Medical and family history, external inspection, as well as measures of visual acuity with the Lea Hyvarinen symbols chart and stereoacuity with Frisby plates were recorded. Results were compared with a gold standard examination that included full orthoptic and ophthalmologic evaluations. One hundred forty-one (79%) children underwent the gold standard examination. Agreement between screening and gold standard examinations was studied. RESULTS: Data showed increased concordance between screening and gold standard examination results with increasing age up to 41 months. Negative predictive value (NPV) and specificity also improved when data were separated by this age. In children <41 months old, the screening test NPV was 90%, specificity, 68%, and sensitivity, 75%. In comparison, children >/=41 months old had screening test NPV of 96%, specificity, 95%, and sensitivity, 50%. Specificity was higher in the older age group ( P < 0.001). Sensitivity was lower ( P = 0.004). CONCLUSION: This study's vision screening protocol appears better suited for children 41 months and older. They had better pass/fail reproducibility than children <41 months. The test's simplicity allows easy use by non-eye-care professionals. It could potentially lower the reliable screening age of children by 13 months, from 54 months of age (4[1\2] years old) to 41 months. This screening may miss some refractive errors and microtropia/monofixation syndrome, despite normal visual acuity, stereoacuity, and external inspection.     

MDS clinical diagnostic criteria for Parkinson's disease

This document presents the Movement Disorder Society Clinical Diagnostic Criteria for Parkinson's disease (PD). The Movement Disorder Society PD Criteria are intended for use in clinical research but also may be used to guide clinical diagnosis. The benchmark for these criteria is expert clinical diagnosis; the criteria aim to systematize the diagnostic process, to make it reproducible across centers and applicable by clinicians with less expertise in PD diagnosis. Although motor abnormalities remain central, increasing recognition has been given to nonmotor manifestations; these are incorporated into both the current criteria and particularly into separate criteria for prodromal PD. Similar to previous criteria, the Movement Disorder Society PD Criteria retain motor parkinsonism as the core feature of the disease, defined as bradykinesia plus rest tremor or rigidity. Explicit instructions for defining these cardinal features are included. After documentation of parkinsonism, determination of PD as the cause of parkinsonism relies on three categories of diagnostic features: absolute exclusion criteria (which rule out PD), red flags (which must be counterbalanced by additional supportive criteria to allow diagnosis of PD), and supportive criteria (positive features that increase confidence of the PD diagnosis). Two levels of certainty are delineated: clinically established PD (maximizing specificity at the expense of reduced sensitivity) and probable PD (which balances sensitivity and specificity). The Movement Disorder Society criteria retain elements proven valuable in previous criteria and omit aspects that are no longer justified, thereby encapsulating diagnosis according to current knowledge. As understanding of PD expands, the Movement Disorder Society criteria will need continuous revision to accommodate these advances. © 2015 International Parkinson and Movement Disorder Society     

Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.