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.     

Management of nucleus and IOL drop

Background: Thirty six cases of lenticular nucleus drop following phacoemulsification and 43 cases of posterior dislocation of intraocular lens (IOL) inclusive of two paediatric cases were managed by a modified vitrectomy procedure without using perfluorocarbon liquid (PFCL).     

用电化学检测器的高效液相色谱法测定血清及尿中速尿的含量

本文报告了用电化学检测器的高效液相色谱法测定血清及尿中速尿含量的方法。样品予处理方法:血清用乙腈除蛋白,尿用蒸馏水稀释50倍。采用作者合成的FD-Val-OH作为内标物。色谱条件为:反相柱,以含35%乙醇的5mmol/L四丁基铵水溶液为流动相(pH7.50),流速1.0ml/min;用电化学检测器,检测电压0.90V:速尿及内标物的保留时间分别为10和15min。通过计算速尿对内标物的峰高比求得速尿含量。血清及尿中的最低检测浓度分别为16和9ng/ml。标准曲线在0.25～5ng/μl(血清)、0.5～10ng/μl(尿)的浓度范围内呈线性关系。血清及尿中回收率分别为100.5%和100.6%。变异系数在4.6%以下。     

Current concepts of children's perceptions of control

Beliefs about control have been identified as an important aspect of the occupational therapy evaluation process because of their potential influence over the course of action chosen. This paper reviews the current status of research and theory in this area as it relates to children. Issues addressed include the drawbacks of the downward extension of adult theoretical models and tests to children, the multidimensionality of perceived control, and the relation between cognitive development and changes in beliefs. Recent revisions in theoretical models and new measures that provide more differentiated information on children's beliefs about causality and personal efficacy are reviewed. The implications for the selection of instruments and interpretation of results for the assessment of children's perceived control in clinical practice are discussed.     

Immunoblotting characterization of neutrophil antigenic targets in autoimmune neutropenia

We characterized neutrophil autoantigens using an immunoblotting technique with antibodies obtained from patients with autoimmune neutropenia. These results were correlated with serologic characterization of the antibodies, using indirect immunofluorescence and leukoagglutination. Of the 17 sera immunoblotted, 16 showed discrete bands in the molecular weight range of 30 to 112. Three patients with Felty's syndrome reacted with an antigenic target of 80 to 84 Kd molecular mass, a finding not seen in any of the other patients studied. By serologic testing, none of the autoimmune sera showed serologic specificity for any known neutrophil-specific alloantigen. Using an anti-NA-1 serum, we identified antigenic targets at 40, 50, and 101 Kd in both NA-1-positive and NA-1-negative neutrophils. Ten of 17 autoimmune sera showed reactivity in this corresponding range. These studies demonstrate that immunoblotting may be used to identify antigenic targets in autoimmune neutropenia and may suggest a specificity of these antibodies not definable by serologic techniques. Correlation of immunoblot reactivity with disease states associated with immune neutropenia may be useful in the study of the pathogenesis of the different forms of autoimmune neutropenia.     

The impact of health needs assessment and prioritisation on District Health Board planning in New Zealand

Objectives District Health Boards (DHBs) in New Zealand are mandated to assess and prioritise the health needs of their resident populations. This paper evaluates the impact of those health needs assessments (HNAs) and prioritisation practices on health service planning and purchasing in the first 3 years of the DHBs (2001–2003). Methods DHB HNAs, 5‐yearly strategic plans, and annual plans were evaluated using document analysis to determine the impact of needs assessments on prioritisation and planning by boards. Key informant interviews with DHB senior managers were used to identify differences between boards. Results HNAs had relatively little influence on the direction of planning and purchasing. HNAs conducted in DHBs that focussed on planning at the service level and in relation to population subgroups using a ‘mixed‐scanning’ approach and ‘service planning groups’ had a greater impact on planning and purchasing than more comprehensive approaches. DHBs found prioritisation difficult due to the level of control exercised by central government over their actions. Conclusions HNAs in New Zealand need to be less ambitious, more focussed and more closely institutionally linked to prioritisation, service planning and purchasing processes if they are to have an impact on the allocation of resources. Copyright © 2009 John Wiley & Sons, Ltd.     

Adult Reye's syndrome: a review with new evidence for a generalized defect in intramitochondrial enzyme processing

A 42-year-old woman developed a flu-like illness and died 8 days later with Reye's syndrome (RS). There are 26 other cases of adult-onset RS reported. Biochemical, immunologic, and molecular studies of liver, brain, and skeletal muscle revealed a non-uniform decrease in several mitochondrial residual enzyme activities in liver and brain. Pyruvate carboxylase activity was negligible. Cross-reacting material was present in normal abundance in isolated liver mitochondria for several enzymes that had reduced catalytic activity including pyruvate carboxylase. Subunit II (encoded by mitochondrial DNA) and subunit IV (encoded by nuclear DNA) of cytochrome c oxidase also were present in normal abundance with normal electrophoretic mobility. These observations, combined with pertinent findings of other investigators, allow us to speculate that the intramitochondrial matrix chemical environment is disturbed by preceding pathophysiologic events resulting in a lowered ATP/ADP ratio. The lowered intramitochondrial energetic state interferes with the refolding and assembly of imported mitochondrial proteins, causing a loss of the catalytic efficiency of these enzymes. This explains the selective vulnerability of mitochondria in RS and the non-uniform, disproportionate loss of enzyme activity.