An evaluation of the trends in analytical performance of international apolipoprotein A-1 and B assays

Two international apolipoprotein A-1 and B (apoA-1 and apoB) surveys were conducted in 1983 and 1986 with use of a lyophilized serum measured by 55 and 91 participants, respectively. The chief source of variability in both surveys was among laboratories, but among-method variation was significant for apoB. Comparing the 1986 with the 1983 findings, we saw a 60% decrease in apoA-1 variability among laboratories, and a similar decrease (53%) was found for apoB. Evaluation of individual measurements suggests that some collaborators may have adjusted their calibrators toward the consensus values published in 1983.     

Activation dependence and kinetics of force and stiffness inhibition by aluminiofluoride,a slowly dissociating analogue of inorganic phosphate,in chemically skinned fibres from rabbit psoas muscle

Summary To examine the mechanism by which aluminiofluoride, a tightly binding analogue of inorganic phosphate, inhibits force in single, chemically skinned fibres from rabbit psoas muscle, we measured the Ca²⁺-dependence of the kinetics of inhibitor dissociation and the kinetics of actomyosin interactions when aluminiofluoride was bound to the crossbridges. The relation between stiffness and the speed of stretch during small amplitude ramp stretches (< 5 nm per h.s.) was used to characterize the kinetic properties of crossbridges attached to actin; sarcomere length was assessed with HeNe laser diffraction. During maximum Ca²⁺-activation at physiological ionic strength (pCa 4.0, 0.2 m /2), stiffness exhibited a steep dependence on the rate of stretch; aluminiofluoride inhibition at pCa 4.0 (0.2 m /2) resulted in an overall decrease in stiffness, with stiffness at high rates of stretch (10³–10⁴ nm per h.s. per s) being disproportionately reduced. Thus the slope of the stiffness-speed relation was reduced during aluminiofluoride inhibition of activated fibres. Relaxation of inhibited fibres (pCa 9.2, 0.2 m /2) resulted in aluminiofluoride being trapped and was accompanied by a further decrease in stiffness at all rates of stretch which was comparable to that found in control relaxed fibres. In relaxed, low ionic strength conditions (pCa 9.2, 0.02 m /2) which promote weak crossbridge binding, stiffness at all rates of stretch was significantly inhibited by aluminiofluoride trapped in the fibre. To determine the Ca²⁺-dependence of inhibitor dissociation, force was regulated independent of Ca²⁺ using an activating tropinin C (aTnC). Results obtained with aTnC-activated fibres confirmed that there is no absolute requirement for Ca²⁺ for recovery from force inhibition by inorganic phosphate analogues in skinned fibres; the only requirement is thin filament activation which enables active crossbridge cycling. These results indicate that aluminiofluoride preferentially inhibits rapid equilibrium or weak crossbridge attachment to actin, that aluminiofluoride-bound crossbridges attach tightly to the activated thin filament, and that, at maximal (or near-maximal) activation, crossbridge attachment to actin prior to inorganic phosphate analogue dissociation is the primary event regulated by Ca²⁺.     

Kv11.1 channel subunit composition includes MinK and varies developmentally in mouse cardiac muscle

The Kv11.1 (also ERG1) K(+) channel underlies cardiac I(Kr), a current that contributes to repolarization in mammalian heart. In mice, I(Kr) current density decreases with development and studies suggest that changes in the structure and/or properties of the heteromultimeric I(Kr)/Kv11.1 channel are responsible. Here, using immunohistochemistry, we report that total Kv11.1 alpha subunit protein is more abundant in neonatal heart and is distributed throughout both adult and neonatal ventricles with greater abundance in epicardia. Immunoblots reveal that the alpha subunit alternative splice variant, Kv11.1a, is more abundant in adult heart while the Kv11.1b variant is more abundant in neonatal heart. Additionally, MinK channel subunit protein is shown to co-assemble with Kv11.1 protein and is more abundant in neonatal heart. In summary, Kv11.1/I(Kr) channel composition varies developmentally and the higher I(Kr) current density in neonatal heart is likely attributable to higher abundance of Kv11.1/I(Kr) channels, more specifically, the Kv11.1b splice variant.     

Efficacy response of inhaled beclomethasone dipropionate in asthma is proportional to dose and is improved by formulation with a new propellant

BACKGROUND: This study tested the hypothesis that there would be improved asthma control with increasing doses of beclomethasone dipropionate (BDP) formulated in hydrofluoroalkane-134a (HFA-BDP) and the standard chlorofluorocarbon propellants (CFC-BDP). Because HFA-BDP has improved lung deposition compared with CFC-BDP, this study also tested the hypothesis that HFA-BDP would provide more effective control of asthma than CFC-BDP. METHODS: In this multicenter, randomized, parallel-group blinded study, asthmatic subjects who had deterioration in asthma control after discontinuation of inhaled corticosteroids were randomized to receive one of 6 possible treatments: 100 microg/d, 400 microg/d, or 800 microg/d of HFA-BDP or 100 microg/d, 400 microg/d, or 800 microg/d of CFC-BDP for 6 weeks. Changes in spirometry, daytime asthma symptom and nighttime asthma-related sleep disturbance scores, morning and evening peak expiratory flows, and daily use of inhaled beta-agonist for symptom control on diary cards were assessed over 6 weeks of treatment. RESULTS: Three hundred twenty-three patients were randomized to the 6 treatment groups, which had similar demographics and baseline lung function. There were significantly larger changes from baseline at week 6 in FEV(1) percent predicted with increasing doses of both HFA-BDP and CFC-BDP. The FEV(1) percent predicted dose-response curve for HFA-BDP was shifted to the left compared with the dose-response curve for CFC-BDP. By using the Finney bioassay method, it was calculated that 2.6 times as much CFC-BDP would be required to achieve the same improvement in FEV(1) percent predicted as HFA-BDP (95% confidence interval, 1.1-11.6). All treatment groups except the 100 microg/d CFC-BDP group tolerated study drug well. Ten (17%) of 59 patients in this group reported an acute asthma episode, increased asthma symptoms (6 of the 8 reports of increased asthma symptoms were classified as severe), or both, and 8 patients withdrew from the study (3 for adverse events related to asthma). CONCLUSIONS: Increasing doses of inhaled corticosteroids lead to improved lung function and asthma control. Moreover, the reformulation of BDP in HFA enables effective asthma control at much lower doses than CFC-BDP.     

Development and evaluation of a microtiter plate enzyme immunoassay for antibodies to 3,3'-dichlorobenzidine

The authors obtained and evaluated antisera from rabbits injected with a derivative of a potent bladder carcinogen, dichlorobenzidine (DCB), conjugated to bovine serum albumin (BSA). A 14C-radioimmunoassay (RIA) was able to detect the presence of DCB antibodies, but its relative insensitivity led to the development of a more sensitive enzyme immunoassay (EIA). The EIA test was a "sandwich" method in which a second antibody, labeled with an enzyme (horseradish peroxidase), was used to measure antibody binding to transferrin (Tf)-conjugated DCB immobilized on a microtiter plate. Antibody titers measured by RIA were approximately 1:40; when measured by EIA, they were approximately 1:40,000. Antibody specificity was assessed by comparing the antibody binding activities of DCB, BSA, Tf, BSA-conjugated to DCB, and a number of N-substituted aromatic compounds that included benzidine (Bz). Among the compounds tested, the rabbit antiserum reacted only with DCB and the carrier protein, BSA. Moreover, antibody binding activity to Tf-conjugated DCB was significantly inhibited by unconjugated DCB concentrations between 30 and 500 ng/mL. The precision of antibody binding activities as a function of DCB concentration (expressed by the CV) ranged from 9% for low (30 ng/mL) DCB levels to 12% for higher (500 ng/mL) levels. This evaluation suggests that the antiserum obtained would be appropriate for detecting DCB levels at the ng/mL level.     

What is the Level of Evidence Substantiating Commercial Payers’ Coverage Policies for Total Joint Arthroplasty?

BackgroundThe prevalence of total joint arthroplasty (TJA) in the United States has drawn the attention of health care stakeholders. The payers have also used a variety of strategies to regulate the medical necessity of these procedures. The purpose of this study was to examine the level of evidence of the coverage policies being used by commercial payers in the United States.MethodsThe references of the coverage policies of four commercial insurance companies were reviewed for type of document, level of evidence, applicability to a TJA population, and success of nonoperative treatment in patients with severe degenerative joint disease.Results282 documents were reviewed. 45.8% were primary journal articles, 14.2% were level I or II, 41.2% were applicable to patients who were candidates for TJA, and 9.9% discussed the success of nonoperative treatment in patients who would be candidates for TJA.ConclusionMost of the references cited by commercial payers are of a lower level of scientific evidence and not applicable to patients considered to be candidates for TJA. This is relatively uniform across the reviewed payers. The dearth of high-quality literature cited by commercial payers reflects the lack of evidence and difficulty in conducting high level studies on the outcomes of nonoperative versus operative treatment for patients with severe, symptomatic osteoarthritis. Patients, surgeons, and payers would all benefit from such studies and we encourage professional societies to strive toward that end through multicenter collaboration.     

Fragile X-related protein and VIG associate with the RNA interference machinery

RNA interference (RNAi) is a flexible gene silencing mechanism that responds to double-stranded RNA by suppressing homologous genes. Here, we report the characterization of RNAi effector complexes (RISCs) that contain small interfering RNAs and microRNAs (miRNAs). We identify two putative RNA-binding proteins, the Drosophila homolog of the fragile X mental retardation protein (FMRP), dFXR, and VIG (Vasa intronic gene), through their association with RISC. FMRP, the product of the human fragile X locus, regulates the expression of numerous mRNAs via an unknown mechanism. The possibility that dFXR, and potentially FMRP, use, at least in part, an RNAi-related mechanism for target recognition suggests a potentially important link between RNAi and human disease.     

The trouble with kidneys derived from the non heart-beating donor: a single center 10-year experience

BACKGROUND: The demand for renal transplantation has increasingly outstripped the supply of donor organs especially over the past 10 years. Although related and unrelated live donation is being promoted as one option for increasing the donor pool, it is unlikely that this will in itself be able to bridge the gap. Non-heart beating donors (NHBD) can provide an alternative supply of organs, which should substantially increase the donor pool. METHODS: In Newcastle, NHBD kidneys have been used for transplantation for a period of 10 years. In the early period (1988-1993) excellent results were obtained (90.5% success); however, these donors were controlled NHBD, Maastricht category III. In the second phase (1994-1998) increasing numbers of donors were obtained from the Accident and Emergency Department unit. These were failed resuscitation for cardiac arrest (category II). The rates of success in this period were poor (45.5% success) and the program was halted. The third phase of the program used machine perfusion of the kidneys and glutathione S transferase enzyme analysis to assess viability. RESULTS: Using such approaches renal transplants from largely category II donors produced a success rate of 92.3% which was significantly better than the phase II period of the program (P=0.023, Fisher two-tail test). CONCLUSION: Machine perfusion and viability assessment of NHB kidneys in phase III of the program has increased our donor pool as well as improved the graft survival. This is particularly relevant for the use of the category II NHB donor where the incidence of primary nonfunction was high, illustrated by phase II where machine perfusion/viability assessment was not used.