Antigenic determinants in idiopathic thrombocytopenic purpura

Serum platelet bindable immunoglobulin (SPBIg) was determined in a group of 23 idiopathic thrombocytopenic purpura (ITP) patients and compared to 20 normal, healthy controls. The mean SPBIg of the ITP group was 16.1 (+/- 17.9 SD) fg/platelet, while the normals were substantially lower, 4.0 (+/- 1.2) fg/platelet. Sera from patients of both groups were then incubated with platelet fractions immobilized on nitrocellulose membrane strips (Western Blotting) to detect platelet antigen specificity using a peroxidase labelled indicator antibody. The normal patient sera did not react with platelet fractions on the nitrocellulose strips. However, 21 of 23 ITP sera bound to one or more platelet fractions with large variations in the number and molecular weights of the platelet fractions identified by ITP antibody. These observations suggest the presence of multiple antigenic binding sites for platelet specific immunoglobulin in ITP sera. This variation may reflect heterogeneous antibodies binding to diverse antigens or homogeneous antibodies to a limited number of antigenic determinants shared by several discrete platelet molecules.     

Optimization of electroporation conditions for Mycobacterium avium

Successful transformation and subsequent genetic manipulation of Mycobacterium avium requires suitable vectors, efficient transformation systems, and reliable selectable markers. A systematic analysis of the parameters involved in the transformation of M. avium was performed to optimize DNA transfer. Factors examined included the composition of the growth medium, growth medium additives, variations in washing of the bacteria prior to electroporation, and conditions of electroporation. Of the parameters assayed, the frequency of transformation (defined as the number of transformants per 10(6) transformed bacteria) showed the greatest increase with the addition of 1.5% glycine to the M. avium culture medium and the use of higher concentrations of plasmid DNA. The addition of 0.5 M sucrose to the growth medium and wash solution yielded a modest increase in transformation frequency, but more importantly afforded greater consistency of results between different batches of cells with no decrease in transformation yields following freezing and thawing. We also confirmed that gfp could be used as a selective marker for M. avium, even as a single copy integrant, and allowed for rapid discrimination between false and true transformants. Using this protocol, we were able to transform nine of 11 clinical strains of M. avium.     

Extrusion of testosterone pellets: a randomized controlled clinical study

BACKGROUND: It has previously been shown that testosterone implantation is an effective and well accepted form of androgen replacement therapy, but that pellet extrusion was the most frequent side-effect. The present study aimed to reduce the extrusion rate. OBJECTIVE: To determine whether the washing of testosterone pellets to remove potentially surface-adherent particles decreased the rate of extrusion of pellet implants. DESIGN: Prospective, randomized parallel group design in a single centre with consecutive procedures to be randomized (1 : 1) into a wash or control group. PATIENTS: The study included 251 testosterone implantation procedures in men with known androgen deficiency. MEASUREMENTS: The primary endpoint, extrusion rate per procedure, was evaluated prospectively by telephone contact at 1 week and then 3 and 6 month intervals. Secondary end-points included peri-procedure adverse events (bleeding, skin reaction, excessive discomfort) noted at the time of implant. Bruising, bleeding and infection were also evaluated as later adverse events by telephone and personal follow-up. Explanatory variables recorded as possible covariables included the number of implants used, production batch number of the implants, the operator, as well as other demographic and medical factors. RESULTS: In the wash group, the extrusion rate was 12% per procedure (19 pellets from 15 subjects) whereas in the control group, the extrusion rate was 11.1% per procedure (18 pellets from 14 subjects), indicating no evidence of any benefit of the wash procedure (OR = 1. 09 [95% CI 0.47-2.6] per procedure). There was no evidence of benefit in secondary endpoints including total adverse events (7.1%, OR 1.28 [0.44-3.9], bleeding/bruising (8.8%, 1.23 [0.47-3.3]) and infection (4.0%, 1.54 [0.35-7.6]) per procedure. Among men reporting an infection requiring antibiotic treatment according to their own general practitioners, six/ten (60%) subsequently experienced an extrusion. There were no significant differences in extrusion rate between four different operators (P = 0.24) nor among 12 different batches of pellets used during the course of the study (P = 0.77). CONCLUSIONS: The pellet washing procedure used during implantation does not reduce the subsequent extrusion rate. The higher rate of both primary and secondary adverse events in this prospective study compared with the previous retrospective survey may reflect either more rigorous follow-up or a secular trend.     

Detection of recombinant human erythropoietin abuse in athletes utilizing markers of altered erythropoiesis

BACKGROUND AND OBJECTIVES: The detection of recombinant human erythropoietin (r-HuEPO) abuse by athletes remains problematic. The main aim of this study was to demonstrate that the five indirect markers of altered erythropoiesis identified in our earlier work were reliable evidence of current or recently discontinued r-HuEPO use. A subsidiary aim was to refine weightings of the five markers in the initial model using a much larger data set than in the pilot study. A final aim was to verify that the hematologic response to r-HuEPO did not differ between Caucasian and Asiatic subjects. DESIGN AND METHODS: Recreational athletes resident in Sydney, Australia (Sydney, n = 49; 16 women, 33 men) or Beijing, China (Beijing, n=24; 12 women, 12 men) were randomly assigned to r-HuEPO or placebo groups prior to a 25 day administration phase. Injections of r-HuEPO (or saline) were administered double-blind at a dose of 50 IU/kg three times per week, with oral iron (105 mg) or placebo supplements taken daily by all subjects. Blood profiles were monitored during and for 4 weeks after drug administration for hematocrit (Hct), reticulocyte hematocrit (RetHct), percent macrocytes (%Macro), serum erythropoietin (EPO) and soluble transferrin receptor (sTfr), since we had previously shown that these five variables were indicative of r-HuEPO use. RESULTS. The changes in Hct, RetHct, %Macro, EPO and sTfr in the Sydney trial were qualitatively very similar to the changes noted in our previous administration trial involving recreational athletes of similar genetic origin. Statistical models developed from Fisher's discriminant analysis were able to categorize the user and placebo groups correctly. The same hematologic response was demonstrated in Beijing athletes also administered r-HuEPO. INTERPRETATION AND CONCLUSIONS: This paper confirms that r-HuEPO administration causes a predictable and reproducible hematologic response. These markers are disturbed both during and for several weeks following r-HuEPO administration. This work establishes an indirect blood test which offers a useful means of detecting and deterring r-HuEPO abuse. Ethnicity did not influence the markers identified as being able to detect athletes who abuse r-HuEPO.     

A CD4+ proliferation of large granular lymphocytes expresses the protease activated receptor-1

The platelet-type thrombin receptor was the first member to be identified in a family of protease activated receptors (PARs) and has been designated PAR-1. We recently reported that the large granular lymphocytes (LGLs) in patients with proliferations of CD8⁺ cells co-expressed PAR-1 and the expression of PAR-1 correlated with the expression of CD57. Here we show, by three-colour immunofluorescence, that the LGLs from a patient with a rare CD4⁺ CD57⁺ monoclonal expansion also expressed PAR-1. Northern blot analysis confirmed the presence of high levels of mRNA for PAR-1 in these LGLs.     

Selection of women at high risk of breast cancer for initial screening

Selective breast cancer screening refers to the intentional restriction of screening to only a high-risk subgroup of the total population of women at risk. Using data from the Canadian National Breast Screening Study, we explored methods of defining such subgroups. Discriminants were based on risk factor information collected prior to screening and were constructed using a training group of 77 cases and 400 controls. They were then tested on a separate group of 38 cases and 200 controls. Both simple risk factor counts and logistic models were utilized and separate analyses were performed for pre- and post-menopausal women. Using a logistic model, we were able to define a high-risk subgroup encompassing less than 40% of the test controls and over 85% of the test cases. Such a selection strategy, if implemented, might reduce initial visit mammography rates by up to 60% with only a small reduction in case detection. Other uses as determining the optimal age for initiation of screening are also discussed.     

Hypocellular acute leukemia

We examined the clinical features and therapeutic response of a group of patients with acute leukemia and hypocellular bone marrow. Therapists have generally avoided, delayed or modified therapy because of hypocellularity. We demonstrated not only that aggressive therapy is possible, but also that the remission rate is high (complete remission = 73 percent) and survival prolonged (x? > 40 months).     

Evaluation of susceptibility testing methods for Burkholderia cepacia complex: a comparison of broth microdilution,agar dilution,gradient strip and EUCAST disc diffusion

ObjectivesTo evaluate the accuracy and reproducibility of antimicrobial susceptibility testing methods in Burkholderia cepacia complex (BCC).MethodsMinocycline, ciprofloxacin, trimethoprim/sulphamethoxazole, meropenem, ceftazidime and chloramphenicol were tested against 155 BCC strains using broth microdilution at 35 ± 1°C (BMD₃₅) in triplicate, then BMD at 30 ± 1°C (BMD₃₀), agar dilution at 30°C and 35°C (AD₃₀ and AD₃₅), gradient strip (GS) and EUCAST standardized disc diffusion (DD) testing methods once.ResultsBMD₃₅ reproducibility ranged from 70% to 84.5% for all agents. Correlations of MICs from BMD₃₅ with BMD₃₀ ranged from 63% to 85%, with AD₃₅ from 32.9% to 87% and with GS methods from 36% to 83.9%. Essential agreement (EA) of MICs by GS with BMD₃₅ ranged from 62.6% (trimethoprim-sulphamethoxazole) to 83.9% (minocycline). EA of EUCAST DD zone diameters using CLSI breakpoint criteria was between 85.8% and 97.4%, however Very Major Errors (VME) for trimethoprim/sulphamethoxazole were 31%.ConclusionsBMD at 35 ± 1°C was poorly reproducible for most agents and no method showed acceptable performance. Of particular concern were the GS results. Although this is the most commonly used method for determining MICs in laboratories, there was poor correlation with BMD₃₅ for meropenem and trimethoprim/sulphamethoxazole. EUCAST DD correlated poorly with BMD₃₅ MICs. This study confirms that no susceptibility method is capable of providing reproducible and accurate MICs when testing BCC.