Flow cytometry analysis of platelet antibodies

We have devised assays to detect both circulating alloantibodies to platelets (indirect assay) and platelet-association IgG and IgM (direct assay) using a flow cytometric technique. A variety of patients with immune thrombocytopenia were studied. Employment of a confocal lens in the flow cytometer increased the discrimination power of the instrument. Patients with autoimmune thrombocytopenia (idiopathic thrombocytic purpura [ITP], systemic lupus erythematosus (SLE), lymphoma, leukemia, and drug-induced thrombocytopenia showed a significant increase in platelet-associated antibody. Circulating antibodies to platelets (alloantibodies) were demonstrated in cases of platelet refractoriness and neonatal isoimmune purpura. Day-today precision of the assays ranged from 3% to 6% (coefficient of variation). No interference was shown in the presence of hemoglobin (5 g/L), triglycerides (10 g/L), or polyclonal and monoclonal immunoglobulinemia (50 g/L: IgG, IgA, IgM). The sensitivity of the direct assay was 500 attograms of IgG or IgM platelet.     

A cluster of bacterial genes for anaerobic benzene ring biodegradation

A reductive benzoate pathway is the central conduit for the anaerobic biodegradation of aromatic pollutants and lignin monomers. Benzene ring reduction requires a large input of energy and this metabolic capability has, so far, been reported only in bacteria. To determine the molecular basis for this environmentally important process, we cloned and analyzed genes required for the anaerobic degradation of benzoate and related compounds from the phototrophic bacterium, Rhodopseudomonas palustris. A cluster of 24 genes was identified that includes twelve genes likely to be involved in anaerobic benzoate degradation and additional genes that convert the related compounds 4-hydroxybenzoate and cyclohexanecarboxylate to benzoyl-CoA. Genes encoding benzoyl-CoA reductase, a novel enzyme able to overcome the resonance stability of the aromatic ring, were identified by directed mutagenesis. The gene encoding the ring-cleavage enzyme, 2-ketocyclohexanecarboxyl-CoA hydrolase, was identified by assaying the enzymatic activity of the protein expressed in Escherichia coli. Physiological data and DNA sequence analyses indicate that the benzoate pathway consists of unusual enzymes for ring reduction and cleavage interposed among enzymes homologous to those catalyzing fatty acid degradation. The cloned genes should be useful as probes to identify benzoate degradation genes from other metabolically distinct groups of anaerobic bacteria, such as denitrifying bacteria and sulfate-reducing bacteria.     

Ocular findings in the chromosome 22q11.2 deletion syndrome.

PURPOSE: To identify the ocular features of the chromosome 22q11.2 deletion syndrome and to provide ophthalmologic examination recommendations for affected patients. METHODS: Ocular abnormalities were evaluated prospectively in patients with 22q11.2 deletion at the Children's Hospital of Philadelphia between 1997 and 1999. RESULTS: Ninety patients with confirmed 22q11.2 deletion were examined. Posterior embryotoxon was found in 49%, tortuous retinal vessels in 34%, eyelid hooding in 20%, strabismus in 18%, ptosis in 4%, amblyopia in 4%, and tilted optic nerves in 1%. CONCLUSIONS: The high incidence of ocular conditions that can potentially affect visual development suggest that children with 22q11.2 deletion should undergo a comprehensive eye examination upon diagnosis of the condition with follow-up as indicated by the findings in each case. In addition, knowledge of the ocular findings, in conjunction with certain cardiac, otolaryngologic, immunologic, and other systemic findings, may alert physicians to the possibility of a chromosome 22q11.2 deletion.     

Phospholipase A2-catalyzed conversion of hepatic cytochrome P450 to P420 in microsomal membranes prepared from young and old rats

Previous studies from this Laboratory have revealed few significant age-related changes in either hepatic microsomal cytochrome P₄₅₀ content or NADPH-cytochrome P₄₅₀ reductase activity, prompting the suggestion that the nearly ubiquitous age-associated decline in the hepatic metabolism of xenobiotics by the mixed-function oxidases is a result of alterations in the lipid matrix in which the macromolecular components are organized. In order to examine this postulate, the membrane microenvironment surrounding hepatic microsomal cytochrome P₄₅₀ was enzymatically digested with snake venom phospholipase A₂, and the kinetics of conversion of cytochrome P₄₅₀ to cytochrome P₄₂₀ determined for preparations obtained from rats of varying age. There were no significant differences in the kinetics of conversion of cytochrome P₄₅₀ to cytochrome P₄₂₀, suggesting that there are no significant age-related changes in the lipid microenvironment immediately adjacent to cytochrome P₄₅₀. However, there was a slight difference, independent of age, in the rate of conversion of cytochrome P₄₅₀ to cytochrome P₄₂₀ between smooth and rough microsomes. The results are discussed in terms of general experimental approaches toward understanding drug metabolism in senescent organisms.     

The alarming trend of substance abuse in anesthesia providers.

The role of the anesthesia provider requires a high level of awareness and constant vigilance. Literature indicates, however, that the substance abuse rate in certified registered nurse anesthetists (CRNAs) and anesthesiologists has reached staggering levels. The literature also shows that there has been a change in which controlled drugs are being misused. It is imperative that perianesthesia nurses be aware of the current problem and take steps when indicated to protect both providers and patients. This article discusses the current trends of addiction in anesthesia providers, treatment, and reentry, as well as the role of the perianesthesia nurse in recognizing, reporting, and preventing substance abuse.     

Physical activity patterns in chronic hemodialysis patients: comparison of dialysis and nondialysis days.

OBJECTIVE: To determine physical activity patterns in chronic hemodialysis patients with a specific emphasis on the difference between dialysis and nondialysis days. Design A cross-sectional single-center study. SETTING: Vanderbilt University Outpatient Dialysis Unit. PATIENTS: Twenty current chronic hemodialysis patients: 10 male, 10 female; 15 black, 5 white; mean age, 50.1 +/- 9.9 years; height, 164.5 +/- 10.9 cm; weight, 82.5 +/- 15.4 kg; length on dialysis, 57.3 +/- 45.3 months. METHODS: Minute-by-minute physical activity was assessed over a 7-day period using a triaxial accelerometer, which consists of raw numbers or counts calculated by the 3 axes of the accelerometer (PA counts). PA counts were extrapolated on a daily and hourly basis. Physical functioning tests included: sit-to-stand, 6-minute walk, and 1-repetition maximal leg press exercise. Laboratory values for serum concentrations of albumin, prealbumin, C-reactive protein, and cholesterol were also collected. MAIN OUTCOME MEASURE: PA counts. RESULTS: Total PA counts were significantly lower on dialysis days when compared with nondialysis days (128,279 +/- 74,009 versus 168,744 +/- 95,168, respectively, P = .025). The average PA counts during the 4-hour dialysis time period were significantly lower on dialysis days when compared with nondialysis days (3,086 +/- 3,749 versus 11,070 +/- 7,695, respectively, P = .001). At postdialysis hours 1 and 2, PA counts on dialysis days were significantly higher than on nondialysis days (11,410 +/- 5,340 versus 9,082 +/- 6,646, P = .008, and 14,048 +/- 9,728 versus 8,662 +/- 6,433, P = .016, respectively). By postdialysis hour 4, PA counts on dialysis days had significantly decreased when compared with nondialysis days (6,068 +/- 6,268 versus 10,512 +/- 7,420 PA counts, P = .01, respectively). From postdialysis hours 5 to 20, there was no significant difference in PA counts between dialysis and nondialysis days. CONCLUSION: This study shows that physical activity is lower on dialysis days when compared with nondialysis days, and this decrease is caused by the lack of activity during the 4-hour hemodialysis procedure. New behavior modification strategies involving physical activity, both during hemodialysis and on nondialysis days, must be examined in this patient population.     

Clinical risk factors for fractures in multi-ethnic women: the Women's Health Initiative.

To identify risk factors for fractures in multi-ethnic women, we studied 159,579 women enrolled in the Women's Health Initiative. In general, risk factors for fractures were similar across ethnic groups. However, irrespective of their ethnicity, women with multiple risk factors have a high risk of fracture. Targeting these high-risk women for screening and intervention could reduce fractures. INTRODUCTION: Fracture rates tend to be lower in minority women, but consequences may be greater. In addition, the number of fractures is expected to increase in minority women because of current demographic trends. There are limited prospective data on risk factors for fractures in minority women. MATERIALS AND METHODS: We studied 159,579 women 50-79 yr of age enrolled in the Women's Health Initiative. Information on risk factors was obtained by questionnaire or examination. Nonspine fractures that occurred after study entry were identified over an average follow-up of 8 +/- 2.6 (SD) yr. RESULTS: Annualized rates (%) of fracture in whites, blacks, Hispanics, Asians, and American Indians were 2.0, 0.9, 1.3, 1.2, and 2.0, respectively. Significant predictors [HR (95% CI)] of fractures by ethnic group were as follows: blacks: at least a high school education, 1.22 (1.0, 1.5); (+) fracture history, 1.7 (1.4, 2.2); and more than two falls, 1.7 (1.9, 2.0); Hispanics: height (>162 cm), 1.6 (1.1, 2.2); (+) fracture history, 1.9 (1.4, 2.5); more than two falls, 1.8 (1.4, 2.3); arthritis, 1.3 (1.1, 1.6); corticosteroid use, 3.9 (1.9, 8.0); and parental history of fracture, 1.3 (1.0, 1.6); Asians: age (per 5 yr), 1.2 (1.0, 1.3); (+) fracture history, 1.5 (1.1, 2.0); current hormone therapy (HT), 0.7 (0.5, 0.8); parity (at least five), 1.8 (1.1, 3.0); more than two falls, 1.4 (1.1, 1.9); American Indian: (+) fracture history, 2. 9 (1.5, 5.7); current HT, 0.5 (0.3, 0.9). Women with eight or more risk factors had more than a 2-fold higher rate of fracture compared with women with four or fewer risk factors. Two ethnicity x risk factor interactions were identified: age and fall history. CONCLUSIONS: Irrespective of their ethnicity, women with multiple risk factors have a high risk of fracture. Targeting these high-risk women for screening and intervention could reduce fractures.