Efficiency of mixed RASTs in serological screening of possibly allergic patients

OBJECTIVE: To determine the age distribution of results of serological allergy screening using mixed-allergen radioallergoabsorbent tests (RASTs), and to determine the cost-effectiveness of mix RASTs. DESIGN: Retrospective. SETTING: University Hospital of Rotterdam; Dijkzigt Hospital and Sophia Children's Hospital, the Netherlands. METHODS: An analysis was made of the results of all determinations requested in a period of 3 years with use of the CAP Phadiatop test (aimed at IgE against a mixture of inhalant allergens) and the CAP f x 5 food mix test (aimed at IgE against a mixture of food allergens). RESULTS: The CAP Phadiatop test was positive most strongly and most frequently in patients aged 7 to 30 years. From the age of 2 years, there was a sharp rise of the number of positive CAP Phadiatop tests, but IgE against inhalant allergens was also found in over 10% of the children aged 0 and 1 year. Up to and including the age category of 6 years, over one-third of the sera submitted had a positive CAP f x 5 food mix test. From a budget point of view, preliminary screening with a mix RAST is the more advantageous the lower the allergy prevalence and the higher the mean number of CAP-RASTs to be requested per serum. CONCLUSION: A substantial saving of laboratory costs can be achieved by having mix RASTs such as CAP Phadiatop and CAP f x 5 food mix tests precede determinations of specific IgE against separate allergens; these savings will be the higher the smaller the proportion of positive results and the higher the mean number of separate RASTs to be requested per serum.     

Fasting and nonfasting iodine-123-idophenylpentadecanoic acid myocardial SPECT imaging in coronary artery disease

Iodine-123-labeled idophenylpentadecanoic acid (IPPA) metabolic imaging has been shown to be clinically useful for the identification of myocardial viability in patients with coronary artery disease and left ventricular dysfunction. Imaging is usually performed under fasting conditions since nonfasting conditions may affect myocardial uptake of 123I-IPPA. The purpose of this study was to examine the impact of dietary condition on 123I-IPPA metabolic imaging. METHODS: Forty patients with stable coronary artery disease underwent, in randomized order and on separate days, 123I-IPPA SPECT myocardial imaging under fasting and nonfasting conditions. Patients were injected with 123I-IPPA (4-5 mCi) at rest with imaging performed at 4 (initial) and 30 (delay) min. For each image (initial and delay images), 10 segments were analyzed by three experienced observers without knowledge of patient identity or dietary condition using a 5-point grading system (O = no uptake to 4 = normal uptake). A summed global score was obtained for each image by adding the scores for all 10 segments. Image quality was assessed using a 3-point grading system. RESULTS: Visual agreement for normal and abnormal segments between fasting and nonfasting conditions was 82% (kappa = 0.63). There were no significant differences in the summed global scores for both conditions. Image quality was equivalent for both conditions in 65% of cases and superior under the nonfasting condition in 25% of cases. CONCLUSION: Image quality as well as the presence, location and severity of defects are similar under fasting and nonfasting conditions with 123I-IPPA. Therefore, fasting is not necessary before 123I-IPPA SPECT imaging for the assessment of myocardial viability.     

Multimodal cerebral monitoring in comatose head-injured patients

We isolated and characterized the ERCC1 coding sequence from three Chinese hamster ovary (CHO) parental (CHO-AA8, CHO-AT3-2 and CHO-9) and 10 ERCC1 mutant cell lines. Two general classes of mutations were observed: two mutant cell lines exhibited nucleotide additions or deletions to produce frameshift mutations and seven mutant cell lines exhibited point mutations that resulted in transitions or transversions, including nonsense mutations and mutations that generated intron/exon splicing errors. One mutant (UV201) which had been provisionally assigned to ERCC1 complementation group 1 (CG1) had no detectable mutation in its coding sequence. Of the nine ERCC1 mutant alleles characterized two mutations were identified in the XpA binding region of the Ercc1 protein; no mutations were found in the N-terminal portion of the Ercc1 protein. Results of Northern hybridization analysis showed that the relative levels of ERCC1 mRNA differed significantly both among the parental cell lines and among the mutant cell lines derived from each parental cell line. Western analysis with a CHO Ercc1-specific antibody detected Ercc1 protein in each of the parental cell lines and also in UV201. The marked reduction in Ercc1 protein levels observed in all the other mutants examined supports the hypothesis that ERCC1 mutations may destabilize this polypeptide.     

Cortical variability and asymmetry in normal aging and Alzheimer's disease

The onset of Alzheimer's disease (AD) is accompanied by a complex and distributed pattern of neuroanatomic change, difficult to distinguish clinically from dynamic alterations in normal aging. Extreme variations in the sulcal patterns of the human cortex have made it difficult to identify diffuse and focal variations in cortical structure in neurodegenerative disease. We report the first comprehensive 3D statistical analysis of deep sulcal structure in vivo, in both normal aging and dementia. High-resolution 3D T1-weighted fast SPGR (spoiled GRASS) MRI volumes were acquired from 10 patients diagnosed with AD (NINCDS-ARDRA criteria; age: 71.9 +/- 10.7 years) and 10 normal subjects matched for age (72.9 +/- 5.6 years), gender, educational level and handedness. Scans were digitally transformed into Talairach stereotaxic space. To determine specific patterns of cortical variation in dementia patients, 3D average and probabilistic maps of primary deep sulci were developed for both normal and AD groups. Major sulci (including supracallosal, cingulate, marginal, parieto-occipital, anterior and posterior calcarine sulci, and Sylvian fissures) were modeled as complex systems of 3D surfaces using a multi-resolution parametric mesh approach. Variations and asymmetries in their extents, curvature, area and surface complexity were evaluated. Three-dimensional maps of anatomic variability, structural asymmetry and local atrophy indicated severe regionally selective fiber loss in AD. A midsagittal area loss of 24.5% at the corpus callosum's posterior midbody (P < 0.025) matched increases in structural variability in corresponding temporo-parietal projection areas. Confidence limits on 3D cortical variation, visualized in 3D, exhibited severe increases in AD from 2 to 4 mm at the callosum to a peak SD of 19.6 mm at the posterior left Sylvian fissure. Normal Sylvian fissure asymmetries (right higher than left; P < 0.0005), mapped for the first time in three dimensions, were accentuated in AD (P < 0.0002), and were greater in AD than in controls (P < 0.05). Severe AD-related increases in 3D variability and asymmetry may reflect disease-related disruption of the commissural system connecting bilateral temporal and parietal cortical zones, regions known to be at risk of early metabolic dysfunction, perfusion deficits and selective neuronal loss in AD.     

Ca-activated chloride current and Na-Ca exchange have different timecourses during sarcoplasmic reticulum Ca release in ferret ventricular myocytes

The aim of this work was to measure membrane currents activated by Ca release from the cardiac sarcoplasmic reticulum (s.r.). Intracellular Ca concentration ([Ca2+]i) was measured using fluo-3 in patch clamped cells. Calcium release from the s.r. (whether occurring spontaneously or evoked by caffeine) produced changes of membrane current which could be separated into a Ca-activated Cl current which was inhibited by DIDS or Cl removal and a Na-Ca exchange current. Both these currents had different time courses from the measured [Ca2+]i. Furthermore the Ca-activated Cl current decayed more quickly than did Na-Ca exchange. Possible explanations for the different kinetics of these two Ca-sensitive currents are discussed.     

Initial characterization of a protochordate histocompatibility locus

The colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction which is controlled by a single, highly polymorphic locus called the Fu/HC. We are using map-based cloning to identify Fu/HC gene(s), and have currently delineated their location to an approximately 1-cM region of the B. schlosseri genome. The Fu/HC physical map currently consists of 85 sequence-tagged sites mapped on a minimum tiling path of 800 kb which consists of five contigs, with four gaps remaining to be crossed, and is estimated to be 75% completed. Approximately half this region has been sequenced throughout the locus, allowing the first analysis of a metazoan histocompatibility locus outside of vertebrates. This has resulted in the identification of 18 predicted genes, a number of which have been found to be expressed. Several of these genes are well conserved among the chordates; however, none of the predicted or expressed genes are linked within the genome of any organism in the databases. In addition, the Fu/HC is one of the most polymorphic loci ever described, and physical mapping has revealed that the locus is quite dynamic, and includes features such as hotspots of recombination.     

Deamidation and isoaspartate formation during in vitro aging of recombinant tissue plasminogen activator

When incubated at pH 7.3, 37 degrees C, human recombinant tissue plasminogen activator accumulated 0.77 mol of isoaspartate per mol of plasminogen activator over a 14-day period. Isoaspartate was detected by enzymatic transfer of 3H-labeled methyl groups from S-adenosyl-L-methionine in a reaction catalyzed by protein L-isoaspartyl methyltransferase. Analysis of tryptic peptides derived from aged plasminogen activator revealed that the two major sites of isoaspartate accumulation resulted from deamidation of Asn58 in the sequence -FNGG- and Asn177 in the sequence -GNSD-. Significant levels of isoaspartate also accumulated via deamidation of Asn37 in the sequence -CNSG-. All three sites occur in sequences predicted from studies with synthetic peptide to be unstable. All three sites appear to be on the surface of the protein, and all three occur in regions of the protein predicted to have higher than average chain mobility. These findings add support to the idea that sequence and flexibility play major roles in determining susceptibility to deamidation and peptide bond isomerization at Asn and Asp sites under mild conditions. These studies also illustrate the utility of enzymatic methylation for characterizing sites of deamidation in a large protein that contains numerous disulfide bonds and several sites of glycosylation.     

Two-dimensional separations of peptides and proteins by comprehensive liquid chromatography-capillary electrophoresis

Analysis of biological samples is problematic because of their complex composition. Reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), and, more recently, capillary zone electrophoresis (CZE) are routinely used for the analysis of these samples, but are eventually limited because they are one-dimensional (1-D) methods. As sample complexity increases, the separation efficiency necessary to resolve a large number of sample components in one dimension becomes prohibitively high. A solution to this problem has been to use a two-dimensional (2-D) approach. Each dimension in a 2-D separation relies on a different separating mechanism. By expanding the separation into two dimensions, sample components unresolved in the first dimension can often be separated in the second. This circumvents the requirement for extremely high efficiencies in either dimension. Two-dimensional slab gel electrophoresis has been used successfully in this area, but a more instrumental approach is desired. In this paper we describe three coupled-column approaches to 2-D separations. First, microcolumn SEC-CZE is explored as a means of 2-D protein analysis. Next, RPLC-CZE is investigated for analysis of peptides in tryptic maps. Finally, RPLC is coupled with fast CZE (FCZE), a unique form of CZE analysis, for fast 2-D analysis of peptides. Details of the instrumentation used in these 2-D systems will be presented along with the results of some typical 2-D analyses.     

The effects of dietary protein restriction and blood-pressure control on the progression of chronic renal disease. Modification of Diet in Renal Disease Study Group

BACKGROUND: Restricting protein intake and controlling hypertension delay the progression of renal disease in animals. We tested these interventions in 840 patients with various chronic renal diseases. METHODS: In study 1, 585 patients with glomerular filtration rates of 25 to 55 ml per minute per 1.73 m2 of body-surface area were randomly assigned to a usual-protein diet or a low-protein diet (1.3 or 0.58 g of protein per kilogram of body weight per day) and to a usual- or a low-blood-pressure group (mean arterial pressure, 107 or 92 mm Hg). In study 2, 255 patients with glomerular filtration rates of 13 to 24 ml per minute per 1.73 m2 were randomly assigned to the low-protein diet (0.58 g per kilogram per day) or a very-low-protein diet (0.28 g per kilogram per day) with a keto acid-amino acid supplement, and a usual- or a low-blood-pressure group (same values as those in study 1). An 18-to-45-month follow-up was planned, with monthly evaluations of the patients. RESULTS: The mean follow-up was 2.2 years. In study 1, the projected mean decline in the glomerular filtration rate at three years did not differ significantly between the diet groups or between the blood-pressure groups. As compared with the usual-protein group and the usual-blood-pressure group, the low-protein group and the low-blood-pressure group had a more rapid decline in the glomerular filtration rate during the first four months after randomization and a slower decline thereafter. In study 2, the very-low-protein group had a marginally slower decline in the glomerular filtration rate than did the low-protein group (P = 0.07). There was no delay in the time to the occurrence of end-stage renal disease or death. In both studies, patients in the low-blood-pressure group who had more pronounced proteinuria at base line had a significantly slower rate of decline in the glomerular filtration rate. CONCLUSIONS: Among patients with moderate renal insufficiency, the slower decline in renal function that started four months after the introduction of a low-protein diet suggests a small benefit of this dietary intervention. Among patients with more severe renal insufficiency, a very-low-protein diet, as compared with a low-protein diet, did not significantly slow the progression of renal disease.