A PCR-based method of screening individuals of all ages, from neonates to the elderly, for familial hyperaldosteronism type I

Aim: Unless specifically treated (glucocorticoids in low doses), Familial Hyperaldosteronism Type I(FH-I) may result in early death from stroke. We report the successful application of a rapid, polymerase chain reaction (PCR)-based method of detecting the 'hybrid' 11 beta-hydroxylase (11 beta-OHase)/aldosterone synthase (AS) gene as a screening test for FH-I. Methods: 'Long-PCR' was used to amplify, concurrently, a 4 kb fragment of AS gene (both primers AS-specific) and a 4 kb fragment of the hybrid gene (5' primer 11 beta-OHase-specific, 3'primer AS-specific) from DNA extracted from blood either collected locally or transported from elsewhere. Sample collection and transport were straightforward. This 4 kb fragment contains all the currently recognised hybrid gene 'crossover' points. Results: Within a single family, long-PCR identified all 21 individuals known to have FH-I. Hypertension was corrected in all 11 treated with glucocorticoids. Nine with normal blood pressure are being closely followed for development of hypertension. Long-PCR cord blood analysis excluded FH-I in three neonates born to affected individuals. Long-PCR newly identified two other affected families: (1) a female (60 years) with a personal and family history of stroke and her normotensive daughter (40 years), and (2) a female (51 years) previously treated for primary aldosteronism with amiloride, her two hypertensive sons (14 and 16 years) and her hypertensive mother (78 years). No false negative or false positive results have yet been encountered. At least seven other centres have successfully performed this test. Conclusion: Long-PCR is a reliable method of screening individuals of all ages for FH-I.

Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Development of CD8 alpha beta CTL epitope-based vaccines requires an effective strategy capable of co-delivering large numbers of CTL epitopes, Here we describe a DNA plasmid encoding a polyepitope or polytope protein, which contained multiple contiguous minimal murine CTL epitopes, Mice vaccinated with this plasmid made MHC-restricted CTL responses to each of the epitopes, and protective CTL were demonstrated in recombinant vaccinia virus, influenza virus, and tumor challenge models, CTL responses generated by polytope DNA plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering a gene for granulocyte-macrophage CSF, and appeared to be induced in the absence of CD4 T cell-mediated help, The ability to deliver large numbers of CTL epitopes using relatively small polytope constructs and DNA vaccination technology should find application in the design of human epitope-based CTL vaccines, in particular in vaccines against EBV, HIV, and certain cancers.

Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H, curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

Towards a validation of multiple features in the assessment of emotions

This study extends previous attempts to assess emotion with single adjective descriptors, by examining semantic as well as cognitive, motivational, and intensity features of emotions. The focus was on seven negative emotions common to several emotion typologies: anger, fear, sadness, shame, pity, jealousy, and contempt. For each of these emotions, seven items were generated corresponding to cognitive appraisal about the self, cognitive appraisal about the environment, action tendency, action fantasy, synonym, antonym, and intensity range of the emotion, respectively. A pilot study established that 48 of the 49 items were linked predominantly to the specific emotions as predicted. The main data set comprising 700 subjects' ratings of relatedness between items and emotions was subjected to a series of factor analyses, which revealed that 44 of the 49 items loaded on the emotion constructs as predicted. A final factor analysis of these items uncovered seven factors accounting for 39% of the variance. These emergent factors corresponded to the hypothesized emotion constructs, with the exception of anger and fear, which were somewhat confounded. These findings lay the groundwork for the construction of an instrument to assess emotions multicomponentially.

A method of analysing stream channel response to environmental change: gauge data for the Tully River

Gauging data are available from numerous streams throughout Australia, and these data provide a basis for historical analysis of geomorphic change in stream channels in response to both natural phenomena and human activities. We present a simple method for analysis of these data, and a briefcase study of an application to channel change in the Tully River, in the humid tropics of north Queensland. The analysis suggests that this channel has narrowed and deepened, rather than aggraded: channel aggradation was expected, given the intensification of land use in the catchment, upstream of the gauging station. Limitations of the method relate to the time periods over which stream gauging occurred; the spatial patterns of stream gauging sites; the quality and consistency of data collection; and the availability of concurrent land-use histories on which to base the interpretation of the channel changes.

Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes

Chimeric papillomavirus (PV) virus-like particles (VLPs) based on the bovine papillomavirus type 1 (BPV-1) L1 protein were constructed by replacing the 23-carboxyl-terminal amino acids of the BPV1 major protein L1 with an artificial polytope minigene, containing known CTL epitopes of human PV16 E7 protein, HIV IIIB gp120 P18, Nef, and reverse transcriptase (RT) proteins, and an HPV16 E7 linear B epitope. The CTL epitopes were restricted by three different MHC class 1 alleles (H-2(b), H-2(d), HLA-A*0201). The chimeric L1 protein assembled into VLPs when expressed in SF-9 cells by recombinant baculovirus. After immunization of mice with polytope VLPs in the absence of adjuvant, serum antibodies were detected which reacted with both polytope VLPs and wild-type BPV1L1 VLPs, in addition to the HPV16E7 linear B cell epitope. CTL precursors specific for the HPV16 E7, HIV P18, and RT CTL epitopes were also detected in the spleen of immunized mice. Polytope VLPs can thus deliver multiple B and T epitopes as immunogens to the MHC class I and class II pathways, extending the utility of VLPs as self-adjuvanting immunogen delivery systems. (C) 2000 Academic Press.

Reliability of multiple frequency bioelectrical impedance analysis: An intermachine comparison

The technical reliability (i.e., interinstrument and interoperator reliability) of three SEAC-swept frequency bioimpedance monitors was assessed for both errors of measurement and associated analyses. In addition, intraoperator and intrainstrument variability was evaluated for repeat measures over a 4-hour period. The measured impedance values from a range of resistance-capacitance circuits were accurate to within 3% of theoretical values over a range of 50-800 ohms. Similarly, phase was measured over the range 1 degrees-19 degrees with a maximum deviation of 1.3 degrees from the theoretical value. The extrapolated impedance at zero frequency was equally well determined (+/-3%). However, the accuracy of the extrapolated value at infinite frequency was decreased, particularly at impedances below 50 ohms (approaching the lower limit of the measurement range of the instrument). The interinstrument/operator variation for whole body measurements were recorded on human volunteers with biases of less than +/-1% for measured impedance values and less than 3% for phase. The variation in the extrapolated values of impedance at zero and infinite frequencies included variations due to operator choice of the analysis parameters but was still less than +/-0.5%. (C) 1997 Wiley-Liss, Inc.

Direct evidence for the expression of multiple endogenous retroviruses in the synovial compartment in rheumatoid arthritis

Objective. Circumstantial evidence links retroviruses (RVs) with human autoimmune diseases, The aim of the present study was to obtain direct evidence of RV gene expression in rheumatoid arthritis (RA). Methods. Synovial samples were obtained from patients with RA, patients with osteoarthritis (OA), and normal control subjects, Reverse transcription-polymerase chain reaction (RT-PCR) was performed using synovial RNA and primers to conserved sequences in the polymerase (pol) genes of known RVs. Results. PCR products (n = 857) were cloned and sequenced, Multiple pol transcripts, many with open reading frames, were expressed in every sample, Sequences were aligned and classified into 6 families (F1-F6) that contained 33 groups of known and unknown endogenous RVs (ERVs), each distinguished by a specific, deduced peptide motif, The frequency of sequences in each family was similar between RA, OA, and normal synovial tissue, but differed significantly in RA synovial fluid cells, F1 sequences (undefined, but related to murine and primate type C RVs) were lower in frequency, F2 (ERV-9-related), F4 (HERV-K-related), and F6 (HERV-L-related) sequences were higher in frequency, and F3 (RTVL-H-related) sequences were not detected, in the RA synovial fluid cells compared with the RA synovial tissues. Conclusion. Multiple ERVs are expressed in normal and diseased synovial compartments, but specific transcripts can be differentially expressed in RA.

Use of multiple channel pH monitoring for evaluation of ultra-distal reflux in patients after fundoplication for treatment of Barrett`s esophagus

Dysplasia and esophageal adenocarcinoma may arise in patients with Barrett`s esophagus after fundoplication esophageal pH monitoring showing no acid in esophagus. This suggests the need to develop methodology to evaluate the occurrence of ultra-distal reflux (1 cm above the LES). The objective of the study was to compare acid exposition in three different levels: 5 cm above the upper border of the LES, 1 cm above the LES and in the intrasphincteric region. Eleven patients with Barrett`s esophagus after Nissen fundoplication with no clinical, endoscopic and radiologic evidence of reflux were selected. Four-channel pH monitoring took place: channel A, 5 cm above the upper border of the LES; channel B, 1 cm above the LES; channel C, intrasphincteric; channel D, intragastric. The results of channels A, B and C were compared. There was significant increase in number of reflux episodes and a higher fraction of time with pH <4.0 in channel B compared to channel A. There was significant decrease in fraction of time with pH <4.0 in channel B compared to channel C. Two cases of esophageal adenocarcinoma were diagnosed in the studied patients. The region 1 cm above the upper border of the LES is more exposed to acid than the region 5 cm above the upper border of the LES, although this exposure occurred in reduced levels. The region 1 cm above the upper border of the LES is less exposed to acid than the intrasphincteric region.

Combination of Multiple Molecular Markers Can Improve Prognostication in Patients With Locally Advanced and Lymph Node Positive Bladder Cancer

Purpose: We tested whether the combination of 4 established cell cycle regulators (p53, pRB, p21 and p27) could improve the ability to predict clinical outcomes in a large multi-institutional collaboration of patients with pT3-4N0 or pTany Npositive urothelial carcinoma of the bladder. We also assessed whether the combination of molecular markers is superior to any individual biomarker. Materials and Methods: The study comprised 692 patients with pT3-4N0 or pTany Npositive urothelial carcinoma of the bladder treated with radical cystectomy and bilateral lymphadenectomy (median followup 5.3 years). Scoring was performed using advanced cell imaging and color detection software. The base model incorporated patient age, gender, stage, grade, lymphovascular invasion, number of lymph nodes removed, number of positive lymph nodes, concomitant carcinoma in situ and adjuvant chemotherapy. Results: Individual molecular markers did not improve the predictive accuracy for disease recurrence and cancer specific mortality. Combination of all 4 molecular markers into number of altered molecular markers resulted in significantly 1 higher predictive accuracy than any single biomarker (p < 0.001.). Moreover addition of number of altered molecular markers to the base model significantly improved the predictive accuracy for disease recurrence (3.9%, p < 0.001) and cancer specific mortality (4.3%, p < 0.001). Addition of number of altered molecular markers retained statistical significance for improving the prediction of clinical outcomes in the subgroup of patients with pT3N0 (280), pT4N0 (83) and pTany Npositive (329) disease (p < 0.001). Conclusions: While the status of individual molecular markers does not add sufficient value to outcome prediction in patients with advanced urothelial carcinoma of the bladder, combinations of molecular markers may improve molecular staging, prognostication and possibly prediction of response to therapy.

The rise of multiple births in Brazil

Aim: The aim of this study was to assess the rise in multiple births and its influence on trends of low birth weight (LBW) rates in Porto Alegre, Brazil. Methods: This is a registry-based study of live births from 1994 to 2005 obtained from the national live birth information system. Chi-square tests for trends were assessed for LBW and multiple birth rates. The impact of multiple births on LBW trends was assessed by sequential modelling, including year and further adjustment for multiple births. Risk factors for multiple births were assessed using the Poisson regression. Results: A total of 263 252 live births were studied. The LBW rate increased from 9.70% to 9.88% (p < 0.001) and the multiple birth rate rose from 1.95% to 2.53% (p < 0.001). LBW rate increased among twins, from 57.14% to 63.46% (p = 0.001). The twin birth rate rose by 24.7%, while the rate of triplets or higher-order increased by 150%. Multiple births may be responsible for 23.9% of the increase in the LBW rate over the period. Mothers with higher levels of schooling, older mothers and mothers delivering in private hospitals were more likely to deliver multiple births. Conclusions: It seems that both the increase in multiple births and in the LBW among multiple births contributed to this rise in overall LBW rate.

Effect of Multiple Cranial Burr Hole Surgery on Prevention of Recurrent Ischemic Attacks in Children with Moyamoya Disease

Moyamoya disease (MMD) is an uncommon cerebrovascular disorder characterized by progressive stenosis of the terminal portion of the internal carotid artery and its main branches. Direct and indirect bypass techniques have been devised with the aim of promoting neoangiogenesis. The current study aimed to investigate the role of multiple cranial burr hole (MCBH) operations in the prevention of cerebral ischemic attacks in children with MMD. Seven children suffering from progressive MMD were submitted to the MCBH and arachnoid opening technique. Ten to 20 burr holes were drilled in the fronto-temporo-parieto-occipital area of each hemisphere in each patient, depending on the site and extent of the disease. All patients were evaluated pre- and postoperatively by means of Barthel index (BI), CT, MR, angio-MR, and angiography. Patients had no recurrence of ischemic attacks postoperatively. Neoangiogenesis was observed in both hemispheres. One patient developed a persistent subdural collection after surgery, thus requiring placement of a subdural-peritoneal shunt. Postoperative BI was statistically significantly improved (P = 0.02). This report suggests that MCBH for revascularization in MMD is a simple procedure with a relatively low risk of complications and effective for preventing cerebral ischemic attacks in children. In addition, MCBH may be placed as an adjunct to other treatments for MMD.

Patient based method of assessing adverse events in clinical trials in rheumatology: the revised Stanford Toxicity Index

We describe the progress towards developing a patient rated toxicity index that meets all of the patient-important attributes defined by the OMERACT Drug Safety Working Party, These attributes are frequency, severity. importance to patient, importance to the clinician, impact on economics, impact on activities, and integration of adverse effects with benefits. The Stanford Toxicity Index (STI) has been revised to collect all attributes with the exception of impact on activities. However, since the STI is a part of the Health Assessment Questionnaire (HAQ). impact on activities is collected by the HAQ. In particular, a new question asks patients to rate overall satisfaction, taking into consideration both benefits and adverse effects. The nest step in the development of this tool is to ensure that the STI meets the OMERACT filter of truth, discrimination, and feasibility. Although truth and feasibility have been confirmed by comparisons within the ARAMIS database, discrimination needs to be assessed in clinical trials.