Novel antibody coating of a magnetizable solid phase for use in enzyme immunoassays.

Novel kinds of magnetizable particles have been prepared using the interaction between the complexing groups of phosphonic acid and polyvalent metal ions on the surface of Fe3O4 particles. After modification of monoclonal and polyclonal antibodies by the bifunctional chelating agent 2-(4-diazophenyl)-1-hydroxyethane-1,1-bisphosphonic acid, antibodies were immobilized at a ratio of 5-10 mg antibody molecules per ml Fe3O4 particles without loss of immunological reactivity. Very sensitive and fast immunoenzymometric assays for the quantitative determination of human interferon-alpha 1 and mouse immunoglobulins were developed using such particles. The advantages of the method include immobilization of proteins on magnetizable carriers without chemical modification of the carrier and the short assay time compared to conventional immunoenzymometric assays.     

The independence of hyperactivity from conduct disorder: methodological considerations

It has been claimed that the childhood behavioural factors "hyperactivity" and "conduct disorder" are highly correlated. The fact that hyperactive symptoms load heavily on the conduct disorder factor has also been used to support the notion that hyperactivity is not an independent behavioural dimension. The present study employs a large sample of combined clinic and normal children to demonstrate that both of these observations are artifacts of methodological technique. When factor score coefficients are used to interpret factors, the hyperactive symptoms do not load on the conduct disorder factor. If factor scores are defined by the use of unit weights, as in previous studies, then the intercorrelation between the hyperactive and conduct disorder factors is high. The use of factor score coefficients to define factors, on the other hand, produces uncorrelated factors. The results support the idea that hyperactivity and conduct disorder are independent behavioural dimensions.     

Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.     

The prognostic value of hypocholesterolemia in hospitalized patients

Clinical observations show that severe illness often leads to hypocholesterolemia. To verify this finding and to define the relationship between serum cholesterol and a patient's prognosis, a study was conducted in two large hospital populations. Of 24,000 and 61,463 adult patients (populations I and II) an average of 3.8% and 3.6% died in hospital, respectively. The mean serum cholesterol levels of patients who died was significantly lower than that of those who survived (163.6 mg/dl versus 217.8 mg/dl;P < 0.0001). The average cholesterol of surviving patients was similar to that of 6,543 healthy controls. During hospitalization serum cholesterol levels of 100 mg/dl were encountered in 1.2% and 3.6% of patients of populations I and II, respectively. The mortality of these hypocholesterolemic patients was about tenfold higher than average and showed a strong, inverse, linear relationship with serum cholesterol concentrations. Patients whose serum cholesterol level dropped to less than 45 mg/dl did not survive. These data show that in severely ill patients serum cholesterol may decline to very low concentrations, and the prognosis is reflected by the degree of hypocholesterolemia, which thus may serve as a clinically useful prognostic parameter. Correspondence to: E. Windler     

Vortex Shaped Current Sources in a Physical Torso Phantom

Recent studies reported differential information in human magnetocardiogram and in electrocardiogram. Vortex currents have been discussed as a possible source of this divergence. With the help of physical phantom experiments, we quantified the influence of active vortex currents on the strength of electric and magnetic signals, and we tested the ability of standard source localization algorithms to reconstruct vortex currents. The active vortex currents were modeled by a set of twelve single current dipoles arranged in a circle and mounted inside a phantom that resembles a human torso. Magnetic and electric data were recorded simultaneously while the dipoles were switched on stepwise one after the other. The magnetic signal strength increased continuously for an increasing number of dipoles switched on. The electric signal strength increased up to a semicircle and decreased thereafter. Source reconstruction with unconstrained focal source models performed well for a single dipole only (less than 3-mm localization error). Minimum norm source reconstruction yielded reasonable results only for a few of the dipole configurations. In conclusion active vortex currents might explain, at least in part, the difference between magnetically and electrically acquired data, but improved source models are required for their reconstruction.     

Comparative Evaluation of Ligation-Mediated PCR and Spoligotyping as Screening Methods for Genotyping of Mycobacterium tuberculosis Strains

Spoligotyping has been suggested as a screening test in multistep genotyping of Mycobacterium tuberculosis strains. Relying on restriction fragment length polymorphism (RFLP) analysis with IS6110 (IS6110 RFLP analysis) as a "gold standard," we performed a comparative evaluation of spoligotyping and ligation-mediated PCR (LMPCR), a recently described PCR-based typing method, as rapid screening tests for fingerprinting of 158 M. tuberculosis strains collected in Verona, Italy. LMPCR seemed to be comparable to spoligotyping in terms both of feasibility with rapidly extracted DNA and of generation of software-analyzable images. Moreover, LMPCR grouped considerably fewer strains than spoligotyping (38 versus 67%) and was found to reduce the cluster overestimation rate (26.3 versus 58%) and to give a better discriminatory index (0.992 versus 0.970) compared to spoligotyping. In our geographical region, where there was no evidence of clustered strains carrying fewer than six IS6110 copies, LMPCR was found to be more discriminatory than spoligotyping. We also evaluated two models of three-step typing strategies, involving the use of spoligotyping and LMPCR as screening methods and IS6110 RFLP analysis as a further supporting test. LMPCR proved to be a more effective first-step test than spoligotyping, significantly reducing the need for subtyping. LMPCR should be considered an alternative to spoligotyping as a rapid screening method for M. tuberculosis fingerprinting, particularly in areas with a low prevalence of M. tuberculosis strains carrying few copies of IS6110.     

Circulating CD26 is negatively associated with inflammation in human and experimental arthritis

Dipeptidyl peptidase IV (DPPIV, CD26), a protease-cleaving N-terminal X-Pro dipeptide from selected proteins including some chemokines, is expressed both as a soluble form in plasma and on the cell surface of various immune and nonimmune cell types. To gain insights into the pathophysiological role of CD26 in arthritis, we explored DPPIV/CD26 expression during murine antigen-induced arthritis (AIA), an experimental model of arthritis. AIA induction led to reduced plasma DPPIV activity. In CD26-deficient mice, the severity of AIA was increased as assessed by enhanced technetium uptake and by increased histological parameters of inflammation (synovial thickness and exudate). We demonstrated that CD26 controls the in vivo half-life of the intact active form of the proinflammatory chemokine stromal cell-derived factor-1 (SDF-1). CD26-deficient mice exhibited increased levels of circulating active SDF-1, associated with increased numbers of SDF-1 receptor (CXCR4)-positive cells infiltrating arthritic joints. In a clinical study, plasma levels of DPPIV/CD26 from rheumatoid arthritis patients were significantly decreased when compared to those from osteoarthritis patients and inversely correlate with C-reactive protein levels. In conclusion, decreased circulating CD26 levels in arthritis may influence CD26-mediated regulation of the chemotactic SDF-1/CXCR4 axis.