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Objectives Multiple 25(OH)D assays have recently been aligned to improve comparibility. In this study we investigated the performance of these assays using both native single-donor sera with target values certified by a reference method as well as single donor sera from a heterogeneous patient population. Design & methods 25(OH)D levels were measured in twenty reference samples (Ref!25OHD; Labquality, Finland) using five automated methods (Lumipulse, Liaison, Cobas, iSYS and Access) and one aligned ID-XLC-MS/MS method (slope: 1,00; intercept: 0,00; R = 0,996). Furthermore, 25(OH)D concentrations measured in 50 pregnant women and 52 random patients using the 5 automated assays were compared to the ID-XLC-MS/MS. In addition, Vitamin D binding protein (DBP) was measured. Results Most automated assays showed significant differences in 25(OH)D levels measured in reference samples. Slopes varied from 1,00 to 1,33, intercepts from − 5.48 to − 15,81 nmol/L and the R from 0,971 to 0,997. This inaccuracy was even more prominent in a heterogeneous patient population. Slopes varied from 0,75 to 1,35, intercepts from − 9.02 to 11,51 nmol/L and the R from 0,840 to 0,949. For most assays the deviation in 25(OH)D concentration increased with elevating DBP concentrations suggesting that DBP might be one of the factors contributing to the inaccuracy in currently used automated 25(OH)D methods. Conclusions Despite the use of standardized assays, we observed significant differences in 25(OH)D concentrations in some automated methods using reference material obtained from healthy single donor sera. In sera of a patient population this inaccuracy was even worse which is highly concerning as patient samples are being investigated in clinical laboratories.

OBJECTIVE: To compare vitamin D status in women with PCOS versus fertile women and subsequently evaluate the association between vitamin D status and metabolic disturbances in PCOS women.METHODS: We conducted a cross-sectional comparison study of 639 women with PCOS and 449 fertile women. Serum 25-hydroxyvitamin D (25(OH)D) was stratified into a severe deficient (< 25 nmol/l), insufficient (25-50 nmol/l), moderate (50-75 nmol/l) and adequate (> 75 nmol/l) status. The main outcome measures were the difference in vitamin D status between PCOS and fertile women, and the association between serum 25(OH)D and metabolic disturbances in PCOS women only.RESULTS: Serum 25(OH)D was significantly lower in PCOS women compared to fertile controls (mean 25(OH)D of 49.0 nmol/l versus 64.5 nmol/l). An adjusted significant difference was seen between serum 25(OH)D and homeostasis model assessment (HOMA-IR) (β = 0.76; 95% CI: 0.63-0.91; p < 0.01), HDL-cholesterol (β = 0.20; 95% CI: 0.05-0.60, p < 0.01) and apolipoprotein A1 (β = 26.2; 95% CI: 7.5-45.0, p < 0.01) between the highest vitamin D group compared to the lowest vitamin D group.CONCLUSIONS: This study demonstrates that women with PCOS have a significantly lower serum 25(OH)D compared to fertile controls. A compromised vitamin D status in PCOS women is associated with a higher HOMA-IR and an unfavourable lipid profile. Large randomized controlled trials are necessary to explore the causality of this linkage.

We describe the phenotypic and functional properties of B lineage cells developing in fetal liver organ cultures (FLOC) of mouse embryos at day 14 or 15 of gestation which contain pro/pre-B-I cells. FLOC B cell development proceeds to mature IgM+, IgD+ and CD23+ lipopolysaccharide-reactive B cells within a culture period of 5-6 days. The phenotypes and relative proportions of pro/pre-B-I, pre-B-II, immature and mature B cells from FLOC were similar to that seen in livers freshly isolated from age-matched, i.e. newborn, mice. More importantly, the numbers of cells recovered in the different B lineage subpopulations from FLOC were close to those developed in vivo. Hence, in contrast to single-cell suspension cultures of fetal liver, FLOC allow the proliferative expansion of pre-B cell receptor-expressing pre-B-II cells. FLOC from embryos of mice with targeted mutations in the RAG-2 and lambda5 genes, which cannot expand by proliferative expansion of their pre-B-II compartment in vivo because they cannot express a pre-B cell receptor on their surface, show this same defect in vitro. FLOC are accessible to the action of mAb and cytokines. Thus, addition of anti-IL-7 receptor mAb to FLOC of normal mice inhibits B cell development at the transition of pro/pre-B-I to pre-B-II cells. This inhibition is reversed by addition of excess rIL-7. Addition of IL-7 alone stimulates the proliferation of pro/pre-B-I cells and inhibits their differentiation to pre-B-II and immature B cells, as it does in single-cell suspension cultures. FLOC should be useful to study the effects of other mAb, cytokines, ligands and other molecules on early B cell development.

Membrane-bound preBCR of wild-type mice, and probably also preBCR-like V(preB) muH chain complexes in lambda5-deficient mice, signal allelic exclusion so that < 0.1% of all preB-II cells and all subsequent B lineage cells express two muH chains on their surface. On the other hand a large number of muH chains which are originally generated at the transition of preB-I to preB-II cells cannot pair with surrogate L chains, cannot form a preBCR on the surface and, hence, allow two H chain alleles to be productively rearranged in one B-lineage cell. By contrast membrane-bound BCR on immature B cells does not signal allelic or isotypic exclusion Of Ig kappaL and lambdaL chain gene loci. This allows the rearrangement machinery to remain active, and secondary L chain rearrangements on one kappaL chain allele are frequently observed. Rapid selection of fitting H/L chain pairs, forming BCR on the surface, allows B-lineage cells to enter the mature B cell pool where the rearrangement machinery is shut off, securing allelic exclusion of L chain loci in most B cells.

Bone marrow of both normal and rearrangement-deficient mice contains a small population of B220(CD45R)+ cells, which do not express the B lineage marker CD19. Instead, part of this population coexpresses the surface marker CD43 and lacks or expresses very low levels of heat stable antigen (HSA) and BP-1, thus representing a part of Hardy's fraction A (B220(+)-CD43+HSA-, BP-1-) of B lineage development. However, some 20-40% of these B220(+)-CD19- cells also coexpress the NK1.1 surface molecule and do not express genes like VpreB or B29 restricted to the B cell lineage. These cells respond to recombinant interleukin 2 in vitro, and develop into killer cells that can lyse the prototypic NK target tumor cell, YAC-1, as well as syngeneic normal lipopolysaccharide or concanavalin A blasts, providing they lack the surface expression of major histocompatibility complex class I molecules. The implications of these findings for studies on B lymphopoiesis are discussed. It is suggested that the CD19-specific monoclonal antibody is more reliable, as in humans, than B220(CD45R) to detect B lineage cells in mice.

Neutralizing monoclonal antibodies specific for human interleukin-6 (IL-6) bind two distinct sites on the IL-6 protein (sites I and II). Their interference with IL-6 receptor binding suggested that site I is a receptor-binding site of IL-6, whereas site II is important for signal transduction. Mutagenesis of site II could therefore result in the isolation of IL-6 receptor antagonists. To test this hypothesis, a panel of IL-6 mutant proteins was constructed that did not bind to a site II-specific monoclonal antibody. One such site II mutant protein (with double substitution of Gln-160 with Glu and Thr-163 with Pro) was found to be an antagonist of human IL-6. It was inactive on human CESS cells, weakly active on human HepG2 cells, but active on mouse B9 cells. It could specifically antagonize the activity of wild-type IL-6 on CESS and HepG2 cells. The binding affinity of this variant for the 80-kDa IL-6 receptor was similar to that of wild-type IL-6. High affinity binding to CESS cells, however, was abolished, suggesting that the mutant protein is inactive because the complex of the 80-kDa IL-6 receptor and the mutant protein cannot associate with the signal transducer gp130. The human IL-6 antagonist protein may be potentially useful as a therapeutic agent., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Shortened activated partial thromboplastin time (aPTT) values are associated with enhanced coagulation activation. However, the clinical relevance of shortened aPTTs is not well defined. The aim of this study was to determine the in-hospital mortality rate in subjects with shortened aPTTs and the effects of polymorphism in plasminogen activator inhibitor (PAI)-, t-PA- and factor XIII gene on the coagulation status. D-dimer, C-reactive protein (CRP) and glucose, markers that have been related with increased mortality, were tested.We found that a shortened aPTT on admission was associated with an increased risk of in-hospital mortality (OR=2.6, 95% CI: 2.1-3.5). Non-survivors with short aPTTs had significantly higher plasma D-dimer, CRP and glucose levels compared with survivors. Subjects homozygous for PAI-1 5G and t-PA I alleles showed higher plasma D-dimer levels compared with 4G/4G PAI-1 (p=0.02) and D/D t-PA (p=0.001) homozygotes, respectively.These results suggest that PAI-1 4G/5G and t-PA I/D polymorphisms determine plasma D-dimer levels in patients with shortened aPTT values. Preliminary results show that, among patients with short aPTTs, homozygosity for the hyperfibrinolytic PAI-1 5G or tPA I alleles are at increased risk of in-hospital mortality compared with 4G/4G PAI-1 and D/D tPA homozygotes (OR=2.6, 95% CI: 1.3-5.5 and OR=5.5, 95% CI: 1.3-24.5, respectively).

Single cell PCR assays have been further developed that detect over 80% of all VkappaJkappa, VkappaRS, and VlambdaJlambda rearrangements at efficiencies between 70% and 90%. These IgL chain gene rearrangement assays were used with small pre-BII cells that develop in comparably high numbers in the bone marrow of wild-type, Ckappa-deficient, and JCkappa-deficient homozygous and heterozygous mice. In all of these mice, only 15%-25% of all small pre-BII cells carry VlambdaJlambda rearrangements. These results confirm that lambdaL chain gene rearrangements occur independently of kappaL chain gene rearrangement and expression. They also show that a large part of the small pre-BII cells that express the rearrangement machinery can develop without IgL chain gene rearrangements.

The repertoires of B cell receptor (Ig)-expressing B cells are generated during B cell development in a stepwise fashion (Tonegawa 1983). First, DH segments are rearranged to JH segments on the H chain locus at the transition from B220+CD19- proB cells to B220+CD19+ckit+ preB-I cells. In most preB-I cells both H chain alleles are DHJH-rearranged (Rolink et al. 1995). PreB-I cells, as proB cells, express the VpreB and λ5 proteins of the surrogate L chain (Melchers et al. 1993) and the preB cell — and B cell — receptor-anchoring proteins Igα and Igβ (Reth 1994). Next, VH segments are rearranged to DHJH segments (Alt et al. 1984) at the transition from preB-I to preB-II cells (ten Boekel et al. 1995). Whenever this rearrangement occurs in-frame, a μH chain can be expressed. Productive VHDHJH- rearranged H chain alleles are found in large, cycling ckit- CD25+ preB-II cells which, like all subsequent stages of B cell development, all express an H chain in their cytoplasm (Rolink et al. 1994). One fifth of all large preB-II cells express the μH chains as complexes with surrogate L chains as so-called preB cell receptors on their surface (Winkler et al. 1995). In these cells the L chain loci remain in germline configuration, and are not transcribed. The other portion of the large preB-II cells appears further progressed in development. They no longer express the preB cell receptor, in fact, they have the expression of the surrogate L chain genes downregulated, and begin to transcribe L chain loci (Grawunder et al. 1995). A small fraction of preB-II cells expressing μH chains in their cytoplasm continues to express ckit and surrogate L chain, but fails to express preB cell receptors on their surface. Their properties are described below.