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ObjectiveTo 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.MethodsWe 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.ResultsSerum 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.ConclusionsThis 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.

Background: Thyroid-stimulating hormone (TSH) and subsequent free thyroxine (FT4) concentrations outside the reference interval (RI) are used to diagnose thyroid diseases. Most laboratories do not provide age-specific RIs for TSH and FT4 beyond childhood, although TSH concentrations vary with age. Therefore, we aimed to establish TSH and FT4 age-specific RIs throughout life and aimed to determine whether using these RIs would result in reclassification of thyroid disease diagnoses in adults. Methods: This multicenter retrospective cross-sectional study used big data to determine indirect RIs for TSH and FT4. These RIs were determined by TMC and refineR-analysis, respectively, using four different immunoassay platforms (Roche, Abbott, Siemens, and Beckman Coulter). Retrospective data (2008-2022) from 13 Dutch laboratories for general practitioners and local hospitals were used. RIs were evaluated per manufacturer. Age groups were established from 2 to 20 years by 2-year categories and decade categories between 20 and 100 years. Results: We included totally 7.6 million TSH and 2.2 million FT4 requests. TSH upper reference limits (URLs) and FT4 lower reference limits were higher in early childhood and decreased toward adulthood. In adulthood, TSH URLs increased from 60 years in men, and from 50 years in women, while FT4 URLs increased from 70 years onward. Using adult age-specific RIs resulted in a decrease in diagnoses of subclinical and overt hypothyroidism in women above 50 and men above 60 years in our Roche dataset. Conclusion: This study stressed the known importance of using age-specific RIs for TSH and FT4 in children. This study also showed the clinical relevance of using age-specific RIs for TSH in adulthood to reduce diagnoses of subclinical hypothyroidism in older persons. Therefore, implementation of adult TSH age-specific RIs should be strongly considered. Data are less uniform regarding FT4 age-specific RIs and more research should be performed before implementing these in clinical practice.

Objectives: The aim of the current study was to explore the changes in lipid and NT-proBNP levels in rheumatoid arthritis (RA) patients through different phases of the disease: from the pre-clinical stage and RA onset up to the treatment phase with biological disease-modifying anti-rheumatic drugs (bDMARDS)., Methods: Thirty-eight consecutive patients, initially with arthralgia and rheumatoid factor and/or anti-citrullinated protein antibodies without arthritis, who later developed RA and eventually started treatment with bDMARDs, were included. Lipid spectrum and N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were measured longitudinally from several months before diagnosis through treatment with bDMARDs., Results: From baseline, C-reactive protein (CPR) initially increased sharply, decreasing with the start of biological treatment. Low-density lipoprotein-cholesterol (LDL-c) remained stable, high-density lipoprotein-cholesterol (HDL-c) increased, apolipoprotein A1 (ApoA1 and lipoprotein (a) (Lp(a)), and total cholesterol (TC)/HDL-c ratio and apolipoprotein B (ApoB) decreased during follow-up. NT-proBNP closely followed progression of CRP. TC, LDL-c, TC/HDL-c ratio, ApoA and ApoB inverse correlated with CRP, while Lp(a) positively correlated. HDL-c and triglycerides showed no correlation., Conclusion: Changes in the lipid profile and NT-proBNP in RA patients seem to be related to inflammation, with changes reflecting an increase in CVD risk occurring along with rises in CRP levels. These changes seem to already be present at diagnosis, indicating the need for timely control of inflammation., (Copyright © 2023 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

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., Competing Interests: JSE has received research grants from the following pharmaceutical companies (in alphabetical order): Ferring, Merck Serono, Merck Sharpe and Dome, Organon, Shering Plough and Serono. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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 -15,81nmol/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 11,51nmol/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., (Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Objective: Low vitamin D status has been associated with impaired glycemic control in patients with type 2 diabetes. The purpose of our study was to evaluate the effect of vitamin D supplementation on glycemic control in patients with type 2 diabetes., Research Design and Methods: This randomized, double-blind, placebo-controlled trial was conducted in 275 adult patients with type 2 diabetes without insulin treatment. Patients were randomly assigned to receive either vitamin D3 (50,000 IU/month) or placebo for 6 months. To assess the primary outcome of the study, change in HbA(1c), we performed a linear regression analysis., Results: Mean baseline serum 25-hydroxyvitamin D [25(OH)D] increased from 60.6 ± 23.3 to 101.4 ± 27.6 nmol/L and 59.1 ± 23.2 to 59.8 ± 23.2 nmol/L in the vitamin D and placebo group, respectively. Mean baseline HbA(1c) was 6.8 ± 0.5% (51 ± 6 mmol/mol) in both groups. After 6 months, no effect was seen on HbA(1c) (mean difference: β = 0.4 [95% CI -0.6 to 1.5]; P = 0.42) and other indicators of glycemic control (HOMA of insulin resistance, fasting insulin, and glucose) in the entire study population. Subgroup analysis in patients with a serum 25(OH)D <50 nmol/L or an HbA(1c) level >7% (53 mmol/mol) did not differ the results., Conclusions: In a well-controlled group of patients with type 2 diabetes, intermittent high-dose vitamin D supplementation did not improve glycemic control., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Background: Besides the classical role of vitamin D on calcium and bone homeostasis, vitamin D deficiency has recently been identified as a contributing factor in the onset of insulin resistance in type 2 diabetes mellitus. However, it is uncertain whether vitamin D deficiency and poor glycaemic control are causally interrelated or that they constitute two independent features of type 2 diabetes mellitus. There are limited clinical trials carried out which measured the effect of vitamin D supplementation on glycaemic control.The objective of this study is to investigate the effect of vitamin D supplementation on glycaemic control and quality of life in patients with type 2 diabetes mellitus., Methods/design: In a randomised double-blind placebo-controlled trial conducted in five general practices in the Netherlands three hundred patients with type 2 diabetes mellitus treated with lifestyle advises or metformin or sulphonylurea-derivatives are randomised to receive either placebo or 50,000 IU Vitamin D3 at monthly intervals. The primary outcome measure is the change in glycated haemoglobin level between baseline and six months. Secondary outcome measures include blood pressure, anthropometric parameters, lipid profile, insulin resistance, quality of life, advanced glycation end products and safety profiles. Quality of life will be measured by The Short Form (SF-36) Health Survey questionnaire. Advanced glycation end products are measured by an AGE-reader., Discussion: This trial will be the first study exploring the effect of vitamin D supplementation on both glycaemic control and quality of life in patients with type 2 diabetes mellitus. Our findings will contribute to the knowledge of the relationship between vitamin D status and insulin resistance in patients with type 2 diabetes mellitus., Trial Registration: The Netherlands trial register: NTR3154.

Objectives: Hypothyroidism is associated with normocytic anaemia. Indeed, a limited number of studies have shown significant associations between free thyroxin (T4) and erythrocyte indices. These studies did not include vitamin B12, folic acid, iron and renal function in the analyses. We therefore studied the association between thyroid hormones and erythrocyte indices in a population-based cohort of older euthyroid subjects, with adjustment for major confounding parameters., Design: Data, including thyroid hormones and erythrocyte indices, are from the Longitudinal Aging Study Amsterdam (LASA), an ongoing cohort study on predictors and consequences of changes in health in the ageing population in the Netherlands. Multivariable linear regression analyses were applied to study the cross-sectional associations between free T4, thyroid stimulating hormone (TSH) and erythrocyte indices (haemoglobin content, haematocrit, mean cell volume (MCV) and erythrocyte count) in a euthyroid sub-sample. The final models were adjusted for vitamin B12, folic acid, iron levels and renal function., Results: In 708 euthyroid older subjects, an increase of 5pmol/L free T4 was associated with a mean increase of 0.12mmol/L or 0.19g/dL of haemoglobin, 0.068 10(12)/L erythrocytes and 0.006L/L haematocrit (P=0.007, P=0.005, P=0.001, respectively). Free T4 was not significantly associated with MCV (P>0.05). TSH appeared not to be associated with any of the erythrocyte indices (all P>0.05)., Conclusions: In a cohort of older subjects, free T4, but not TSH, was associated with erythrocyte indices, confirming the role of thyroid hormones in the regulation of erythropoiesis., (Copyright © 2012 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)

Background: Plasma cells are one of the end products of the B-lymphocyte mediated immune response. These cells normally reside in the bone marrow or some peripheral lymphoid tissues. Increased numbers of plasma cells in the blood, usually indicates pathology such as infection, auto-immunity or haematological malignancy. Therefore, the ability to measure plasma cells (PCs) on an automated cell analyzer might be advantageous., Methods: The performance of the Sysmex XE-5000 leukocyte differential channel [high fluorescent lymphocyte count (HFLC) area] was evaluated for the ability to detect plasma cells in peripheral blood and compared to the detection of plasma cells by flow cytometric analyses., Results: Our results show that the HFL count from the XE-5000 correlates (R(2)=0.8) with the number of PCs in peripheral blood, but detects PCs with moderate to good sensitivity (88.9%) and specificity (87.8%)., Conclusions: The Sysmex XE-5000 is suitable for screening blood samples for the presence of elevated number of plasma cells in peripheral blood, but the actual quantification needs to be confirmed by flow cytometry.

Background: In this study, we compared the B.R.A.H.M.S Kryptor procalcitonin (PCT) assay with the newly developed ADVIA Centaur B.R.A.H.M-S PCT assay. Furthermore, the long-term stability of PCT at - 20 degrees C was assessed., Methods: Samples from 97 patients with lower respiratory tract infections were retested on both systems and compared with Passing-Bablok regression over two clinically relevant cutoff ranges for PCT, 0 - 2.0 microg/L and > 2.0 microg/L., Results: After storage for 2.5 to 4 years, PCT levels in patient sera declined only 3.7%. Passing-Bablok regression analysis of the total sample range (n = 97) showed that both methods correlated well (r = 0.9944), although with a deviation from the line of identity (y = 0.880x - 0.025 microg/L). Comparison of both methods within the clinically important interval of 0 - 2.0 microg/L showed acceptable correlation (y = 0.943x + 0.010 microg/L)., Conclusions: The ADVIA Centaur B.R.A.H.M.S PCT assay showed good correlation with the established Kryptor method. Therefore, this new technique can be used in clinical routine with the same clinical interpretation.

The number of CD27++ plasma cells (PCs) in peripheral blood may be a valuable biomarker for systemic lupus erythematosus (SLE) disease management. More insights into the behavior of the PC population are, however, required to validate CD27 as a reliable biomarker. In the current study, we have monitored the PC compartment of patients with acute bacterial infections and patients with SLE and, in addition, examined the relationship between the presence of serum antinuclear antibodies (ANAs) and the number of peripheral PCs. Kinetic analyses in patients with bacterial infection revealed a 10-60-fold expansion of the CD27++ PC compartment that peaked at day 2-5 and returned toward normal values at day 7-9 after hospital admission. The transient expansion of the PC population appeared to be a late phenomenon in the process of recovering from a bacterial infection. SLE subjects had significantly increased frequencies of PCs compared with patients suspected of a connective tissue disease and healthy controls. In patients suspected of a connective tissue disease, no relationship was found between the presence of serum ANAs and the number of CD27++ PCs. Additionally, the presence of serum ANAs was not associated with abnormalities in other peripheral B-cell subsets. It remains to be established at which stage of SLE development the expansion of the PC compartment is initiated.

Introduction: Abnormally shortened activated partial thromboplastin times (aPTT) are associated with significantly increased risk of thrombotic disorders and in-hospital mortality. Shortened aPTTs have been related to increased levels of factor (F) VIII and thrombin-antithrombin complex (TAT). In the current study, four different commercial aPTT reagents were evaluated for their performance to detect shortened aPTTs., Materials and Methods: aPTT of 400 patients was determined using Actin-FS (Dade Behring), APTT-SP (Instrumentation Laboratory), Automated-APTT (bioMerieux) and Platelin-LS (bioMerieux) reagents. FVIII, FIX, FXI and TAT levels were measured in shortened and normal aPTT samples., Results: An association between shortened aPTTs and elevated levels of coagulation factors (FVIII, FIX and FXI) and thrombin generation (TAT) was found with all tested aPTT reagents. Method-comparison studies demonstrated good agreement between Instrumentation Laboratory and bioMerieux reagents. However, 53 to 59% of the patients with a shortened aPTT measured with Actin-FS reagent was determined as a normal aPTT with APTT-SP, Automated-APTT and Platelin-LS reagents. These patients had increased levels of FVIII, FIX and FXI and moderately increased levels of TAT., Conclusion: Overall, an acceptable agreement between the different commercial reagents was found with respect to detection of short aPTTs. However, a disparity between some of reagents existed. Actin-FS reagent appeared to be more sensitive in inducing shortened aPTT reactions than APTT-SP, Automated-APTT and Platelin-LS reagents.

The diagnostic approach and the clinical management of critically ill patients is challenging. The recognition of biomarkers related to in-hospital mortality is of importance for identification of patients at increased risk of death. Many prediction models assessing the severity of illness and likelihood of hospital survival were developed using logistic regression analyses. These models include several laboratory parameters, such as white blood cell counts, serum bilirubin, serum albumin, blood glucose, serum electrolytes and markers which reflect acid-base disturbances. Recently, several other biomarkers, including troponin, B-type natriuretic peptide (BNP), N-terminal proBNP, C-reactive protein, procalcitonin, cholesterol and coagulation related markers have emerged as clinically useful tools for risk stratification and mortality prediction of heterogeneous and more specific subgroups of critically ill patients. More investigations are required to verify whether risk stratification based on mortality-related biomarkers may translate into targeted treatment strategies to improve clinical outcome of the critical illness. Biomarkers which are related to in-hospital mortality are highlighted in the current review.

Background: 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., Results: 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., Conclusions: 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).

Abnormally short activated partial thromboplastin times (APTTs) are associated with an increased risk of thrombotic disorders. We have examined the status of coagulation activity in subjects with short APTTs. In addition, the presence of the thrombotic risk factors G1691A-factor V, G20210A-prothrombin gene mutation and factor VIII coagulant activity (FVIII:C) was determined. Plasma levels of TAT, F1+2, D-dimer and FVIII:C were markedly higher in subjects with short APTTs compared with subjects with normal APTTs. APTTs were inversely related to TAT, F1+2, D-dimer and FVIII:C levels. The prevalence of G1691A-factor V and G20210A-prothrombin gene mutation between the group with short APTTs and the control group was not significantly different. Hence, these gene polymorphisms do not contribute to the increased risk of thrombosis associated with short APTTs. In conclusion, short APTTs are indicative of marked coagulation activity and elevated FVIII:C levels. Elevated FVIII:C levels may play a pathogenic role in the increased risk of thrombosis associated with abnormally short APTTs., (Copyright 2002 S. Karger AG, Basel)

During B-cell development the surrogate light (SL) chain is selectively expressed in progenitor and precursor B cells during the developmental stages of D(H) to J(H) and V(H) to D(H)J(H) rearrangements. Approximately half of all muH chains produced by these rearrangements cannot pair with SL chains and cannot form a pre-B-cell receptor (pre-BCR). A spectrum of affinities between VpreB and individual V(H) domains generates preB cells with pre-BCR of different fitness which, in turn, determines the extent of the pre-B II-cell proliferation and the fidelity of allelic exclusion of the H chain locus. Once pre-BCR is expressed, SL chain expression is turned off. As pre-B II cells proliferate, SL is diluted out, thus limiting pre-BCR formation. As a consequence, pre-B II cells stop proliferating, become small and resting and begin to rearrange the L chain loci. Multiple rearrangements of the kappaL chain alleles are often detected in wild-type small pre-B II cells. Around 20% of the muH chain-expressing small pre-B II cells also express L chains but do not display the Ig on the surface. Hence, it is likely that not all L chains originally generated in resting pre-B II cells can pair with the muH chain previously present in that cell. The best fitting ones are selected preferentially to generate sIg+ B cells. Furthermore, the transition of immature B cells from the bone marrow to spleen and their development to mature cells appear as two separate steps controlled by different genes.

The combined analysis of the expression of receptor tyrosine kinases c-Kit and Flt3/Flk-2 and of the human CD25 gene expressed as a transgene under the regulation of the mouse lambda5 promoter in the bone marrow of 1-week-old mice allows us to identify three stages of B lymphocyte development before the CD19(+)c-Kit(+) pre-B-I cells. Single-cell PCR analysis of the rearrangement status of the Ig heavy chain alleles allows us to order these early stages of B cell development as follows: (i) B220(+)CD19(-)c-Kit(lo)Flt3/Flk-2(hi)lambda5(-), (ii) B220(+)CD19(-)c-Kit(lo)Flt3/Flk-2(hi)lambda5(+) and (iii) B220(+)CD19(+)c-Kit(lo)Flt3/Flk-2(lo)lambda5(+) before B220(+)CD19(+)c-Kit(lo)Flt3/Flk-2(-)lambda5(+) pre-B-I cells. All these progenitors are clonable on stromal cells in the presence of IL-7 and can differentiate to CD19(+)c-Kit(-) B-lineage cells. A combination of stem cell factor, Flt3 ligand and IL-7 was also able to support the proliferation and differentiation of the progenitors in a suspension culture. Furthermore, the analyses indicate that the onset of D(H)J(H) rearrangements precedes the expression of the lambda5 gene. These progenitor populations were characteristic of juvenile mice and could not be detected in the bone marrow of adult mice. Hence the expression pattern, and probably the function, of the receptor tyrosine kinases in early B cell differentiation appears to be different in juvenile and adult mice.

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.

PCR analyses of the kappaL chain locus in single B-lineage cells of wild-type, Ckappa-, or JCkappa-deficient homozygous or heterozygous mice often detect multiple in- and out-of-frame rearrangements at the kappaL and lambdaL loci. They are most frequent in small pre-BII cells and equally so in wild-type and kappaL chain-deficient cells. Hence, kappaL chain production appears not to inhibit secondary rearrangements. Around 20% of all small preBII cells express IgL chains in their cytoplasm. Cells with a first productive rearrangement on one allele are favored to enter the immature B cell compartment. Thus, allelic exclusion might be secured by control of accessibility of IgL chain loci for rearrangement and by rapid selection of cells with a fitting over those with a nonfitting IgL chain.

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.

A common variable immunodeficiency (CVID) patient, who carries mutations on both alleles of the gene encoding the surrogate light chain component lambda 5/14.1, shows a similar phenotype of B-cell deficiency as the lambda 5-deficient mutant mouse. As discussed here by Paolo Ghia and colleagues, this points to a remarkably similar developmental pathway of B cells in humans and mice.

Within the pools of muH chain-producing precursor and mature B cells from normal and lambda5-defective mice, the frequency of cells in which both H chain alleles were productively VHDJH rearranged was determined. An equally high percentage (2%-4%) of cells carrying two productively VHDJH-rearranged H chain loci was found in precursor and mature B cell pools of both mouse strains. In all of these cells, one allele encodes a muH chain incapable of forming a surface-expressed pre-B cell receptor. Hence, allelic exclusion is maintained at the level of pre-B cell receptor expression. The surprising conservation of H chain allelic exclusion in lambda5-defective B cells suggests that an alternative form of pre-B cell receptor might function to ensure this allelic exclusion.

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.

The V(H) repertoire on both H chain alleles of normal and lambda5-deficient B lineage cells were analyzed by single-cell PCR. The mu H chains were tested for their capacity to form a pre-B cell receptor. In bone marrow, D-proximal V(H) genes were found preferentially expressed in lambda5-deficient pre-B cells and in a newly identified early c-kit+ cytoplasmic mu H chain+ pre-B cell population of normal mice. Only half of the mu H chains expressed in these cells have the capacity to form a pre-B cell receptor. Representation of the D-proximal V(H) genes was found suppressed on the productive but not on the nonproductive V(H)DJ(H) rearranged alleles of c-kit preB-II cells and splenic lambda5-deficient B cells. More than 95% of the mu H chains expressed in preB-II cells can form a pre-B cell receptor. These results demonstrate that the pre-B cell receptor in normal mice and the B cell receptor in lambda5-deficient mice mediate a shift in the V(H) repertoire.

CD19+CD10+ human B lineage bone marrow cells were separated into cycling or resting cells, which differ in their expression of CD34, VpreB, recombination activating gene (RAG-1), and terminal deoxynucleotidyl transferase (TdT). Polymerase chain reaction analyses developed for DHJH and VkJk, VkJkK(de) and VkK(de) rearrangements with DNA of single cells and a comparison with B lineage cell development in mouse bone marrow, allow to delineate the human B lymphocyte pathway of development as follows: CD34+VpreB+RAG-1+TdT+, DHJH-rearranged, kL germline cycling pre-B I cells-->CD34-VpreB+microH chain+ (pre-B receptor+) RAG-1-TdT-, VHDHJH-rearranged, kL germline, cycling pre-B II cells-->CD34-VpreB-, intracytoplasmic microH chain+ (pre-B receptor-) RAG-1+/-TdT-, VHDHJH-rearranged, mainly kL germline cycling pre-B II cells-->CD34-VpreB-intracytoplasmic microH chain+, RAG-1+TdT-, VHDHJH-rearranged, VkJk-rearranged, IgM-, resting pre-B II cells CD34+VpreB-, sIgM+, RAG-1+TdT-, VHDHJH- and VkJk-rearranged IgM+ immature B cells-->CD34-, CD10-, sIgM+/sIgD+ mature B cells. This order, for the first time established for human B lineage cells, shows striking similarities with that established for mouse B lineage cells in bone marrow.

The surrogate light chain is composed of two polypeptides, VpreB and lambda 5. In the mouse there are two VpreB genes which are 99% identical within the coding regions. Extensive restriction enzyme mapping and sequencing of these two genes showed that only the coding region and immediate 5' and 3' flanking sequences exhibited such high homology. More distal sequences have diverged considerably. The region 5' of the respective gene directed transcription of a reporter gene in a pre-B cell line, indicating that it contained promoter, and perhaps enhancer function. The VpreB2 gene is functional, as it directed the production in COS cells of a 16-kDa protein that assembled with lambda 5 and was recognized by a VpreB-specific monoclonal antibody. Using transfected COS cells expressing either VpreB1 or VpreB2, a PCR assay was developed to examine the steady state level of transcripts from each gene. When this assay was applied to a number of cell lines representing early stages of B cell differentiation, co-expression of the two genes was observed in every case. VpreB1 and VpreB2 were co-expressed in the fetal liver of CB17 mice, where peak expression of each gene occurred at days 16-17 of gestation. Similarly, adult bone marrow from several strains of mice expressed both genes. In sorted bone marrow cells expression of both VpreB genes was detected in pro-B/pre-BI and large pre-BII cells, while the RNA steady state levels were at least 100-fold lower in small pre-BII and immature/mature B cells. Finally, single-cell reverse transcriptase-polymerase chain reaction on such sorted bone marrow cells detected VpreB1 and VpreB2 expression in at least 30% of all pro-B/pre-BI cells and large Ig heavy chain, surrogate light chain (pre-B receptor) expressing pre-BII cells. These results demonstrate that the control of expression of the two VpreB genes overlaps during development. They suggest that both VpreB1 and VpreB2 polypeptides can assemble with lambda 5 and mu to form pre-B cell receptor complexes.

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.

Mutagenesis of a region of human interleukin (IL)-6 which is important for triggering signal transduction via the IL-6 receptor beta-chain (gp130) has lead to the isolation of a variant of human IL-6 (IL-6.Q160E/T163P), which could antagonize the biological activity of wild type IL-6 on the human EBV transformed B cell line CESS and the human hepatoma cell line HepG2. Surprisingly this antagonistic IL-6 variant had an agonistic effect on the human myeloma cell line XG-1, albeit at a 1000-fold higher concentration than wild type IL-6. This residual activity of the mutant arose from triggering gp130, because it could be inhibited by a gp130 specific mAb. Extensive mutagenesis of residues between Q153 and H165 of human IL-6, a region which is partly homologous in cytokines which also signal via gp130 (oncostatin M, ciliary neurotrophic factor, leukaemia inhibitory factor, IL-11), did result in the isolation of a second antagonist for IL-6 activity on CESS and HepG2 cells. However on XG-1 cells this variant was active as well. These results suggest that (an) additional region(s) of the IL-6 molecule might be involved in gp130 triggering. Recently we indeed found that residues Lys42-Ala57 are also important for gp130 triggering. Inhibition experiments with neutralizing IL-6R alpha-chain specific mAb show that this region can be functionally separated from the Q153-H165 region. These findings have important implications for the development of receptor antagonists of IL-6 and IL-6 family members.

Differential expression of c-kit, CD25 (TAC), surrogate L chain and cytoplasmic muH chain, and surface expression of IgM and IgD allows the separation of B220 (CD45+) B cell subpopulations. PCR analyses with DNA of single cells developed by others and by us have been used to monitor the conformation of the Ig H and L chain gene loci in these different B lineage subpopulations. The results of these analyses indicate that B220+/c-kit+/CD25- cells are the precursors of large B220+/CD25+/sIgM- which, in turn, are the precursors of small B220+/CD25+/sIgM- cells. The majority of B220+/c-kit+/CD25- cells are DHJH-rearranged, with L chain loci in germline configuration and are thus pre-B I cells. More than 90% of all large B220+/CD25+/sIgM- cells have at least one H chain locus VHDHJH rearranged; half of them have also the second locus VHDHJH rearranged and are thus large pre-B II cells. Rearrangements of at least one allele of the kappa L chain loci become detectable in 65% of the small B220+/CD25+/sIgM- cells, 67% of the immature B and > 75% of the mature B cells. The ratio of kappa L to lambda L gene rearrangements in all three subpopulations is approximately 10:1, indicating that the kappa L/lambda L ratio is established as soon as rearrangements are made.

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.