Complex biological pattern of fertility hormones in children and adolescents: a study of healthy children from the CALIPER cohort and establishment of pediatric reference intervals.

BACKGROUND: Pediatric endocrinopathies are commonly diagnosed and monitored by measuring hormones of the hypothalamic-pituitary-gonadal axis. Because growth and development can markedly influence normal circulating concentrations of fertility hormones, accurate reference intervals established on the basis of a healthy, nonhospitalized pediatric population and that reflect age-, gender-, and pubertal stage-specific changes are essential for test result interpretation. METHODS: Healthy children and adolescents (n = 1234) were recruited from a multiethnic population as part of the CALIPER study. After written informed parental consent was obtained, participants filled out a questionnaire including demographic and pubertal development information (assessed by self-reported Tanner stage) and provided a blood sample. We measured 7 fertility hormones including estradiol, testosterone (second generation), progesterone, sex hormone-binding globulin, prolactin, follicle-stimulating hormone, and luteinizing hormone by use of the Abbott Architect i2000 analyzer. We then used these data to calculate age-, gender-, and Tanner stage-specific reference intervals according to Clinical Laboratory Standards Institute C28-A3 guidelines. RESULTS: We observed a complex pattern of change in each analyte concentration from the neonatal period to adolescence. Consequently, many age and sex partitions were required to cover the changes in most fertility hormones over this period. An exception to this was prolactin, for which no sex partition and only 3 age partitions were necessary. CONCLUSIONS: This comprehensive database of pediatric reference intervals for fertility hormones will be of global benefit and should lead to improved diagnosis of pediatric endocrinopathies. The new database will need to be validated in local populations and for other immunoassay platforms as recommended by the Clinical Laboratory Standards Institute.

Standardized Procedural Practices of the Ontario Prenatal Screening Program for aneuploidies and open neural tube defects.

BACKGROUND/OBJECTIVES: The Ontario Prenatal Screening Program (OPSP) follows internationally recognized standardized procedures for laboratories and genetics clinics. However, it has been found that some procedures are subject to interpretation, so the current procedures are designed to facilitate a unified approach in the interpretation of literature recommendations. In Ontario, the OPSP offers multiple screening modalities with integrated prenatal screening (including both first and second trimester markers) being the most commonly chosen option. Other screening modalities include first trimester screening, second trimester quad screening, serum integrated screening, and NT-Quad. METHODS: The standardization was based on a literature review and on current practices in Ontario. RESULTS/DISCUSSION: The main finding of the review was a paucity of published data relating to the procedures and the decision-making processes involved in prenatal screening. The purpose of this publication is to provide the most up-to-date and pertinent information for clinical laboratory professionals involved with prenatal screening for Down syndrome, trisomy 18 and open neural tube defects.

Analysis of the role of IL-21 in development of murine B cell progenitors in the bone marrow.

IL-21 plays a key role in the late stage of B cell development, where it has been shown to induce growth and differentiation of mature B cells into Ig-secreting plasma cells. Because IL-21R has also been reported on bone marrow (BM) B cell progenitors, we investigated whether IL-21R influenced earlier stages of B cell development. IL-21R is functional as early as the pro-B cell stage, and the strength of receptor-mediated signaling increases as cells mature. The addition of IL-21 to B cell progenitors in cell culture resulted in the accelerated appearance of mature B cell markers and was associated with the induction of Aid, Blimp1, and germline transcripts. We also found that stimulation of both IL-21R and CD40 was sufficient to induce the maturation of early B cell progenitors into IgM- and IgG-secreting cells. Consistent with a role for IL-21 in promoting B cell differentiation, the number of B220(+)CD43(+)IgM(-) pro-B cells was increased, and the number of mature IgM(hi)IgD(hi) cells was decreased in BM of IL-21R-deficient mice. We also report in this paper that IL-21 is expressed by BM CD4(+) T cells. These results provide evidence that IL-21R is functional in B cell progenitors and indicate that IL-21 regulates B cell development.

Interleukin-21 regulates expression of the immediate-early lytic cycle genes and proteins in Epstein-Barr Virus infected B cells.

The switch between the latent and the lytic cycle of Epstein-Barr Virus (EBV) infection is characterized by the induction of viral transcriptional activators Zta and Rta. CD4(+) T cell-derived cytokines are likely to regulate expression of these proteins in EBV-infected B cells. Here we show that interleukin-21 decreases constitutive expression of Rta and its target EA-D in EBV-infected B cell lines during the first 3 days of culture. In some cell lines this is followed by a strong increase in the expression of Zta, Rta, and EA-D during prolonged culture. Additionally, we provide evidence that IL-21-mediated JAK/STAT signals regulate increase in the Zta expression.

Structure-activity relationships of orotidine-5'-monophosphate decarboxylase inhibitors as anticancer agents.

A series of 6-substituted and 5-fluoro-6-substituted uridine derivatives were synthesized and evaluated for their potential as anticancer agents. The designed molecules were synthesized from either fully protected uridine or the corresponding 5-fluorouridine derivatives. The mononucleotide derivatives were used for enzyme inhibition investigations against ODCase. Anticancer activities of all the synthesized derivatives were evaluated using the nucleoside forms of the inhibitors. 5-Fluoro-UMP was a very weak inhibitor of ODCase. 6-Azido-5-fluoro and 5-fluoro-6-iodo derivatives are covalent inhibitors of ODCase, and the active site Lys145 residue covalently binds to the ligand after the elimination of the 6-substitution. Among the synthesized nucleoside derivatives, 6-azido-5-fluoro, 6-amino-5-fluoro, and 6-carbaldehyde-5-fluoro derivatives showed potent anticancer activities in cell-based assays against various leukemia cell lines. On the basis of the overall profile, 6-azido-5-fluoro and 6-amino-5-fluoro uridine derivatives exhibited potential for further investigations.

Interleukin-21 regulates expression of key Epstein-Barr virus oncoproteins, EBNA2 and LMP1, in infected human B cells.

Epstein-Barr virus (EBV) persists for the life of the host by accessing the long-lived memory B cell pool. It has been proposed that EBV uses different combinations of viral proteins, known as latency types, to drive infected B cells to make the transition from resting B cells to memory cells. This process is normally antigen-driven. A major unresolved question is what factors coordinate expression of EBV latency proteins. We have recently described novel type III latency EBV+ B cell lines (OCI-BCLs) that were induced to differentiate into late plasmablasts/early plasma cells in culture with interleukin-21 (IL-21), mimicking normal B cell development. The objective of this study was to determine whether IL-21-mediated signals also regulate the expression of key EBV latent proteins during this window of development. Here we show that IL-21-reduced gene and protein expression of growth-transforming EBV nuclear antigen 2 (EBNA2) in OCI-BCLs. By contrast, the expression of CD40-like, latent membrane protein 1 (LMP1) strongly increased in these cells suggesting an EBNA2-independent mode of regulation. Same results were also observed in Burkitt's lymphoma line Jijoye and B95-8 transformed lymphoblastoid cell lines. The effect of IL-21 on EBNA2 and LMP1 expression was attenuated by a pharmacological JAK inhibitor indicating involvement of JAK/STAT signalling in this process. Our study also shows that IL-21 induced transcription of ebna1 from the viral Q promoter (Qp).

Identification of cellular intermediates and molecular pathways induced by IL-21 in human B cells.

The complex process of B cell development is controlled by multiple factors from the surrounding microenvironment including cytokines. IL-21 is a recently identified type I cytokine, mainly produced by activated CD4(+) T cells. It has been shown to promote differentiation of human primary B cells into Ig-secreting plasma cells. The objective of our study was to describe cellular intermediates that exist during IL-21-induced transition from an activated B cell to an Ig-secreting cell and to identify molecular mechanisms involved in this process. Novel Epstein-Barr Virus-positive human B cell lines with phenotypes characteristic of Ag-activated IgG(+) B cell blasts were used as a model system to study IL-21 effects in vitro. We show that IL-21 increased both proliferation and survival of B cell lines during the first 3 days of in vitro culture. This process was associated with CD38(low/int)CD23(int)HLA-DR(high)CD19(high)CD20(int) cell surface phenotype. Continued culture with IL-21 resulted in accumulation of cells in G(0)/G(1) stage of the cell cycle and increased apoptosis. This coincided with differentiation into small, CD38(high)CD23(low/-)HLA-DR(int)CD19(int)CD20(low) late plasmablasts/early plasma cells that expressed lower levels of c-Myc protein, and secreted greater amounts of Ig than the control cells. Partial inhibition of IL-21-induced JAK/STAT signaling by the low-dose pharmacological agent, JAK inhibitor I, did not prevent the initial increase in proliferation. However, decrease in c-Myc protein expression and subsequent differentiation to late plasmablasts/early plasma cells were strongly inhibited. Our study is the first to show the link between IL-21-induced JAK/STAT signaling, c-Myc regulation, and differentiation of human B cells.