Analytical Characterization of an Enzyme-Linked Immunosorbent Assay for the Measurement of Transforming Growth Factor ß1 in Human Plasma.

BACKGROUND: The transforming growth factor ß (TGF-ß)-signaling pathway has emerged as a promising therapeutic target for many disease states including hepatocellular carcinoma (HCC). Because of the pleiotropic effects of this pathway, patient selection and monitoring may be important. TGF-ß1 is the most prevalent isoform, and an assay to measure plasma levels of TGF-ß1 would provide a rational biomarker to assist with patient selection. Therefore, the objective of this study was to analytically validate a colorimetric ELISA for the quantification of TGF-ß1 in human plasma. METHODS: A colorimetric sandwich ELISA for TGF-ß1 was analytically validated per Clinical and Laboratory Standards Institute protocols by assessment of precision, linearity, interfering substances, and stability. A reference range for plasma TGF-ß1 was established for apparently healthy individuals and potential applicability was demonstrated in HCC patients. RESULTS: Precision was assessed for samples ranging from 633 to 10822 pg/mL, with total variance ranging from 28.4% to 7.2%. The assay was linear across the entire measuring range, and no interference of common blood components or similar molecules was observed. For apparently healthy individuals, the average TGF-ß1 level was 1985 ± 1488 pg/mL compared to 4243 ± 2003 pg/mL for HCC patients. Additionally, the TGF-ß1 level in plasma samples was demonstrated to be stable across all conditions tested, including multiple freeze-thaw cycles. CONCLUSIONS: The ELISA described in this report is suitable for the quantification of TGF-ß1 in human plasma and for investigational use in an approved clinical study.

Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA.

OBJECTIVES: Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. METHODS: Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. RESULTS: The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10(-4), OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10(-7), OR 0.71; case-only pmeta=1.9×10(-4), OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. CONCLUSIONS: These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications.

Massive mobilization of dendritic cells during influenza A virus subtype H5N1 infection of nonhuman primates.

Highly pathogenic avian influenza virus infection is characterized by a marked inflammatory response, but the impact of infection on dendritic cells (DCs) is unknown. We show that influenza A virus subtype H5N1 infection rapidly and profoundly impacts DCs in cynomolgus macaques, increasing the number of blood myeloid and plasmacytoid DCs by 16- and 60-fold, respectively. Infection was associated with recruitment, activation, and apoptosis of DCs in lung-draining lymph nodes; granulocyte and macrophage infiltration in lungs was also detected, together with expression of CXCL10. This degree of DC mobilization is unprecedented in viral infection and suggests a potential role for DCs in the pathogenesis of highly pathogenic avian influenza virus.