Genome-Wide Association Study Identifies Genetic Loci Associated with Iron Deficiency

The existence of multiple inherited disorders of iron metabolism in man, rodents and other vertebrates suggests genetic contributions to iron deficiency. To identify new genomic locations associated with iron deficiency, a genome-wide association study (GWAS) was performed using DNA collected from white men aged ≥25 y and women ≥50 y in the Hemochromatosis and Iron Overload Screening (HEIRS) Study with serum ferritin (SF) ≤ 12 µg/L (cases) and iron replete controls (SF>100 µg/L in men, SF>50 µg/L in women). Regression analysis was used to examine the association between case-control status (336 cases, 343 controls) and quantitative serum iron measures and 331,060 single nucleotide polymorphism (SNP) genotypes, with replication analyses performed in a sample of 71 cases and 161 controls from a population of white male and female veterans screened at a US Veterans Affairs (VA) medical center. Five SNPs identified in the GWAS met genome-wide statistical significance for association with at least one iron measure, rs2698530 on chr. 2p14; rs3811647 on chr. 3q22, a known SNP in the transferrin (TF) gene region; rs1800562 on chr. 6p22, the C282Y mutation in the HFE gene; rs7787204 on chr. 7p21; and rs987710 on chr. 22q11 (GWAS observed P<1.51×10−7 for all). An association between total iron binding capacity and SNP rs3811647 in the TF gene (GWAS observed P = 7.0×10−9, corrected P = 0.012) was replicated within the VA samples (observed P = 0.012). Associations with the C282Y mutation in the HFE gene also were replicated. The joint analysis of the HEIRS and VA samples revealed strong associations between rs2698530 on chr. 2p14 and iron status outcomes. These results confirm a previously-described TF polymorphism and implicate one potential new locus as a target for gene identification

Isolation and characterization of novel single-chain Fv specific for human granzyme B

Granzyme B, a neutral serine protease, has been demonstrated to be a pivotal molecule for protective immunity against viral infection and cellular malignant transformation. To facilitate monitoring of granzyme B levels, we have recently applied phage display technology to produce single-chain Fv antibodies specific for granzyme B, as versatile alternatives and complementary reagents to currently available monoclonal antibodies. Through four rounds of panning on purified human granzyme B-coated on solid phase, three unique clones were isolated. Expressed soluble scFv antibodies demonstrated specific immunological applications including ELISA, Western blotting, immunoprecipitation and intracellular staining. Based on sequence analyses and structural modeling, one scFv, Fv17, may have overlapping antigen binding specificity with monoclonal antibodies 2C5/F5 and GB11. Owing to the availability of its DNA sequence and large scale production capability, Fv17 should be a superior reagent for monitoring granzyme B expression in natural killer cells and antigen specific CD8+ T cell immunity.link_to_subscribed_fulltex

Evaluating antigen-specific cytotoxic T lymphocyte responses by a novel mouse granzyme B ELISPOT assay

We have established novel ELISA- and ELISPOT-based assays specific for the detection of a potent cytotoxic mediator, granzyme B (GrB), for the assessment of antigen-specific cytotoxic T lymphocyte responses in mice. The sensitivity and specificity of our assays was demonstrated by ELISA using purified mouse GrB and supernatants and cell lysates of cytotoxic lymphocytes derived from GrB-deficient mice. No reactivity was observed by the GrB ELISA in GrB-deficient cells. The mouse GrB ELISPOT was successfully employed to detect antigen-specific effector cell responses of two CTL clones, producing GrB ELISPOT results that correlated strongly with target cell lysis, as assessed by 51Cr-release. Furthermore, we were able to demonstrate direct correlations between GrB ELISPOT and killing by LCMV gp33-specific effector and memory T cells generated in vivo. Thus, the mouse GrB ELISPOT may be used to detect cytotoxic responses, at the single-cell level, for the functional assessment of antigen-specific CD8+ T cell responses in mouse models of infection. © 2005 Elsevier B.V. All rights reserved.link_to_subscribed_fulltex