Whole-blood proliferation assay for autoimmune thyroid disease: comparison to density-gradient separated-peripheral blood lymphocytes.

Autoimmune thyroid diseases feature prominent cellular infiltration of the thyroid gland as well as autoantibody production to thyroid antigens. The most common assay to evaluate cell-mediated immunity is based on incorporation of tritiated thymidine into proliferating T cells after stimulation by the test antigens. In the past, cell proliferation assays of thyroglobulin (Tg) using peripheral blood mononuclear cells (PBMC) of individuals with autoimmune thyroid diseases required large quantities of blood and specialized separation techniques, and have not yielded high counts or high stimulation indices. We therefore developed a proliferation assay using less than 5 mL of whole blood and compared proliferation of cells in whole blood to that using PBMCs separated by density gradient centrifugation. We also determined if responses could be enhanced by addition of interleukin-2 (IL-2) to the cultures. We found that an IL-2-stimulated proliferation assay to Tg using diluted whole blood is superior to the separated cell assay in detecting Tg-specific T-cell proliferation in autoimmune thyroid disease patients. Further refinement of this technique and larger trials may confirm its value for clinical investigation and special diagnostic applications.

The therapeutic action of monoclonal antibodies against a surface glycoprotein of Giardia muris.

Twenty-three monoclonal antibodies (mAbs) against Giardia muris were obtained in two fusions using spleen cells of immunized mice. Similarly, the fusion of mesenteric lymph node (MLN) cells of an infected mouse yielded 15 mAbs. Three mAbs arising from spleen cells and two from MLN (all five were IgM) were able to kill trophozoites in the presence of guinea-pig complement (GPC). They can agglutinate trophozoites and also impair the movement of the flagella in the absence of GPC. I.p. treatment of mice with mAb 45C on Days -1, 1, 3 and 5 of infection significantly reduced the intestinal parasite burden. Indirect immunofluorescence assays on live and fixed trophozoites revealed that the cytotoxic mAbs were directed against antigens located on the periphery of the body, the sucking disc, the flagella and the ventro-lateral flange. In Western blots, our mAbs recognized major 36,200 and 30,300 MW glycoproteins located on the surface of the parasite.

Coordinate transactivation of the interleukin-2 CD28 response element by c-Rel and ATF-1/CREB2.

The interleukin-2 CD28 response element (CD28RE) acts as a composite enhancer, in conjunction with a 3'-12-O-tetradecanoylphorbol-13-acetate response element (TRE)-like element, to confer CD28 receptor-dependent inducibility to the interleukin-2 promoter in T-cells. When inserted as a single copy upstream of a basal promoter, this composite enhancer, termed the CD28RE-TRE, is both highly active and CD28-inducible in transactivation assays. A multicomponent nuclear protein complex that binds the CD28RE-TRE was isolated by DNA affinity chromatography from nuclear extracts of mitogen- and CD28 receptor-costimulated human T-cells. Immunological and biochemical analyses of this complex reveal the presence of c-Rel, ATF-1, and CREB2 as major DNA-binding components. Coexpression of c-Rel in combination with ATF-1, CREB2, or ATF-1/CREB2 leads to synergistic transactivation of a CD28RE-TRE reporter plasmid in quiescent Jurkat T-cells. Furthermore, CD28-dependent transactivation of the CD28RE-TRE is specifically inhibited by cAMP response element-binding protein (CREB) dominant-negative expression vectors. Moreover, mutant promoter constructs in which the internal 5'-CD28RE and 3'-TRE-like sequences have been topologically positioned 180 degrees out of phase with one another show loss of mitogen- and CD28-dependent inducibility. Finally, the addition of the CREB-binding transcriptional coactivator p300 leads to a dramatic CREB-dependent increase in both mitogen- and CD28-mediated transactivation of the CD28RE-TRE. These findings demonstrate that full physiological responsiveness to CD28 receptor stimulation in T-cells is dependent on topologically linked sequences within the CD28RE-TRE composite enhancer and provide strong support of a direct role for the CREB family of transcription factors and p300/CREB-binding protein coactivator proteins in cytokine gene induction during T-cell activation.

Targeting of p300 to the interleukin-2 promoter via CREB-Rel cross-talk during mitogen and oncogenic molecular signaling in activated T-cells.

In this report, we explore the mechanisms of targeting of p300 to the interleukin-2 (IL-2) promoter in response to mitogenic and oncogenic molecular signals. Recruitment of p300 by cAMP-responsive element-binding protein-Rel cross-talk at the composite CD28 response element (CD28RE)-TRE element of the IL-2 promoter is essential for promoter inducibility during T-cell activation, and CD28RE-TRE is the exclusive target of the human T-cell lymphotropic virus type I oncoprotein Tax. The intrinsic histone acetyltransferase activity of p300 is dispensable for activation of the IL-2 promoter, and the N-terminal 743 residues contain the minimal structural requirements for synergistic transactivation of the CD28RE-TRE, the IL-2 promoter, and endogenous IL-2 gene expression. Mutational analysis of p300 reveals differential structural requirements for the N-terminal p300 module by individual cis-elements within the IL-2 promoter. These findings provide evidence that p300 assembles at the IL-2 promoter to form an enhanceosome-like signal transduction target that is centrally integrated at the CD28RE-TRE element of the IL-2 promoter through specific protein module-targeted associations in activated T-cells.

Immunoreactivity of multiple molecular forms of human thyroglobulin.

Human thyroglobulin (Tg) was purified from thyroids of normal individuals and of patients with Graves' disease using gel filtration (Sephacryl S-400) and ion-exchange (DEAE) column chromatography. We isolated five protein peaks of Tg from the DEAE column, using a step gradient, characterized them for protein and iodine content, and assessed their immunological properties by reactivity to polyclonal and monoclonal antibodies (mAbs). Normal and Graves' Tgs differed in the relative protein content of these five protein peaks from the DEAE column. In the case of normal Tg, the majority of Tg was eluted in peak 2 but in the Graves' Tg, most of the protein was eluted in peak 1 of this column. The immunoreactivity of these five protein peaks of Tg was studied using 11 mouse mAbs prepared against human Tg, sera from patients with autoimmune thyroiditis and polyclonal antibody from a rabbit immunized with human Tg. All of five protein peaks of Tg reacted equally with rabbit antibody. The sera of five thyroiditis patients showed greater binding to peak 1 of Graves' Tg than peak 1 of normal Tg. Similarly, most mAbs showed greater binding to peak 1 of Graves' Tg than the peak 1 of normal Tg. Of particular interest was one mAb (42C3) which reacted only with Tgs containing iodine. The immunoreactivity of this mAb paralleled the iodine content of Tg. This mAb might be useful for evaluating the role of iodine in the antigenicity of human Tg.