David Sehy

Previous studies have implicated T cell production of IL-17 in resistance to Toxoplasma gondii as well as the development of immune-mediated pathology during this infection. Analysis of C57BL/6 and C57BL/6 RAG(-/-) mice challenged with T. gondii-identified NK cells as a major innate source of IL-17. The ability of soluble Toxoplasma Ag to stimulate NK cells to produce IL-17 was dependent on the presence of accessory cells and the production of IL-6, IL-23, and TGF-beta. In contrast, these events were inhibited by IL-2, IL-15, and IL-27. Given that IL-6 was one of the most potent enhancers of NK cell production of IL-17, further studies revealed that only a subset of NK cells expressed both chains of the IL-6R, IL-6 upregulated expression of the Th17-associated transcription factor RORgammat, and that IL-6(-/-) mice challenged with T. gondii had a major defect in NK cell production of IL-17. Together, these data indicate that many of the same cytokines that regulate Th17 cells are part of a conserved pathway that also control innate production of IL-17 and identify a major role for IL-6 in the regulation of NK cell responses.

In this work we introduce a novel microfluidic enzyme linked immunoassays (ELISA) microplate as the next generation assay platform for unparalleled assay performances. A combination of microfluidic technology with standard SBS-configured 96-well microplate architecture, in the form of microfluidic microplate technology, allows for the improvement of ELISA workflows, conservation of samples and reagents, improved reaction kinetics, and the ability to improve the sensitivity of the assay by multiple analyte loading. This paper presents the design and characterization of the microfluidic microplate, and its application in ELISA.

In studies of the regulation of hematopoiesis, increasing attention has focused on the role of bone marrow stromal cells as rich sources of various cytokines. Present studies indicate that marrow stromal cells and monocytes produce activin A, implicating this new cytokine in the paracrine control of hematopoiesis. Activin A, which was initially recognized as a beta A beta A dimeric gonadal protein, was found to potentiate the proliferation and differentiation of erythroid progenitors; both purified erythroid colony-forming units (CFU-E) and K562 cells possess high affinity receptors specific for activin A. Present studies using Western and Northern blots demonstrate the presence of beta A subunits of activin A in the conditioned medium of monocytes and stromal cells and its RNA transcripts in these cells. The presence of functional and homodimeric beta A beta A activin molecule was confirmed through bioassay with or without a blocking antiserum against activin A or an activin binding protein, follistatin; its presence is further supported by a specific enzyme-linked immunosorbent assay (ELISA) in which a monoclonal antibody reacted only with the beta A beta A dimeric form of this molecule. In other experiments, the production of activin A was found to be regulated by various cytokines and regulators. The production of activin A in monocytes was stimulated more than ninefold by treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). Activin A expression was also stimulated, albeit less potently, by bacterial lipopolysaccharide (LPS) and gamma-interferon. On the other hand, the expression of activin A in marrow stromal cells was upregulated by incubation with tumor necrosis factor-alpha (TNF-alpha), LPS, and interleukin 1 alpha (IL-1 alpha). Therefore, we propose that the local production of activin A in the microenvironment within bone marrow may fine tune the regulation of steady-state hematopoiesis. In addition, this factor may normally be produced at minimal levels, but under certain situations may be further induced to provide important biological functions.