Contributions of the T Cell Receptor–associated CD3γ–ITAM to Thymocyte Selection

The immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 chains associated with the T cell receptor (TCR) are crucial for TCR signaling. To probe the role of the CD3γ–ITAM in T cell development, we created knock-in mice in which the CD3γ chain of the TCR complex is replaced by a mutant signaling-deficient CD3γ chain, lacking the CD3γ–ITAM. This mutation results in considerable impairment in positive selection in the polyclonal TCR repertoire. When CD3γ–ΔITAM mice are crossed to mice expressing transgenic F5 TCRs, their thymocytes are completely unable to perform positive selection in vivo in response to intrathymic ligands. Also, the in vitro positive selection response of double-positive (DP) thymocytes with F5–CD3γ–ΔITAM mutant receptors to their agonist ligand and many of its variants is severely impaired or abrogated. Yet, the binding and dissociation constants of agonist ligands for the F5 receptor are not affected by the CD3γ–ΔITAM mutation. Furthermore, DP thymocytes with mutant receptors can respond to agonist ligand with normal antigen sensitivity and to normal levels, as shown by their ability to induce CD69 up-regulation, TCR down-regulation, negative selection, and ZAP70 and c-Jun NH2-terminal kinase activation. In sharp contrast, induction of extracellular signal-regulated kinase (ERK) activation and linker for activation of T cells (LAT) phosphorylation are severely impaired in these cells. Together, these findings underscore that intrinsic properties of the TCR–CD3 complex regulate selection at the DP checkpoint. More importantly, this analysis provides the first direct genetic evidence for a role of the CD3γ–ITAM in TCR-driven thymocyte selection

Immunomodulation of murine collagen-induced arthritis by N, N-dimethylglycine and a preparation of Perna canaliculus

<p>Abstract</p> <p>Background</p> <p>Rheumatoid arthritis (RA) and its accepted animal model, murine collagen-induced arthritis (CIA), are classic autoimmune inflammatory diseases which require proinflammatory cytokine production for pathogenesis. We and others have previously used N, N-dimethylglycine (DMG) and extracts from the New Zealand green-lipped mussel <it>Perna canaliculus </it>(Perna) as potent immunomodulators to modify ongoing immune and/or inflammatory responses.</p> <p>Methods</p> <p>In our initial studies, we treated lipopolysaccahride (LPS) stimulated THP-1 monocytes <it>in vitro </it>with increasing concentrations of Perna extract or DMG. Additionally, we treated rat peripheral blood neutrophils with increasing concentrations of Perna extract and measured superoxide burst. In subsequent <it>in vivo </it>experiments, CIA was induced by administration of type II collagen; rats were prophylactically treated with either Perna or DMG, and then followed for disease severity. Finally, to test whether Perna and/or DMG could block or inhibit an ongoing pathologic disease process, we induced CIA in mice and treated them therapeutically with either of the two immunomodulators.</p> <p>Results</p> <p>Following LPS stimulation of THP-1 monocytes, we observed dose-dependent reductions in TNF-α and IL-12p40 production in Perna treated cultures. DMG treatment, however, showed significant increases in both of these cytokines in the range of 0.001–1 μM. We also demonstrate that <it>in vitro </it>neutrophil superoxide burst activity is dose-dependently reduced in the presence of Perna. Significant reductions in disease incidence, onset, and severity of CIA in rats were noted following prophylactic treatment with either of the two immunomodulators. More importantly, amelioration of mouse CIA was observed following therapeutic administration of Perna. In contrast, DMG appeared to have little effect in mice and may act in a species-specific manner.</p> <p>Conclusion</p> <p>These data suggest that Perna, and perhaps DMG, may be useful supplements to the treatment of RA in humans.</p

A consolidated approach to analyzing data from high-throughput protein microarrays with an application to immune response profiling in humans

Abstract Motivation: DNA microarrays are a well known and established technology in biological and pharmaceutical research providing a wealth of information essential for understanding biological processes and aiding drug development. Protein microarrays are quickly emerging as a follow up technology, which will also begin to experience rapid growth as the challenges in protein to spot methodologies are overcome. Like DNA microarrays, their protein counterparts produce large amounts of data that must be suitably analyzed in order to yield meaningful information that should eventually lead to novel drug targets and biomarkers. Although the statistical management of DNA microarray data has been well described, there is no available report which offers a successful consolidated approach to the analysis of high throughput protein microarray data. We describe the novel application of a statistical methodology to analyze the data from an immune response profiling assay using human protein microarray with over 5000 proteins on each chip

Perna and DMG reduce IgM (panel A), but not IgG (panel B) anti-type II collagen antibody levels

<p><b>Copyright information:</b></p><p>Taken from "Immunomodulation of murine collagen-induced arthritis by N, N-dimethylglycine and a preparation of "</p><p>http://www.biomedcentral.com/1472-6882/7/20</p><p>BMC Complementary and Alternative Medicine 2007;7():20-20.</p><p>Published online 11 Jun 2007</p><p>PMCID:PMC1899520.</p><p></p> Rats were untreated, treated with Perna, DMG, or the combination of the two. Anti-collagen levels were analyzed from serum by ELISA. * indicates a p-value < 0.05

Perna extract inhibits superoxide production and burst activity by neutrophils

<p><b>Copyright information:</b></p><p>Taken from "Immunomodulation of murine collagen-induced arthritis by N, N-dimethylglycine and a preparation of "</p><p>http://www.biomedcentral.com/1472-6882/7/20</p><p>BMC Complementary and Alternative Medicine 2007;7():20-20.</p><p>Published online 11 Jun 2007</p><p>PMCID:PMC1899520.</p><p></p> Rat neutrophils were isolated from peripheral blood and stimulated with PMA (50 ng/ml) in the presence of increasing concentrations of Perna extract. Percent inhibition was calculated by comparing Perna extracts with untreated cells. * indicates a p-value < 0.05