MHC class I–deficient natural killer cells acquire a licensed phenotype after transfer into an MHC class I–sufficient environment

In MHC class I–deficient hosts, natural killer (NK) cells are hyporesponsive to cross-linking of activation receptors. Functional competence requires engagement of a self–major histocompatability complex (MHC) class I–specific inhibitory receptor, a process referred to as “licensing.” We previously suggested that licensing is developmentally determined in the bone marrow. In this study, we find that unlicensed mature MHC class I–deficient splenic NK cells show gain-of-function and acquire a licensed phenotype after adoptive transfer into wild-type (WT) hosts. Transferred NK cells produce WT levels of interferon-γ after engagement of multiple activation receptors, and degranulate at levels equivalent to WT NK cells upon coincubation with target cells. Only NK cells expressing an inhibitory Ly49 receptor specific for a cognate host MHC class I molecule show this gain-of-function. Therefore, these findings, which may be relevant to clinical bone marrow transplantation, suggest that neither exposure to MHC class I ligands during NK development in the BM nor endogenous MHC class I expression by NK cells themselves is absolutely required for licensing

Ocean and coastal acidification off New England and Nova Scotia

Author Posting. © The Oceanography Society, 2015. This article is posted here by permission of The Oceanography Society for personal use, not for redistribution. The definitive version was published in Oceanography 28, no. 2 (2015): 182-197, doi:10.5670/oceanog.2015.41.New England coastal and adjacent Nova Scotia shelf waters have a reduced buffering capacity because of significant freshwater input, making the region’s waters potentially more vulnerable to coastal acidification. Nutrient loading and heavy precipitation events further acidify the region’s poorly buffered coastal waters. Despite the apparent vulnerability of these waters, and fisheries’ and mariculture’s significant dependence on calcifying species, the community lacks the ability to confidently predict how the region’s ecosystems will respond to continued ocean and coastal acidification. Here, we discuss ocean and coastal acidification processes specific to New England coastal and Nova Scotia shelf waters and review current understanding of the biological consequences most relevant to the region. We also identify key research and monitoring needs to be addressed and highlight existing capacities that should be leveraged to advance a regional understanding of ocean and coastal acidification.This project was supported in part by an appointment to the Internship/Research Participation Program at the Office of Water, US Environmental Protection Agency (EPA), administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the EPA. JS acknowledges support from NASA grant from NNX14AL84G NASA-CCS

t10c12 Conjugated Linoleic Acid Suppresses HER2 Protein and Enhances Apoptosis in SKBr3 Breast Cancer Cells: Possible Role of COX2

BACKGROUND: HER2-targeted therapy with the monoclonal antibody trastuzumab (Herceptin) has improved disease-free survival for women diagnosed with HER2-positive breast cancers; however, treatment resistance and disease progression are not uncommon. Current data suggest that resistance to treatment in HER2 cancers may be a consequence of NF-kappaB overexpression and increased COX2-derived prostaglandin E2 (PGE(2)). Conjugated linoleic acid (CLA) has been shown to have anti-tumor properties and to inhibit NF-kappaB activity and COX2. METHODS: In this study, HER2-overexpressing SKBr3 breast cancer cells were treated with t10c12 CLA. Protein expression of the HER2 receptor, nuclear NF-kappaB p65, and total and phosphorylated IkappaB were examined by western blot and immunofluorescence. PGE(2) levels were determined by ELISA. Proliferation was measured by metabolism of 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and apoptosis was measured by FITC-conjugated Annexin V staining and flow cytometry. RESULTS/CONCLUSIONS: We observed a significant decrease in HER2 protein expression on western blot following treatment with 40 and 80 microM t10c12 CLA (p<0.01 and 0.001, respectively) and loss of HER2 protein in cells using immunoflourescence that was most pronounced at 80 microM. Protein levels of nuclear NF-kappaB p65 were also significantly reduced at the 80 microM dose. This was accompanied by a significant decrease in PGE(2) levels (p = 0.05). Pretreatment with t10c12 CLA significantly enhanced TNFalpha-induced apoptosis and the anti-proliferative action of trastuzumab (p = 0.05 and 0.001, respectively). These data add to previous reports of an anti-tumor effect of t10c12 CLA and suggest an effect on the HER2 oncogene that may be through CLA mediated downregulation of COX2-derived PGE(2)

Signaling in natural killer cells: SHIP, 2B4 and the Kinome

The NK cell is a large granular lymphocyte that plays a key role in protecting the body against numerous pathogens including parasites, intracellular bacteria, viral infections, as well as showing anti-tumor activity and playing a role in the rejection of allogeneic BM. Unlike other lymphocytic cell types, that utilize rearranging receptors, NK cells are regulated by a complex array of germ line encoded activating and inhibitory receptors. NK cells are often described as a front line or rapid defense given their response to stimuli can be immediate, although they also maintain functions that extend their role well into the adaptive immune system. Inhibitory receptors that recognize MHC class I molecules regulate NK cell responses and self-tolerance. Recent evidence indicates self-ligands not present in the MHC locus can also modulate NK function. We previously demonstrated that the NK receptor repertoire is disrupted by SHIP-deficiency. Here we show that an inhibitory receptor, 2B4, that recognizes an MHC-independent ligand is over expressed in NK cells of SHIP-/- mice at all stages of NK development and differentiation. Overexpression of 2B4 compromises key cytolytic NK functions, including killing of allogeneic, tumor and viral targets. These results demonstrate that in SHIP-/- NK cell 2B4 is the dominant inhibitory receptor. We then furthered this finding by examining the molecular basis of 2B4 dominance. We show that in SHIP-/- NK cells there is increased 2B4 expression as well as a strong bias towards the 2B4L isoform. We have also identified a greater than tenfold increase in SHP1 recruitment to 2B4. Consistent with this SHP1 over recruitment,both a broad and a selective SHP1 inhibitor restore SHIP-/- NK killing of complex targets.Through this study we have identified the molecular mechanism of 2B4 receptor dominance as SHP1 over-recruitment.In addition we have utilized protein array technology to explore NK signaling through the determination of the NK kinome. To this end we have been able to identify multiple pathways that may mark crucial differences between the mature and immature NK cell

Comparison of methods for alcohol and drug screening in primary care clinics.

Question: How are commonly used screening methods for alcohol and drug use associated with implementation outcomes among adult patients in primary care clinics, and what is the best approach for implementing electronic health record–integrated screening? Findings: In this quality improvement study implementing systematic screening for alcohol and drug use among 93 114 patients in 6 primary care clinics, 72% of patients completed screening. Screening at any visit (in comparison with screening at annual examinations only) was associated with higher screening rates for alcohol and drug use, and self-administered screening was associated with greater detection of moderate- to high-risk alcohol use compared with staff-administered screening. Meaning: These findings suggest that, to maximize the adoption of substance use screening during primary care visits, clinics can conduct screening at any visit and use self-administered screening tools to increase the detection of unhealthy alcohol use among adult patients

Major remodelling of the murine stem cell kinome following differentiation in the hematopoietic compartment

The changes in signal transduction associated with the acquisition of specific cell fates remain poorly understood. We performed massive parallel assessment of kinase signatures of the radiations of the hematopoietic system, including long-term repopulating hematopoietic stem cells (LT-HSC), short-term repopulating HSC (ST-HSC), immature natural killer (iNK) cells, NK cells, B cells, T cells, and myeloid cells. The LT-HSC kinome is characterized by noncanonical Wnt, Ca2+and classical protein kinase C (PKC)-driven signaling, which is lost upon the transition to ST-HSC, whose kinome signature prominently features receptor tyrosine kinase (RTK) activation of the Ras/MAPK signaling cassette. Further differentiation to iNK maintains signaling through this cassette but simultaneously leads to activation of a PI3K/PKB/Rac signaling, which becomes the dominant trait in the kinase signature following full differentiation toward NK cells. Differentiation along the myeloid and B cell lineages is accompanied by hyperactivation of both the Ras/MAPK and PI3K/PKB/Rac signaling cassette. T cells, however, deactivate signaling and only display residual G protein-coupled pathways. Thus, differentiation along the hematopoietic lineage is associated with major remodelling of cellular kinase signature