Making SharePoint^®Chemically Aware™

Abstract Background The use of SharePoint® collaboration software for content management has become a critical part of today's drug discovery process. SharePoint 2010 software has laid a foundation which enables researchers to collaborate and search on various contents. The amount of data generated during a transition of a single compound from preclinical discovery to commercialization can easily range in terabytes, thus there is a greater demand of a chemically aware search algorithm that supplements SharePoint which enables researchers to query for information in a more intuitive and effective way. Thus by supplementing SharePoint with Chemically Aware™ features provides a great value to the pharmaceutical and biotech companies and makes drug discovery more efficient. Using several tools we have integrated SharePoint with chemical, compound, and reaction databases, thereby improving the traditional search engine capability and enhancing the user experience. Results This paper describes the implementation of a Chemically Aware™ system to supplement SharePoint. A Chemically Aware SharePoint (CASP) allows users to tag documents by drawing a structure and associating it with the related content. It also allows the user to search SharePoint software content and internal/external databases by carrying out substructure, similarity, SMILES, and IUPAC name searches. Building on traditional search, CASP takes SharePoint one step further by providing a intuitive GUI to the researchers to base their search on their knowledge of chemistry than textual search. CASP also provides a way to integrate with other systems, for example a researcher can perform a sub-structure search on pdf documents with embedded molecular entities. Conclusion A Chemically Aware™ system supplementing SharePoint is a step towards making drug discovery process more efficient and also helps researchers to search for information in a more intuitive way. It also helps the researchers to find information which was once difficult to find by allowing one to tag documents with molecular entities and integrating with image recognition software to find information from pdf documents.</p

The Activating KIR Genes 2DS1 and 2DS2 Regulate NK Alloreactivity In Vitro.

Abstract The role of Natural Killer (NK) cells in host protection against viral infection and malignant transformation has been well described. NK cells may also lead to a reduction in post-transplant relapse and improved survival in hematopoietic stem cell transplantation (HSCT) for acute myelogenous leukemia (AML). It has been hypothesized that the genotype for the inhibiting killer immunoglobulin-like receptor (KIR) of the hematopoietic stem cell donor in combination with the HLA class I genotype of the recipient could control NK alloreactivity leading to a reduction in post-transplant complications. The KIR gene family encodes however both activating and inhibiting receptors. Here we test the hypothesis that activating KIRs with ligand specificity for HLA class I may contribute to alloreactivity, and potentially could be a genetic factor of significance in allogeneic HSCT. We tested this hypothesis in studies of two pairs of inhibiting and activating KIRs with highly homologous codon sequences in the extracellular domain, namely KIR2DL2/3-KIR2DS2 and KIR2DL1-KIR2DS1. Both the inhibitory 2DL1 and activating 2DS1 have ligand specificity for HLA-Cw group 2, and 2DL2 and 2DL3, have ligand specificity for HLA-Cw group 1, while the activating 2DS2 does not bind in vitro to C1 group. Using an EBV-transformed B-lymphoblastoid cell line (EBV-BLCL) target cell panel homozygous for HLA Class I alleles, we found that NK cells from donors with KIR haplotypes lacking KIR2DS1 or 2DS2 were not cytotoxic to allogeneic EBV-BLCL, independent of the target HLA class I genotype. Polyclonal NK cells obtained from KIR2DS1 positive and C1 group positive donors mediated NK cytotoxicity against C2 positive targets. In contrast, NK cells from KIR2DS1 positive, C2 group homozygous donors displayed minimal cytotoxicity against the C2 group targets (p&amp;lt;0.01). NK clones generated from 2DS1 positive, C2-group negative individuals were cytotoxic to C2-group target cells, while such NK clones could not be obtained from individuals positive for 2DS1 and cognate ligands. Similar findings were made for the relationship between 2DS2, 2DL2/3 and cognate ligand C1 group. Both polyclonal IL-2 propagated NK cells and NK clones from individuals positive for 2DS2 and homozygous for C2 group displayed specific cytotoxicity against C1 positive target cells. The cytotoxicity of 2DS2 positive, C1 group positive NK cells against the C1 positive BLCLs was minimal (p&amp;lt;0.01). These studies demonstrate that 2DS1 and 2DS2 are activating receptors that can induce an alloantigen response. We also present a model for combinations of KIR and HLA genotypes in which the allogeneic function of KIR2DS1 and 2DS2 is consistently seen in donor NK cells. Activating KIR may therefore play a role in allogeneic HSCT, and could contribute to the balance between activating and inhibiting signals for NK cells in HLA-Cw incompatible donor-recipient combinations. Activating KIR interactions with cognate ligand could potentially also play a role in the innate immune response. In the normal host, the increased affinity of the inhibiting KIR isoforms for HLA class I may prevent auto-reactivity, while the activating isoforms may only function in an HLA restricted pattern in context of specific pathogens or transformed cells. It is possible that the low affinity activating KIR may require additional co-stimulating signals that are up-regulated during cellular stress.</jats:p

Th1 memory cells induce lethal allogeneic disease in recipient bone marrow and spleen (169.43)

Abstract Graft versus host disease (GVHD) represents a major cause of morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (HSCT). We sought to determine if Th1 memory CD4 cells were capable of inducing disease in allogeneic recipients. Studies were performed using the murine MHC Class II-disparate HSCT model, C57BL/6 (B6) to B6.C-H-2bm12 (B6→bm12). Initial studies using naïve CD4 cells in the B6→bm12 model revealed bone marrow aplasia present in diseased mice, in addition to classic GVHD tissue findings. FACS CD4 phenotyping of the transferred cells isolated directly from diseased tissues revealed a marked increase in IFN-γ in the allogeneic mice compared to syngeneic recipients. Using a previously described transgenic Ifng reporter mouse containing the Thy1.1 gene knocked in-frame into the first exon of the Ifng gene, we were able to isolate IFN-γ-producing Th1 cells with magnetic bead separation. Adoptive transfer studies of allogeneic Thy1.1-positive cells into bm12 allogeneic mice resulted in fatality in a dose-dependent fashion. FACS phenotyping of Thy1.1-expressing cells in mice demonstrating illness revealed persistence of the Th1 phenotype and tissue histology showed severe disease of bone marrow and spleen in the absence of classic GVHD findings. Serial blood counts confirmed decreases in all hematologic parameters in allogeneic Th1 recipients. These results have important implications for future studies in graft manipulation for human HSCT.</jats:p

In Vitro Polarized Th17 CD4+ T Cells Home Primarily to Sites of Initial Antigen Encounter

Abstract The Th1 and Th2 lineages of CD4+ T helper cells are essential for control of host infection. Both lineages respond to antigenic stimulation with distinct effector functions and cytokine profiles. Differential homing patterns permit localization within specific tissue sites where these cells interact with other immune cells to promote the immune response. Variability in T helper lineage homing is due, in part, to differing chemokine receptor expression patterns. This laboratory and others recently described another CD4+ T helper lineage, Th17. Following stimulation, Th17 cells also produce a unique cytokine profile, including interleukin (IL)-17, IL-21, and IL-22. The Th17 lineage has now been implicated in the pathogenesis of several human autoimmune diseases, including psoriasis and inflammatory bowel disease, and appears to be critical for the inflammation of both the skin and gastrointestinal tract, respectively, seen in these diseases. It is not well understood whether Th17 cells arise within the inflammatory milieu in these tissues, or whether these cells possess a distinct homing pattern. We have performed studies using in vitro polarized Th17 cells for the study of tissue homing patterns in vivo. Experiments were performed using the well-described HLA Class II-disparate C57BL/6 (B6) to B6.C-H-2bm12 (bm12) model. Previous studies have established CD4+ T cell-dependent inflammation in this model. Naïve CD4+ T cells from B6 mice were polarized to the Th17 lineage in vitro using standard techniques, including IL-6 and TGF-β. FACS analysis of the Th17 cells prior to adoptive transfer revealed IL-17-positive staining in &amp;gt;60% cells and IFN-γ-positivity in &amp;lt;10%. Th17 or Th2-polarized control cells (1 × 106) were transferred into lethally irradiated bm12 mice (or syngeneic B6 control mice). Mice receiving Th17 cells demonstrated weight gain in the initial weeks compared to Th2 control recipients, but less than B6 syngeneic recipients. The Th17 recipients appeared less active, however, and most mice in this group eventually became moribund, requiring euthanasia. Complete necropsy was performed on mice from each group at intervals following transfer. Tissue analysis in the Th17 recipients revealed marked inflammation within the lungs, skin, liver, and gastrointestinal tract. Syngeneic B6 recipients of Th17 cells also demonstrated a similar tissue pattern, but with markedly reduced inflammation. Tissues from the bm12 Th2-polarized cell control mice, as well as T cell depleted marrow alone recipients did not demonstrate significant inflammation. Additional time course experiments revealed the initial target organs affected as the lungs and stomach, with subsequent involvement of other affected organs. FACS analysis of recipient hematopoietic tissues, using CD45.1 isotype distinction, revealed Th17 cell proliferation within the bm12 allogeneic recipients compared to the B6 syngeneic recipient mice (25–35% total cells of donor origin compared to 2–8%, respectively). CD4+ T cell counts performed on recipient spleens confirmed increased proliferation of Th17 cells within the allogeneic recipient compared to Th2 allogeneic and Th17 syngeneic controls (108 total donor-derived cells compared to 106 and 107, respectively). Cytokine analysis was performed by FACS on CD4+ T cells harvested from tissues. In contrast to pre-transfer analysis, the transferred CD4+ T cells harvested from allogeneic bm12 recipients secreted increased amounts of IFN-γ (12–33%) concomitant with a decrease in IL-17 production. Our studies demonstrate that Th17 CD4+ T cells are able to home to mucosal sites of early antigen encounter, in both the allogeneic and syngeneic setting. This pattern is consistent with the known role of IL-17 in innate immune response to infection. In the setting of chronic T cell stimulation, we also observed that Th17 cells can transition to a Th1-like, IFN-γ-producing CD4+ T cell. The skin, lungs, and GI tract are important sites of initial antigen encounter, and understanding the CD4+ Th17 T cell homing and proliferation patterns could have important implications in understanding both innate and adaptive immune responses to acute infection. Ongoing studies are underway to identify the role of specific chemokine receptors responsible for Th17 homing.</jats:p

Human Natural Killer Cells Achieve Tolerance by Preferentially Endowing Functional Competence to Inhibitory Killer Ig-Like Receptors Specific for Self-HLA Class I.

Abstract Natural killer (NK) cells are capable of cytotoxic targeting of virally infected cells and some tumor cells. It has been well-demonstrated that NK cells recognize target cells that have down-regulated MHC class I antigen expression (i.e. “missing self recognition”), and that it is the lack of class I engagement of inhibitory receptors such as the killer Ig-like receptors (KIR) that thereby allows NK activation and effector function. How these same inhibitory receptors achieve self-tolerance and simultaneously avoid autoimmunity in humans has not been clear, as more than 60% of individuals have inhibitory KIR for which they lack the HLA ligand. We demonstrate that mature NK cells achieve self-tolerance by preferentially endowing functional competence to the inhibitory KIRs for which they exhibit the cognate HLA ligands. To allow evaluation of inhibitory KIR and avoid interference from potentially class-I recognizing activating KIR, we analyzed NK cells from 10 individuals with various HLA backgrounds, but who were all homozygous for KIR haplotype-A. KIR haplotype-A contains the inhibitory KIR receptors 2DL3, 2DL1, and 3DL1, specific for HLA-Cw3, -Cw4, and -Bw4 ligands respectively, in addition to at most one other activating KIR whose ligand is unknown. Using 6-color staining and flow cytometric analysis of intracellular IFN-γ production, we evaluated the responsiveness of 30 inhibitory KIR-expressing NK subsets following activation with 721.221, a target cell line deficient in class I expression, with 721.221 transfectants expressing HLA-Cw3, -Cw4, or -Bw4 ligands, and with B-lymphocyte cell lines with diverse HLA phenotypes. NK cells exclusively expressing an inhibitory KIR for self-HLA demonstrated increased IFN-γ when coincubated with target cells lacking the cognate HLA ligand, whereas NK cells exclusively expressing an inhibitory KIR for non-self HLA were hyporesponsive to all targets. In all individuals, NK cells expressing inhibitory KIR specific for self-HLA were significantly more responsive than NK cells expressing inhibitory KIR for non-self HLA (p&lt;0.001). All 3 inhibitory KIR (KIR2DL3, 2DL1, and 3DL1) demonstrated capacity for tolerance, with predictable response patterns based on the HLA background of the individual. NK cells lacking all inhibitory NK receptors (KIR−CD94/NKG2A−ILT2−) were identifiable and were markedly hyporesponsive. KIR−CD94/NKG2A+ILT2+ NK cells were also minimally responsive, comparable to the NK subset expressing inhibitory KIR for non-self HLA. These results demonstrate NK tolerance in humans, consistent with the licensing model proposed in mice: NK cells expressing inhibitory KIR to self-HLA are significantly more responsive than NK cells expressing inhibitory KIR for non-self HLA, and are rendered tolerant to self through inhibition upon binding to self-HLA ligands. NK cells expressing inhibitory KIR for non-self HLA are hyporesponsive and therefore rendered tolerant and incapable of autoreactivity when ligand is not engaged.</jats:p