Screening chemical microarrays: methods and applications.
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Animals↗
Biomedical subjects
Pappanaicken R Kumaresan
Publications and source records attributed to Pappanaicken R Kumaresan.
Mutational analysis of the human 2B4 (CD244)/CD48 interaction: Lys68 and Glu70 in the V domain of 2B4 are critical for CD48 binding and functional activation of NK cells.
Interaction between receptors and ligands plays a critical role in the generation of immune responses. The 2B4 (CD244), a member of the CD2 subset of the Ig superfamily, is the high affinity ligand for CD48. It is expressed on NK cells, T cells, monocytes, and basophils. Recent data indicate that 2B4/CD48 interactions regulate NK and T lymphocyte functions. In human NK cells, 2B4/CD48 interaction induces activation signals, whereas in murine NK cells it sends inhibitory signals. To determine the structural basis for 2B4/CD48 interaction, selected amino acid residues in the V domain of the human 2B4 (h2B4) were mutated to alanine by site-directed mutagenesis. Following transient expression of these mutants in B16F10 melanoma cells, their interaction with soluble CD48-Fc fusion protein was assessed by flow cytometry. We identified amino acid residues in the extracellular domain of h2B4 that are involved in interacting with CD48. Binding of CD48-Fc fusion protein to RNK-16 cells stably transfected with wild-type and a double-mutant Lys(68)Ala-Glu(70)Ala h2B4 further demonstrated that Lys(68) and Glu(70) in the V domain of h2B4 are essential for 2B4/CD48 interaction. Functional analysis indicated that Lys(68) and Glu(70) in the extracellular domain of h2B4 play a key role in the activation of human NK cells through 2B4/CD48 interaction.
Alanine↗
The LLT1 receptor induces IFN-gamma production by human natural killer cells.
Natural killer cell functions are regulated by signals through activating and inhibitory receptors. These receptors belong to the immunoglobulin superfamily or the lectin superfamily. We have previously identified a lectin-like transcript, LLT1, expressed in human NK cells. In the present study, we have generated a monoclonal antibody, L9.7, that specifically binds LLT1 receptor and studied the functional role of LLT1 in human NK cells. Binding of mAb L9.7 to surface LLT1 induced IFN-gamma production, but did not modulate cytotoxicity by YT cells, a human NK cell line. We further demonstrate that in resting NK cells as well as in IL-2 activated NK cells LLT1 induced IFN-gamma production, but not cytotoxicity. Excess amounts of L9.7 mAb failed to increase natural or antibody-dependent cell-mediated cytolytic activity, whereas minimal amounts achieved maximal production of IFN-gamma by YT and activated NK cells. These findings further support the separation of signaling pathways that regulate cytotoxicity and IFN-gamma production in resting as well as activated NK cells.
Antibodies, Monoclonal↗
Effect of C-reactive protein on chemokine expression in human aortic endothelial cells.
Inflammation plays a pivotal role in atherosclerosis. In addition to being a risk marker for cardiovascular disease, much recent data support a role for C-reactive protein (CRP) in atherogenesis. Interleukin-8 (IL-8), a member of the CXC chemokines promotes monocyte-endothelial cell adhesion and arrest and is abundant in atherosclerotic plaques. However, there is a paucity of data examining the effect of CRP on IL-8 secretion in human aortic endothelial cells (HAEC). In this report, we show that incubation of HAEC with CRP resulted in a time and dose-dependent increase in IL-8 protein and mRNA via transcription. In contrast to human umbilical vein endothelial cells, monocyte-chemoattractant protein-1 expression in HAEC was not affected by CRP. Furthermore, CRP upregulated NF-kappa B activity in HAEC and inhibitors of NF-kappa B significantly reversed the upregulation of IL-8 by CRP. Blocking antibodies to IL-8 significantly decreased monocyte-endothelial cell adhesion induced by CRP (31%, P<0.01). In conclusion, this study makes the novel observation that CRP induces IL-8 synthesis and secretion in HAEC via upregulation of NF-kappa B activity.
Aorta↗
CS1, a novel member of the CD2 family, is homophilic and regulates NK cell function.
CS1 is a novel member of the CD2 subset of immunoglobulin superfamily (IgSF) expressed on NK, T and stimulated B cells. The cytoplasmic domain of CS1 contains immunoreceptor tyrosine-based switch motif (ITSM) which is present in 2B4, SLAM and CD84. The signaling adaptor molecule SAP/SH2D1A, the defective gene in X-linked lymphoproliferative disease (XLPD), binds to ITSM and regulates immune cell function. However, recent studies indicate that CS1 may be regulated by a SAP-independent mechanism. In this study, we have examined the ligand specificity of CS1 and the effect of CS1 interaction with its ligand on the cytolytic activity of YT, a human NK cell line. Recombinant fusion protein, CS1-Ig, containing the CS1 extracellular domain and Fc portion of the human IgG bound cells transfected with CS1. CS1-Ig did not show any binding to cells expressing other members of the CD2 family. The cytolytic activity of YT was enhanced in presence of soluble CS1-Ig fusion protein. These results demonstrate that CS1 is a self-ligand and homophilic interaction of CS1 regulates NK cell cytolytic activity.
Amino Acid Motifs↗
Development of tissue plasminogen activator specific "on demand cleavable" (odc) linkers for radioimmunotherapy by screening one-bead-one-compound combinatorial peptide libraries.
New strategies are needed to protect normal organs from radiation in cancer radioimmunotherapy (RIT). This can be achieved by rapid clearance of radiometal in the circulation after accumulation of radioimmunoconjugates (RIC) in the tumor. Our strategy is to place highly efficient and specific cleavable linkers between radiometal chelates and the tumor targeting agents. Such linkers must be resistant to cleavage by enzymes present in the plasma and the tumor. After radiotargeting agents have accumulated in the tumor, a cleaving agent can be administered "on demand" to cleave a specific linker, resulting in the release of radiometal from the circulating RIC in a form that will have rapid renal clearance. We have selected TNKase, a thrombolytic agent approved for patient use, as our model on-demand cleaving agent. To identify TNKase-specific linkers, we screened fluorescent-quenched random "one-bead-one-compound" (OBOC) combinatorial peptide libraries. d-Amino acid containing peptides that were specific for TNKase but were resistant to cleavage by plasma and tumor-associated proteases were identified. One of these peptide substrates (rqYKYkf) was used to link the DOTA chelate to ChL6, a monoclonal antibody known to target breast cancer. This antibody conjugate was stable in plasma for 7 days while preserving the immunoreactivity to intact tumor cells. The addition of TNKase at clinical achievable plasma level (10 mug/mL) resulted in the release of 28% of the radiometal from the radioimmunoconjugate within 72 h. This lead linker, after further optimization to increase its response to TNKase, may be useful in the development of more effective radioimmunotherapeutic and radioimaging agents.
Cellulose↗