技术资料

The DNA damage response (DDR) alerts the immune system to the danger posed by DNA damage through the induction of damage-associated molecular pattern molecules,chemokines,and ligands for activating immune receptors such as lymphocyte function-associated antigen 1 (LFA-1),NKG2D,and DNAX accessory molecule 1 (DNAM-1). Here we provide evidence that OVA(257-264) -pulsed fibroblasts gain the ability to activate naïve OT-I CD8(+) T cells in response to DNA damage. The ability of fibroblasts to activate OT-I CD8(+) T cells depended on the upregulation of ICAM-1 on fibroblasts and DNAM-1 expression of CD8(+) T cells. OVA(257-264) -pulsed fibroblasts were able to induce a protective T-cell response against B16-OVA cells in a DDR-dependent manner. Hence,the DDR may alert the immune system to the presence of potentially dangerous cells by upregulating the expression of ligands that can induce the activation of innate and adaptive immune cells. View Publication

Structural bone allografts are widely used in the clinic to treat critical sized bone defects,despite lacking the osteoinductive characteristics of live autografts. To address this,we generated revitalized structural allografts wrapped with mesenchymal stem/progenitor cell (MSC) sheets,which were produced by expanding primary syngenic bone marrow derived cells on temperature-responsive plates,as a tissue-engineered periosteum. In vitro assays demonstrated maintenance of the MSC phenotype in the sheets,suggesting that short-term culturing of MSC sheets is not detrimental. To test their efficacy in vivo,allografts wrapped with MSC sheets were transplanted into 4-mm murine femoral defects and compared to allografts with direct seeding of MSCs and allografts without cells. Evaluations consisted of X-ray plain radiography,3D microCT,histology,and biomechanical testing at 4- and 6-weeks post-surgery. Our findings demonstrate that MSC sheets induce prolonged cartilage formation at the graft-host junction and enhanced bone callus formation,as well as graft-host osteointegration. Moreover,a large periosteal callus was observed spanning the allografts with MSC sheets,which partially mimics live autograft healing. Finally,biomechanical testing showed a significant increase in the structural and functional properties of MSC sheet grafted femurs. Taken together,MSC sheets exhibit enhanced osteogenicity during critical sized bone defect repair,demonstrating the feasibility of this tissue engineering solution for massive allograft healing. View Publication

Activin A belongs to the superfamily of transforming growth factor beta (TGF$\beta$) and is a critical regulatory cytokine in breast cancer and inflammation. However,the role of activin A in migration of breast cancer cells and immune cells was not well characterized. Here,a microfluidic device was used to examine the effect of activin A on the migration of human breast cancer cell line MDA-MB-231 cells and human blood neutrophils as well as their migratory interaction. We found that activin A promoted the basal migration but impaired epidermal growth factor (EGF)-induced migration of breast cancer cells. By contrast,activin A reduced neutrophil chemotaxis and transendothelial migration to N-Formyl-Met-Leu-Phe (fMLP). Finally,activin A promoted neutrophil chemotaxis to the supernatant from breast cancer cell culture. Collectively,our study revealed the different roles of activin A in regulating the migration of breast cancer cells and neutrophils and their migratory interaction. These findings suggested the potential of activin A as a therapeutic target for inflammation and breast cancers. View Publication

Abnormal glucose metabolism and enhanced oxidative stress accelerate cardiovascular disease,a chronic inflammatory condition causing high morbidity and mortality. Here,we report that in monocytes and macrophages of patients with atherosclerotic coronary artery disease (CAD),overutilization of glucose promotes excessive and prolonged production of the cytokines IL-6 and IL-1β,driving systemic and tissue inflammation. In patient-derived monocytes and macrophages,increased glucose uptake and glycolytic flux fuel the generation of mitochondrial reactive oxygen species,which in turn promote dimerization of the glycolytic enzyme pyruvate kinase M2 (PKM2) and enable its nuclear translocation. Nuclear PKM2 functions as a protein kinase that phosphorylates the transcription factor STAT3,thus boosting IL-6 and IL-1β production. Reducing glycolysis,scavenging superoxide and enforcing PKM2 tetramerization correct the proinflammatory phenotype of CAD macrophages. In essence,PKM2 serves a previously unidentified role as a molecular integrator of metabolic dysfunction,oxidative stress and tissue inflammation and represents a novel therapeutic target in cardiovascular disease. View Publication

Antibody-based therapies,such as rituximab and alemtuzumab,have contributed significantly to the treatment of Chronic Lymphocytic leukaemia (CLL). The CD40 antigen is expressed predominantly on B-cells and represents a potential target for immune-based therapies. SGN-40 is a humanized IgG1 monoclonal antibody currently in Phase I/II clinical trials for indolent lymphomas,diffuse large B cell lymphomas and Multiple Myeloma. Its biological effect on CLL cells has not been studied. The present study demonstrated that SGN-40 mediated modest apoptosis in a subset of patients with secondary cross-linking but did not mediate complement-dependent cytotoxicity. SGN-40 also mediated antibody-dependent cellular cytotoxicity (ADCC) predominantly through natural killer (NK) cells. Previous studies by our group and others have demonstrated that lenalidomide upregulates CD40 expression on primary B CLL cells and activates NK-cells. We therefore examined for the combinatorial effect of lenalidomide and SGN-40 and demonstrated that both enhanced direct apoptosis and ADCC against primary CLL B cells. These data together provide justification for clinical trials of SGN-40 and lenalidomide in combination for CLL therapy. View Publication

IL-27 is formed by the association of a cytokine subunit,p28,with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27R comprises gp130 and WSX-1. The marked difference between EBI3(-/-) and WSX-1(-/-) mice suggests that p28 has functions independent of EBI3. We have identified an alternative secreted complex formed by p28 and the soluble cytokine receptor cytokine-like factor 1 (CLF). Like IL-27,p28/CLF is produced by dendritic cells and is biologically active on human NK cells,increasing IL-12- and IL-2-induced IFN-gamma production and activation marker expression. Experiments with Ba/F3 transfectants indicate that p28/CLF activates cells expressing IL-6Ralpha in addition to the IL-27R subunits. When tested on CD4 and CD8 T cells,p28/CLF induces IL-6Ralpha-dependent STAT1 and STAT3 phosphorylation. Furthermore,p28/CLF inhibits CD4 T cell proliferation and induces IL-17 and IL-10 secretion. These results indicate that p28/CLF may participate in the regulation of NK and T cell functions by dendritic cells. The p28/CLF complex engages IL-6R and may therefore be useful for therapeutic applications targeting cells expressing this receptor. Blocking IL-6R using humanized mAbs such as tocilizumab has been shown to be beneficial in pathologies like rheumatoid arthritis and juvenile idiopathic arthritis. The identification of a new IL-6R ligand is therefore important for a complete understanding of the mechanism of action of this emerging class of immunosuppressors. View Publication

Granulomas are innate sequestration responses that can be modified by superimposed acquired immune mechanisms. The present study examined the innate stage of pulmonary granuloma responses to bead-immobilized Th1- and Th2-inducing pathogen antigens (Ags),Mycobacteria bovis purified protein derivative (PPD) and Schistosoma mansoni soluble egg Ags (SEA). Compared to a nonpathogen Ag,PPD and SEA bead elicited larger lesions with the former showing accelerated inflammation. Temporal analyses of cytokine and chemokine transcripts showed all Ag beads induced tumor necrosis factor-alpha mRNA but indicated biased interleukin (IL)-1,IL-6,and IL-12 expression with PPD challenge. All beads elicited comparable levels of CXCL9,CXL10,CCL2,CCL17,and CCL22 mRNA,but PPD beads caused biased CXCL2 CXCL5,CCL3,and CCL4 expression whereas both pathogen Ags induced CCL7. Immunohistochemical,electron microscopic,and flow cytometric analyses showed that Ag beads mobilized CD11c+ dendritic cells (DCs) of comparable maturation. Transfer of DCs from PPD Ag-challenged lungs conferred a Th1 anamnestic cytokine response in recipients. Surprisingly,transfer of DCs from the helminth SEA-challenged lungs did not confer the expected Th2 response,but instead rendered recipients incapable of Ag-elicited IL-4 production. These results provide in vivo evidence that lung DCs recruited under inflammatory conditions favor Th1 responses and alternative mechanisms are required for Th2 commitment. View Publication