搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

INTRODUCTION Complexins (CPLXs),initially identified in neuronal presynaptic terminals,are cytoplasmic proteins that interact with the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex to regulate the fusion of vesicles to the plasma membrane. Although much is known about CPLX function in neuronal synaptic vesicle exocytosis,their distribution and role in immune cells are still unclear. In this study,we investigated CPLX2 knockout (KO) mice to reveal the role of CPLXs in exocytosis of lymphocytes. METHODS We examined the expression of CPLXs and SNAREs in lymphocytes. To study the effect of CPLXs on the immune system in vivo,we analyzed the immune phenotype of CPLX2 KO mice. Furthermore,antibodies secretion from the peritoneal cavity,spleen,and bone marrow cells of wild-type (WT) and CPLX2 KO mice were determined. RESULTS CPLX2 was detected in B cells but not in T cells,while other CPLXs and SNAREs were expressed at a similar level in both B and T cells. To clarify the function of CPLX2 in B lymphocytes,serum concentrations of immunoglobulin G (IgG),IgA,IgM,and IgE were measured in WT and CPLX2 KO mice using enzyme-linked immunosorbent assay. The level of IgM,which mainly consists of natural antibodies,was higher in KO mice than that in WT mice,while the levels of other antibodies were similar in both types of mice. Additionally,we found that spontaneous secretion of IgM and IgG1 was enhanced from the splenic antibody-secreting cells (ASCs) of CPLX2 KO mice. CONCLUSION Our data suggest that CPLX2 inhibits spontaneous secretion of IgM and IgG1 from splenic ASCs. This study provides new insight into the mechanism of antibody secretion of ASCs. View Publication

Regulatory T cells (Tregs) are crucial immune cells for tissue repair and regeneration. However,their potential as a cell-based regenerative therapy is not yet fully understood. Here,we show that local delivery of exogenous Tregs into injured mouse bone,muscle,and skin greatly enhances tissue healing. Mechanistically,exogenous Tregs rapidly adopt an injury-specific phenotype in response to the damaged tissue microenvironment,upregulating genes involved in immunomodulation and tissue healing. We demonstrate that exogenous Tregs exert their regenerative effect by directly and indirectly modulating monocytes/macrophages (Mo/MΦ) in injured tissues,promoting their switch to an anti-inflammatory and pro-healing state via factors such as interleukin (IL)-10. Validating the key role of IL-10 in exogenous Treg-mediated repair and regeneration,the pro-healing capacity of these cells is lost when Il10 is knocked out. Additionally,exogenous Tregs reduce neutrophil and cytotoxic T cell accumulation and IFN-γ production in damaged tissues,further dampening the pro-inflammatory Mo/MΦ phenotype. Highlighting the potential of this approach,we demonstrate that allogeneic and human Tregs also promote tissue healing. Together,this study establishes exogenous Tregs as a possible universal cell-based therapy for regenerative medicine and provides key mechanistic insights that could be harnessed to develop immune cell-based therapies to enhance tissue healing. Regulatory T cells (Tregs) are known for suppressing inflammatory processes,but their full capacity for tissue regeneration is yet to be harnessed. Here,the authors demonstrate the efficiency of Tregs in facilitating tissue healing in mouse models of bone,muscle,and skin injury,with monocytes/macrophages and interleukin-10 playing a key mechanistic role in the process. View Publication

CD24 expression on pro-B cells plays a role in B cell selection and development in the bone marrow. We previously detected higher CD24 expression and frequency within IgD+ na{\{i}}ve and memory B cells in patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) compared with age-matched healthy controls (HC). Here we investigated the relationship between CD24 expression and B cell maturation. In vitro stimulation of isolated B cells in response to conventional agonists were used to follow the dynamics of CD24 positivity during proliferation and differentiation (or maturation). The relationship between CD24 expression to cycles of proliferation and metabolism in purified B cells from HC was also investigated using phospho-flow (phosphorylation of AMPK-pAMPK) 1proton nuclear magnetic resonance and Mitotracker Far-red (Mitochondrial mass-MM). In vitro in the absence of stimulation there was an increased percentage of CD24+ viable B cells in ME/CFS patients compared to HC (p {\textless} 0.05) following 5 days culture. Following stimulation with B cell agonists percentage of CD24+B cells in both na{\"{i}}ve and memory B cell populations decreased. P {\textless} 0.01). There was a negative relationship between percentage of CD24+B cells with MM (R2 = 0.76; p {\textless} 0.01) which was subsequently lost over sequential cycles of proliferation. There was a significant correlation between CD24 expression on B cells and the usage of glucose and secretion of lactate in vitro. Short term ligation of the B cell receptor with anti-IgM antibody significantly reduced the viability of CD24+ memory B cells compared to those cross-linked by anti-IgD or anti-IgG antibody. A clear difference was found between na{\""{i}}ve and memory B cells with respect to CD24 expression and pAMPK most notably a strong positive association in IgD+IgM+ memory B cells. In vitro findings confirmed dysregulation of CD24-expressing B cells from ME/CFS patients previously suggested by immunophenotype studies of B cells from peripheral blood. CD24-negative B cells underwent productive proliferation whereas CD24+ B cells were either unresponsive or susceptible to cell death upon BCR-engagement alone. We suggest that CD24 expression may reflect variations in energy metabolism on different B cell subsets.""" View Publication

T-cell expression of programmed death receptor-1 (PD-1) down-regulates the immune response against malignancy by interacting with cognate ligands (eg,PD-L1) on tumor cells; however,little is known regarding PD-1 and natural killer (NK) cells. NK cells exert cytotoxicity against multiple myeloma (MM),an effect enhanced through novel therapies. We show that NK cells from MM patients express PD-1 whereas normal NK cells do not and confirm PD-L1 on primary MM cells. Engagement of PD-1 with PD-L1 should down-modulate the NK-cell versus MM effect. We demonstrate that CT-011,a novel anti-PD-1 antibody,enhances human NK-cell function against autologous,primary MM cells,seemingly through effects on NK-cell trafficking,immune complex formation with MM cells,and cytotoxicity specifically toward PD-L1(+) MM tumor cells but not normal cells. We show that lenalidomide down-regulates PD-L1 on primary MM cells and may augment CT-011's enhancement of NK-cell function against MM. We demonstrate a role for the PD-1/PD-L1 signaling axis in the NK-cell immune response against MM and a role for CT-011 in enhancing the NK-cell versus MM effect. A phase 2 clinical trial of CT-011 in combination with lenalidomide for patients with MM should be considered. View Publication

Borrelia (or Borreliella ) burgdorferi,the causative agent of Lyme disease,is a motile and invasive zoonotic pathogen adept at navigating between its arthropod vector and mammalian host. While motility and chemotaxis are well known to be essential for its enzootic cycle,the role of each methyl-accepting chemotaxis proteins (MCPs) in the infectious cycle of B . burgdorferi remains unclear. In this study,we show that mcp5,a gene encoding one of the most abundant MCPs in B . burgdorferi,is differentially expressed in response to environmental signals and at distinct stages of the pathogen’s enzootic cycle. Notably,mcp5 expression is regulated by the Hk1-Rrp1 and Rrp2-RpoN-RpoS pathways,two key regulatory pathways that are critical for the spirochete’s colonization of the tick vector and mammalian host,respectively. Infection experiments with an mcp5 mutant revealed that spirochetes lacking MCP5 were unable to establish infections in either C3H/HeN mice or Severe Combined Immunodeficiency (SCID) mice,which are deficient in adaptive immunity,underscoring MCP5’s critical role in mammalian infection. However,the mcp5 mutant was able to establish infection and disseminate in NOD SCID Gamma (NSG) mice,which are deficient in both adaptive and most innate immune responses,suggesting that MCP5 plays an important role in evading host innate immunity. Moreover,NK cell depletion in C3H and SCID mice restored the infectivity of the mcp5 mutant,further highlighting MCP5’s role in evading NK cell-associated immunity. Co-culture assays with NK cells and macrophages revealed that the mcp5 mutant enhanced interferon-gamma production by NK cells. In the tick vector,the mcp5 mutants survived feeding but failed to transmit to mice. These findings reveal that MCP5,regulated by both the Rrp1 and Rrp2 pathways,is critical for establishing infection in mammalian hosts by evading NK cell-mediated host innate immunity and is important for the transmission of spirochetes from ticks to mammalian hosts. This work provides a foundation for further elucidation of chemotactic signals sensed by MCP5 that facilitate B . burgdorferi in evading host defenses. View Publication

Retinoic acid (RA),a vitamin A metabolite,modulates mucosal T helper cell responses. Here we examined the role of RA in regulating IL-22 production by γδ T cells and innate lymphoid cells in intestinal inflammation. RA significantly enhanced IL-22 production by γδ T cells stimulated in vitro with IL-1β or IL-18 and IL-23. In vivo RA attenuated colon inflammation induced by dextran sodium sulfate treatment or Citrobacter rodentium infection. This was associated with a significant increase in IL-22 secretion by γδ T cells and innate lymphoid cells. In addition,RA treatment enhanced production of the IL-22-responsive antimicrobial peptides Reg3β and Reg3γ in the colon. The attenuating effects of RA on colitis were reversed by treatment with an anti-IL-22 neutralizing antibody,demonstrating that RA mediates protection by enhancing IL-22 production. To define the molecular events involved,we used chromatin immunoprecipitation assays and found that RA promoted binding of RA receptor to the IL-22 promoter in γδ T cells. Our findings provide novel insights into the molecular events controlling IL-22 transcription and suggest that one key outcome of RA signaling may be to shape early intestinal immune responses by promoting IL-22 synthesis by γδ T cells and innate lymphoid cells. View Publication

Mouse embryonic stem (ES) cells cultured as aggregates and exposed to retinoic acid are induced to express multiple phenotypes normally associated with neurons. A large percentage of treated aggregates produce a rich neuritic outgrowth. Dissociating the induced aggregates with trypsin and plating the cells as a monolayer results in cultures in which a sizable percentage of the cells have a neuronal appearance. These neuron-like cells express class III beta-tubulin and the neurofilament M subunit. Induced cultures express transcripts for neural-associated genes including the neurofilament L subunit,glutamate receptor subunits,the transcription factor Brn-3,and GFAP. Levels of neurofilament L and GAD67 and GAD65 transcripts rise dramatically upon induction. Physiological studies show that the neuron-like cells generate action potentials and express TTX-sensitive sodium channels,as well as voltage-gated potassium channels and calcium channels. We conclude that a complex system of neuronal gene expression can be activated in cultured ES cells. This system should be favorable for investigating some of the mechanisms that regulate neuronal differentiation. View Publication

Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem-like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study,we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS,including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs,autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4,a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients,we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas,including putative sCSCs,supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease. View Publication