Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system.
High-titer,HIV-1-based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34(+) cells and clonogenic progenitors very poorly (textless 1%),reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier,we developed a simian immunodeficiency virus (SIV)-based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% +/- 1% of rhesus bulk CD34(+) cells and 75% +/- 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector-mediated stem cell gene transfer in vivo,3 rhesus macaques underwent transplantation with transduced,autologous cytokine-mobilized peripheral blood CD34(+) cells following myeloablative conditioning. Hematopoietic reconstitution was rapid,and an average of 18% +/- 8% and 15% +/- 7% GFP-positive granulocytes and monocytes,respectively,were observed 4 to 6 months after transplantation,consistent with the average vector copy number of 0.19 +/- 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models.
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产品类型：
产品号#：
04434
04444
产品名：
MethoCult™H4434经典
MethoCult™H4434经典
De Palma M et al. (MAR 2005)
Blood 105 6 2307--15
Promoter trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary hematopoietic cells.
Recent reports have indicated that human immunodeficiency virus (HIV) and murine leukemia virus (MLV) vectors preferentially integrate into active genes. Here,we used a novel approach based on genetic trapping to rapidly score several thousand integration sites and found that MLV vectors trapped cellular promoters more efficiently than HIV vectors. Remarkably,1 in 5 MLV integrations trapped an active promoter in different cell lines and primary hematopoietic cells. Such frequency was even higher in growth-stimulated lymphocytes. We show that the different behavior of MLV and HIV vectors was dependent on a different integration pattern within transcribed genes. Whereas MLV-based traps showed a strong bias for promoter-proximal integration leading to efficient reporter expression,HIV-based traps integrated throughout transcriptional units and were limited for expression by the distance from the promoter and the reading frame of the targeted gene. Our results indicate a strong propensity of MLV to establish transcriptional interactions with cellular promoters,a behavior that may have evolved to enhance proviral expression and may increase the insertional mutagenesis risk. Promoter trapping efficiency provides a convenient readout to assess transcriptional interactions between the vector and its flanking genes at the integration site and to compare integration site selection among different cell types and in different growth conditions.
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产品类型：
产品号#：
03434
03444
09600
09650
18757
18757RF
产品名：
MethoCult™GF M3434
MethoCult™GF M3434
StemSpan™ SFEM
StemSpan™ SFEM
EasySep™小鼠CD117（cKIT）正选试剂盒
RoboSep™ 小鼠CD117（cKIT）正选试剂盒含滤芯吸头
Santoni de Sio FR et al. (JUN 2006)
Blood 107 11 4257--65
Proteasome activity restricts lentiviral gene transfer into hematopoietic stem cells and is down-regulated by cytokines that enhance transduction.
The therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture,they display low permissivity to the vector,requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation,we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays,we show that transduction enhancement by cytokines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer,allowing the reach of very high levels of vector integration in their progeny in vivo. Thus,LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly,cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors,highlighting one mechanism by which cytokines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy.
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Foley JF et al. (APR 2005)
Journal of immunology (Baltimore,Md. : 1950) 174 8 4892--900
Roles for CXC chemokine ligands 10 and 11 in recruiting CD4+ T cells to HIV-1-infected monocyte-derived macrophages, dendritic cells, and lymph nodes.
We investigated roles for chemoattractants in dissemination of HIV-1 by examining the induction of T cell-active chemokines in HIV-1-infected human monocyte-derived macrophages and dendritic cells. Of the 12 chemokines analyzed,mRNAs for two,CXCL10 and CXCL11,ligands for the chemokine receptor CXCR3,were up-regulated in both cell types upon infection by HIV-1. Induction of these chemokine genes in infected cultures was dependent on both viral entry and reverse transcriptase activity,but not on the HIV-1 envelope glycoprotein. Conditioned medium from infected cells was chemotactic for freshly isolated human CD4+ T cells,and chemotaxis was abolished by pretreatment with an Ab against CXCR3. A lymph node from an HIV-1-infected individual expressed CXCL10 and CXCL11 mRNAs in the paracortex,including venules,as detected by in situ hybridization,whereas neither mRNA was detected after highly active antiretroviral therapy. Because CCR5 on CD4+ T cells is found predominantly on cells that also express CXCR3,these data implicate CXCL10 and CXCL11 in the recruitment of susceptible T cells to HIV-1-infected lymph nodes,macrophages,and dendritic cells. This recruitment might enhance the sequestration of T cells in infected lymphoid organs and the spread of infection between cells,contributing to the immunopathology of AIDS.
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产品类型：
产品号#：
15022
15062
产品名：
RosetteSep™人CD4+ T细胞富集抗体混合物
RosetteSep™人CD4+ T细胞富集抗体混合物
Wu X et al. (DEC 2008)
Blood 112 12 4675--82
Alternative splicing regulates activation-induced cytidine deaminase (AID): implications for suppression of AID mutagenic activity in normal and malignant B cells.
The mutagenic enzyme activation-induced cytidine deaminase (AID) is required for immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in germinal center (GC) B cells. Deregulated expression of AID is associated with various B-cell malignancies and,currently,it remains unclear how AID activity is extinguished to avoid illegitimate mutations. AID has also been shown to be alternatively spliced in malignant B cells,and there is limited evidence that this also occurs in normal blood B cells. The functional significance of these splice variants remains unknown. Here we show that normal GC human B cells and blood memory B cells similarly express AID splice variants and show for the first time that AID splicing variants are singly expressed in individual normal B cells as well as malignant B cells from chronic lymphocytic leukemia patients. We further demonstrate that the alternative AID splice variants display different activities ranging from inactivation of CSR to inactivation or heightened SHM activity. Our data therefore suggest that CSR and SHM are differentially switched off by varying the expression of splicing products of AID at the individual cell level. Most importantly,our findings suggest a novel tumor suppression mechanism by which unnecessary AID mutagenic activities are promptly contained for GC B cells.
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产品类型：
产品号#：
19054
19054RF
20119
20155
21000
产品名：
EasySep™人B细胞富集试剂盒
RoboSep™ 人B细胞富集试剂盒含滤芯吸头
RoboSep™ 吸头组件抛光剂
RoboSep™分选试管套装（9个塑料管+吸头保护器）
RoboSep™- S
Lambert AA et al. (AUG 2008)
Blood 112 4 1299--307
The C-type lectin surface receptor DCIR acts as a new attachment factor for HIV-1 in dendritic cells and contributes to trans- and cis-infection pathways.
The dynamic interplay between dendritic cells (DCs) and human immunodeficiency virus type-1 (HIV-1) is thought to result in viral dissemination and evasion of antiviral immunity. Although initial observations suggested that the C-type lectin receptor (CLR) DC-SIGN was responsible for the trans-infection function of the virus,subsequent studies demonstrated that trans-infection of CD4(+) T cells with HIV-1 can also occur through DC-SIGN-independent mechanisms. We demonstrate that a cell surface molecule designated DCIR (for DC immunoreceptor),a member of a recently described family of DC-expressing CLRs,can participate in the capture of HIV-1 and promote infection in trans and in cis of autologous CD4(+) T cells from human immature monocyte-derived DCs. The contribution of DCIR to these processes was revealed using DCIR-specific siRNAs and a polyclonal antibody specific for the carbohydrate recognition domain of DCIR. Data from transfection experiments indicated that DCIR acts as a ligand for HIV-1 and is involved in events leading to productive virus infection. Finally,we show that the neck domain of DCIR is important for the DCIR-mediated effect on virus binding and infection. These results point to a possible role for DCIR in HIV-1 pathogenesis by supporting the productive infection of DCs and promoting virus propagation.
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产品类型：
产品号#：
19052
19052RF
产品名：
EasySep™人CD4+ T细胞富集试剂盒
RoboSep™ 人CD4+ T细胞富集试剂盒含滤芯吸头
Cesaro A et al. (SEP 2012)
PLoS ONE 7 9 e45478
An inflammation loop orchestrated by S100A9 and Calprotectin is critical for development of arthritis
OBJECTIVE: The S100A9 and S100A8 proteins are highly expressed by neutrophils and monocytes and are part of a group of damage-associated molecular pattern molecules that trigger inflammatory responses. Sera and synovial fluids of patients with rheumatoid arthritis (RA) contain high concentrations of S100A8/A9 that correlate with disease activity.backslashnbackslashnMETHODS: In this study,we investigated the importance of S100A9 in RA by using neutralizing antibodies in a murine lipopolysaccharide-synchronized collagen-induced arthritis model. We also used an in vitro model of stimulation of human immune cells to decipher the role played by S100A9 in leukocyte migration and pro-inflammatory cytokine secretion.backslashnbackslashnRESULTS: Treatment with anti-S100A9 antibodies improved the clinical score by 50%,diminished immune cell infiltration,reduced inflammatory cytokines,both in serum and in the joints,and preserved bone/collagen integrity. Stimulation of neutrophils with S100A9 protein led to the enhancement of neutrophil transendothelial migration. S100A9 protein also induced the secretion by monocytes of proinflammatory cytokines like TNFα,IL-1β and IL-6,and of chemokines like MIP-1α and MCP-1.backslashnbackslashnCONCLUSION: The effects of anti-S100A9 treatment are likely direct consequences of inhibiting the S100A9-mediated promotion of neutrophil transmigration and secretion of pro-inflammatory cytokines from monocytes. Collectively,our results show that treatment with anti-S100A9 may inhibit amplification of the immune response and help preserve tissue integrity. Therefore,S100A9 is a promising potential therapeutic target for inflammatory diseases like rheumatoid arthritis for which alternative therapeutic strategies are needed.
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产品类型：
产品号#：
03800
03801
03802
03803
03804
03805
03806
15028
15068
产品名：
ClonaCell™-HY 杂交瘤试剂盒
ClonaCell™-HY Medium
ClonaCell™-HY Medium
ClonaCell™-HY Medium
ClonaCell™-HY Medium
ClonaCell™-HY Medium
ClonaCell™-HY PEG (融合)
RosetteSep™ 人单核细胞富集抗体混合物
RosetteSep™人单核细胞富集抗体混合物
Shen H et al. (AUG 2008)
Journal of immunology (Baltimore,Md. : 1950) 181 3 1849--58
Dual signaling of MyD88 and TRIF is critical for maximal TLR4-induced dendritic cell maturation.
TLR4 is a unique TLR because downstream signaling occurs via two separate pathways,as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (TRIF). In this study,we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation,neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs,whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors,of which type I IFNs were one of the active components,played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types,MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore,signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs,with MyD88 playing a larger role than TRIF. In sum,in our experimental systems,TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation.
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S. Cao et al. (mar 2019)
Science advances 5 3 eaav6322
Hybrid nanocarriers incorporating mechanistically distinct drugs for lymphatic CD4+ T cell activation and HIV-1 latency reversal.
A proposed strategy to cure HIV uses latency-reversing agents (LRAs) to reactivate latent proviruses for purging HIV reservoirs. A variety of LRAs have been identified,but none has yet proven effective in reducing the reservoir size in vivo. Nanocarriers could address some major challenges by improving drug solubility and safety,providing sustained drug release,and simultaneously delivering multiple drugs to target tissues and cells. Here,we formulated hybrid nanocarriers that incorporate physicochemically diverse LRAs and target lymphatic CD4+ T cells. We identified one LRA combination that displayed synergistic latency reversal and low cytotoxicity in a cell model of HIV and in CD4+ T cells from virologically suppressed patients. Furthermore,our targeted nanocarriers selectively activated CD4+ T cells in nonhuman primate peripheral blood mononuclear cells as well as in murine lymph nodes,and substantially reduced local toxicity. This nanocarrier platform may enable new solutions for delivering anti-HIV agents for an HIV cure.
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产品类型：
产品号#：
17952
17952RF
100-0696
产品名：
EasySep™人CD4+ T细胞分选试剂盒
RoboSep™ 人CD4+ T细胞分选试剂盒
EasySep™人CD4+ T细胞分离试剂盒
Muthuswamy R et al. (JUL 2008)
Cancer research 68 14 5972--8
Ability of mature dendritic cells to interact with regulatory T cells is imprinted during maturation.
Preferential activation of regulatory T (Treg) cells limits autoimmune tissue damage during chronic immune responses but can also facilitate tumor growth. Here,we show that tissue-produced inflammatory mediators prime maturing dendritic cells (DC) for the differential ability of attracting anti-inflammatory Treg cells. Our data show that prostaglandin E(2) (PGE(2)),a factor overproduced in chronic inflammation and cancer,induces stable Treg-attracting properties in maturing DC,mediated by CCL22. The elevated production of CCL22 by PGE(2)-matured DC persists after the removal of PGE(2) and is further elevated after secondary stimulation of DC in a neutral environment. This PGE(2)-induced overproduction of CCL22 and the resulting attraction of FOXP3(+) Tregs are counteracted by IFN alpha,a mediator of acute inflammation,which also restores the ability of the PGE(2)-exposed DC to secrete the Th1-attracting chemokines: CXCL9,CXCL10,CXCL11,and CCL5. In accordance with these observations,different DCs clinically used as cancer vaccines show different Treg-recruiting abilities,with PGE(2)-matured DC,but not type 1-polarized DC,generated in the presence of type I and type II IFNs,showing high Treg-attracting activity. The current data,showing that the ability of mature DC to interact with Treg cells is predetermined at the stage of DC maturation,pave the way to preferentially target the regulatory versus proinflammatory T cells in autoimmunity and transplantation,as opposed to intracellular infections and cancer.
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EasySep™小鼠TIL（CD45）正选试剂盒
产品手册Isolate Human Immune Cells
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