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

Protective antibody responses to vaccination or infection depend on affinity maturation,a process by which high-affinity germinal center (GC) B cells are selected on the basis of their ability to bind,gather,and present antigen to T follicular helper (Tfh) cells. Here,we show that human GC B cells have intrinsically higher-affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared with na{\{i}}ve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to antigen affinity- and Tfh cell-dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structures was dictated by the intrinsic antigen affinity thresholds of GC B cells. Low-affinity antigens triggered continuous engagement and disengagement of membrane-associated antigens whereas high-affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of na{\"{i}}ve B cells. Thus intrinsic properties of human GC B cells set thresholds for affinity selection.""" View Publication

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. View Publication

Gamma-delta (??) T cells recognize antigens in a major histocompatibility complex (MHC) independent and have cytotoxic capability. Human immunodeficiency virus (HIV) infection reduces the proportion of the V?2 cell subset compared to the V?1 cell subset of ?? T cells in the blood in most infected individuals,except for elite controllers. The capacity of V?2 T cells to kill HIV-infected targets has been demonstrated in vitro,albeit in vivo confirmatory studies are lacking. Here,we provide the first characterization of ?? T cell-HIV interactions in bone marrow-liver-thymus (BLT) humanized mice and examined the immunotherapeutic potential of V?2 T cells in controlling HIV replication in vivo. We demonstrate a reduced proportion of V?2 T cells and an increased proportion of V?1 T cells in HIV-infected BLT humanized mice,like in HIV-positive individuals. HIV infection in BLT humanized mice also impaired the ex vivo expansion of V?2 T cells,like in HIV-positive individuals. Adoptive transfer of activated V?2 T cells did not control HIV replication during cell-associated HIV transmission in BLT humanized mice but instead exacerbated viremia,suggesting that V?2 T cells may serve as early targets for HIV replication. Our findings demonstrate that BLT humanized mice can model ?? T cell-HIV interactions in vivo. View Publication

PURPOSE: Circulating cell-free DNA in the blood of cancer patients harbors tumor-specific aberrations. Here,we investigated whether this DNA might also reflect the presence of circulating tumor cells (CTC). EXPERIMENTAL DESIGN: To identify the source of cell-free DNA in blood,plasma derived from 81 patients with prostate cancer was examined for CTCs and cell-free DNA. An epithelial immunospot assay was applied for detection of CTCs,and a PCR-based fluorescence microsatellite analysis with a panel of 14 polymorphic markers was used for detection of allelic imbalances (AI). RESULTS: The plasma DNA levels significantly correlated with the diagnosis subgroups of localized (stage M0,n = 69) and metastasized prostate cancer (stage M1,n = 12; P = 0.03) and with the tumor stage of these patients (P textless 0.005). AI was found on cell-free DNA in plasma from 45.0% and 58.5% of M0 and M1 patients,respectively. Detection of CTCs showed that 71.0% or 92.0% of the M0 and M1 patients harbored 1 to 40 CTCs in their blood,respectively. The occurrence of CTCs correlated with tumor stage (P textless 0.03) and increasing Gleason scores (P = 0.04). Notably,significant associations of the number of CTCs with the AI frequencies at the markers D8S137 (P = 0.03),D9S171 (P = 0.04),and D17S855 (P = 0.02) encoding the cytoskeletal protein dematin,the inhibitor of the cyclin-dependent kinase CDKN2/p16 and BRCA1,respectively,were observed. CONCLUSIONS: These findings show,for the first time,a relationship between the occurrence of CTCs and circulating tumor-associated DNA in blood,which,therefore,might become a valuable new source for monitoring metastatic progression in cancer patients. View Publication

BACKGROUND: Malignant melanoma is an exceptionally aggressive,drug-resistant and heterogeneous cancer. Recently it has been shown that melanoma cells with high clonogenic and tumourigenic abilities are common,but markers distinguishing such cells from cells lacking these abilities have not been identified. There is therefore no definite evidence that an exclusive cell subpopulation,i.e. cancer stem cells (CSC),exists in malignant melanoma. Rather,it is suggested that multiple cell populations are implicated in initiation and progression of the disease,making it of importance to identify subpopulations with elevated aggressive properties. METHODS AND FINDINGS: In several other cancer forms,Aldehyde Dehydrogenase (ALDH),which plays a role in stem cell biology and resistance,is a valuable functional marker for identification of cells that show enhanced aggressiveness and drug-resistance. Furthermore,the presence of ALDH(+) cells is linked to poor clinical prognosis in these cancers. By analyzing cell cultures,xenografts and patient biopsies,we showed that aggressive melanoma harboured a large,distinguishable ALDH(+) subpopulation. In vivo,ALDH(+) cells gave rise to ALDH(-) cells,while the opposite conversion was rare,indicating a higher abilities of ALDH(+) cells to reestablish tumour heterogeneity with respect to the ALDH phenotype. However,both ALDH(+) and ALDH(-) cells demonstrated similarly high abilities for clone formation in vitro and tumour initiation in vivo. Furthermore,both subpopulations showed similar sensitivity to the anti-melanoma drugs,dacarbazine and lexatumumab. CONCLUSIONS: These findings suggest that ALDH does not distinguish tumour-initiating and/or therapy-resistant cells,implying that the ALDH phenotype is not associated with more-aggressive subpopulations in malignant melanoma,and arguing against ALDH as a universal" marker. Besides� View Publication

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies,but is absent on normal tissues,including hematopoietic progenitor cells,and may therefore be an appropriate candidate for T cell-mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity,PRAME-specific cytotoxic T lymphocytes (CTLs),we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal,PRAME-specific CTL lines and elicited high-avidity CTLs,with a high proportion of cells recognizing a previously uninvestigated HLA-A*02-restricted epitope,P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME(+) hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts,but also against leukemic progenitor cells as assessed by colony-forming-inhibition assays,which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors,indicating that this approach may be of value for immunotherapy of PRAME(+) hematologic malignancies. View Publication

The shortage of human organs and tissues for transplant has led to significant interest in xenotransplantation of pig tissues for human patients. However,transplantation of pig organs results in an acute immune rejection,leading to death of the organ within minutes. The $\$-1,3-galactosyltransferase (GALT) gene has been knocked out in pigs to reduce rejection,yet additional genes need to be modified to ultimately make pig tissue immunocompatible with humans. The development of pig induced pluripotent stem cells (piPSCs) from GALT knockout (GALT-KO) tissue would provide an excellent cell source for complex genetic manipulations (e.g.,gene targeting) that often require highly robust and proliferative cells. In this report,we generated GALT-KO piPSCs by the overexpression of POU5F1,SOX2,NANOG,LIN28,KLF-4,and C-MYC reprogramming genes. piPSCs showed classical stem cell morphology and characteristics,expressing integrated reprogramming genes in addition to the pluripotent markers AP,SSEA1,and SSEA4. GALT-KO piPSCs were highly proliferative and possessed doubling times and telomerase activity similar to human embryonic stem cells. These results demonstrated successful reprogramming of GALT-KO fibroblasts into GALT-KO piPSCs. GALT-KO piPSCs are potentially an excellent immortal cell source for the generation of pigs with complex genetic modifications for xenotransplantation,somatic cell nuclear transfer,or chimera formation. View Publication

Induced pluripotent stem cells (iPSCs) have the ability to differentiate in any specialized somatic cell type,which makes them an attractive tool for a wide variety of scientific approaches,including regenerative medicine. However,their pluripotent state and their growth in compact colonies render them difficult to access and,therefore,restrict delivery of specific agents for cell manipulation. Thus,our investigation focus was set on the evaluation of the capability of Layer-by-Layer (LbL) designed microcarriers to serve as a potential drug delivery system to iPSCs,as they offer several appealing advantages. Most notably,these carriers allow for the transport of active agents in a protected environment and for a rather specific delivery through surface modifications. As we could show,charge and mode of LbL carrier application as well as the size of the iPSC colonies determine the interaction with and the uptake rate by iPSCs. None of the examined conditions had an influence on iPSC colony properties such as colony morphology and size or maintenance of pluripotent properties. An overall interaction rate of LbL carriers with iPSCs of up to 20 % was achieved. Those data emphasize the applicability of LbL carriers for stem cell research. Additionally,the potential use of LbL carriers as a promising delivery tool for iPSCs was contrasted to viral particles and liposomes. The identified differences among those delivery tools have substantiated our major conclusion that LbL carrier uptake rate is influenced by characteristic features of the iPSC colonies (most notably colony size) in addition to their surface charges. View Publication

Despite the high incidence of trophoblast-related diseases,the molecular mechanism of inadequate early trophoblast development is still unclear due to the lack of an appropriate cellular model in vitro. In the present study,we reprogrammed the amniotic cells to be induced pluripotent stem cells (iPSCs) via a non-virus and non-integrated method and subsequently differentiated them into trophoblast-like cells by a modified BMP4 strategy in E6 medium. Compared with the previously studied trophoblast-like cells from ESCs,the iPSCs derived trophoblast-like cells behave similarly in terms of gene expression profiles and biofunctions. Also we confirmed the differentiating tendency from iPSCs to be syncytiotrophoblasts-like cells might be caused by inappropriate differentiating oxygen condition. Additionally,we preliminarily indicated in vitro artificial" differentiation of iPSCs also undergoing a possible trophoblastic stem cell stage as witnessed in vivo. In conclusion we provided an in vitro cellular model to study early trophoblast development for specific individual by using the feasible amnion. View Publication