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

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor,C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells,dendritic cells,or cells from the thymus,lymph node,or spleen of normal mice. Unlike human Th2 cells,mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge,the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection,isolation,and in vivo depletion of eosinophils. View Publication

The translation of laboratory processes into scaled production systems suitable for manufacture is a significant challenge for cell based therapies; in particular there is a lack of analytical methods that are informative and efficient for process control. Here the potential of image analysis as one part of the solution to this issue is explored,using pluripotent stem cell colonies as a valuable and challenging exemplar. The Cell-IQ live cell imaging platform was used to build image libraries of morphological culture attributes such as colony edge� View Publication

Determining how normal and leukemic stem cells behave in vivo,in a dynamic and noninvasive way,remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes,which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context,we evaluated the possibility to use gene transfer-free labeling technologies. The lipophilic dye 3,3,3',3' tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate [DiI],DiD,DiR,and PKH26) can give rise to microenvironmental contamination,including when used in suboptimal concentration,after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually,we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non-genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches. View Publication

Staphylococcal enterotoxins (SE) crosslink the MHC‐II on antigen‐presenting cells (APC) with the T‐cell receptor,inducing a polyclonal T‐cell response. Although APCs are the initial targets of SE and are critical in shaping subsequent T‐cell activation,the effects of SE on APC function remain poorly understood. This study investigates the immunomodulatory effects of staphylococcal enterotoxin A (SEA) on monocytes and their differentiation into monocyte‐derived dendritic cells (moDC) or macrophages (MDM). Transcriptomic analyses of human monocytes via RNA sequencing revealed SEA‐induced enrichment of gene pathways associated with inflammation,infection,and dermatitis,effects that were amplified in the presence of T cells. Phenotypic and functional characterization showed that SEA‐primed monocytes differentiated into MDM with an altered polarization,deviating from classical M1/M2 pathways. SEA‐primed MDM exhibited downregulation of key markers,including HLA‐DR,CD80,CD86,and PD‐L1. Functional assays demonstrated that SEA‐primed MDM pushed hyperinflammatory T‐cell responses,with significantly enhanced proliferation and IFN‐γ secretion. In contrast,following SEA‐priming,moDC retained robust antigen‐presenting capabilities and displayed enhanced expression of molecules involved in T‐cell interactions. These findings provide mechanistic insights into SEA‐mediated immune modulation,illustrating how SEA reprograms MDM functions and amplifies proinflammatory T‐cell responses. This advances our understanding of superantigen‐driven immune interactions,offering a foundation for developing therapeutic strategies to mitigate superantigen‐mediated immune conditions. Staphylococcal enterotoxin A (SEA) alters monocyte differentiation and function,while preserving T cell stimulatory capacity. SEA‐primed macrophages downregulate antigen‐presenting markers yet drive heightened T‐cell proliferation and IFN‐γ secretion. These findings reveal mechanisms of SEA‐mediated immune modulation and superantigen‐driven inflammation. View Publication

The activating NK cell receptor DNAX accessory molecule-1 (DNAM-1) contributes to tumor immune surveillance and plays a crucial role in NK cell-mediated recognition of several types of human tumors,including ovarian carcinoma. Here,we have analyzed the receptor repertoire and functional integrity of NK cells in peritoneal effusions from patients with ovarian carcinoma. Relative to autologous peripheral blood NK cells,tumor-associated NK cells expressed reduced levels of the DNAM-1,2B4,and CD16 receptors and were hyporesponsive to HLA class I-deficient K562 cells and to coactivation via DNAM-1 and 2B4. Moreover,tumor-associated NK cells were also refractory to CD16 receptor stimulation,resulting in diminished Ab-dependent cellular cytotoxicity against autologous tumor cells. Coincubation of NK cells with ovarian carcinoma cells expressing the DNAM-1 ligand CD155 led to reduction of DNAM-1 expression. Therefore,NK cell-mediated rejection of ovarian carcinoma may be limited by perturbed DNAM-1 expression on tumor-associated NK cells induced by chronic ligand exposure. Thus,these data support the notion that tumor-induced alterations of activating NK cell receptor expression may hamper immune surveillance and promote tumor progression. View Publication

The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article,we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced the expression of the vitamin A-converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro,respectively. Furthermore,our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A-converting enzymes in mesenteric lymph node dendritic cells,along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover,we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion,our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system. View Publication

beta 1 Integrins are known to regulate terminal differentiation and morphogenesis in the adult epidermis. We have investigated their role in the embryonic development of keratinocytes by comparing the differentiation of wild-type and beta 1-null mouse embryonal stem (ES) cells. By 12-15 days in culture,differentiation of embryonic or simple epithelial cells occurred in both ES cell populations,as detected by expression of keratins 8,18,and 19. From 21 days,expression of keratins 10 and 14 and of the cornified envelope precursor involucrin indicated that some of the wild-type cells had differentiated into keratinocytes. In contrast,keratinocyte markers were not expressed in beta 1-null cultures. The beta 1-null cells failed to express the alpha 2 and alpha 3 integrin subunits on the cell surface,consistent with the association of these a subunits with beta 1. Furthermore,alpha 6 and beta 4 expression was reduced in the beta 1-null cultures. Although beta 1-null ES cells failed to undergo differentiation into keratinocytes in vitro,they did form keratinocyte cysts expressing alpha 6 beta 4,keratins 1 and 14,and involucrin when allowed to form teratomas by subcutaneous injection in mice; furthermore,beta 1-null keratinocytes were found in the epidermis of a wild-type/beta 1-null chimeric mouse. As judged by immunofluorescence microscopy,extracellular matrix assembly was severely impaired in beta 1-null ES cell cultures,but not in the teratomas or chimeric mouse skin. We therefore speculate that the failure of beta 1-null cells to differentiate into keratinocytes in vitro may reflect an inability to assemble a basement membrane. View Publication

To understand the mechanism of the sequential restriction of multipotency of stem cells during development,we have established culture conditions that allow the differentiation of neuroepithelial precursor cells from embryonic stem (ES) cells. A highly enriched population of neuroepithelial precursor cells derived from ES cells proliferates in the presence of basic fibroblast growth factor (bFGF). These cells differentiate into both neurons and glia following withdrawal of bFGF. By further differentiating the cells in serum-containing medium,the neurons express a wide variety of neuron-specific genes and generate both excitatory and inhibitory synaptic connections. The expression pattern of position-specific neural markers suggests the presence of a variety of central nervous system (CNS) neuronal cell types. These findings indicate that neuronal precursor cells can be isolated from ES cells and that these cells can efficiently differentiate into functional post-mitotic neurons of diverse CNS structures. View Publication

Although oral dendritic cells (DCs) were shown to induce cell-mediated immunity,the identity and function of the various oral DC subsets involved in this process is unclear. In this study,we examined the mechanisms used by DCs of the buccal mucosa and of the lining mucosa to elicit immunity. After plasmid DNA immunization,buccally immunized mice generated robust local and systemic CD8(+) T cell responses,whereas lower responses were seen by lining immunization. A delayed Ag presentation was monitored in vivo in both groups; yet,a more efficient presentation was mediated by buccal-derived DCs. Restricting transgene expression to CD11c(+) cells resulted in diminished CD8(+) T cell responses in both oral tissues,suggesting that immune induction is mediated mainly by cross-presentation. We then identified,in addition to the previously characterized Langerhans cells (LCs) and interstitial dendritic cells (iDCs),a third DC subset expressing the CD103(+) molecule,which represents an uncharacterized subset of oral iDCs expressing the langerin receptor (Ln(+)iDCs). Using Langerin-DTR mice,we demonstrated that whereas LCs and Ln(+)iDCs were dispensable for T cell induction in lining-immunized mice,LCs were essential for optimal CD8(+) T cell priming in the buccal mucosa. Buccal LCs,however,failed to directly present Ag to CD8(+) T cells,an activity that was mediated by buccal iDCs and Ln(+)iDCs. Taken together,our findings suggest that the mechanisms engaged by oral DCs to prime T cells vary between oral mucosal tissues,thus emphasizing the complexity of the oral immune network. Furthermore,we found a novel regulatory role for buccal LCs in potentiating CD8(+) T cell responses. View Publication

Greater than 75% of all hematologic malignancies derive from germinal center (GC) or post-GC B cells,suggesting that the GC reaction predisposes B cells to tumorigenesis. Because GC B cells acquire expression of the highly mutagenic enzyme activation-induced cytidine deaminase (AID),GC B cells may require additional DNA repair capacity. The goal of this study was to investigate whether normal human B cells acquire enhanced expression of DNA repair factors upon AID induction. We first demonstrated that several DNA mismatch repair,homologous recombination,base excision repair,and ATR signaling genes were overexpressed in GC B cells relative to naïve and memory B cells,reflecting activation of a process we have termed somatic hyperrepair (SHR). Using an in vitro system,we next characterized activation signals required to induce AID expression and SHR. Although AID expression was induced by a variety of polyclonal activators,SHR induction strictly required signals provided by contact with activated CD4+ T cells,and B cells activated in this manner displayed reduced levels of DNA damage-induced apoptosis. We further show the induction of SHR is independent of AID expression,as GC B cells from AID-/-mice retained heightened expression of SHR proteins. In consideration of the critical role that CD4+ T cells play in inducing the SHR process,our data suggest a novel role for CD4+ T cells in the tumor suppression of GC/post-GC B cells. View Publication

The germinal center (GC) reaction in Peyer's patches (PP) requires continuous access to antigens,but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7- B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells,we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs,40{\%} of antigen-specific SED B cells bind antigen within 2 h,whereas unspecifc cells do not,indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice,but is unperturbed in mice depleted of classical dendritic cells (DC). Thus,we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses,and should be taken into account when developing mucosal vaccines. View Publication

Human mucosal tissues and skin contain two distinct types of dendritic cell (DC) subsets,epidermal Langerhans cells (LCs) and dermal DCs,which can be distinguished by the expression of C-type lectin receptors,Langerin and DC-SIGN,respectively. Although peripheral blood monocytes differentiate into these distinct subsets,monocyte-derived LCs (moLCs) induced by coculture with GM-CSF,IL-4,and TGF-$\beta$1 coexpress both Langerin and DC-SIGN,suggesting that the environmental cues remain unclear. In this study,we show that LC differentiation is TGF-$\beta$1 dependent and that cofactors such as IL-4 and TNF-$\alpha$ promote TGF-$\beta$1-dependent LC differentiation into Langerin+DC-SIGN- moLCs but continuous exposure to IL-4 blocks differentiation. Steroids such as dexamethasone greatly enhanced TNF-$\alpha$-induced moLC differentiation and blocked DC-SIGN expression. Consistent with primary LCs,dexamethasone-treated moLCs express CD1a,whereas monocyte-derived DCs (moDCs) express CD1b,CD1c,and CD1d. moDCs but not moLCs produced inflammatory cytokines after stimulation with CD1b and CD1d ligands mycolic acid and $\alpha$-galactosylceramide,respectively. Strikingly,CD1a triggering with squalene on moLCs but not moDCs induced strong IL-22-producing CD4+ helper T cell responses. As IL-22 is an important cytokine in the maintenance of skin homeostasis,these data suggest that CD1a on LCs is involved in maintaining the immune barrier in the skin. View Publication