搜索结果: 'easysep direct'

Pathogen induced migration of neutrophils across mucosal epithelial barriers requires epithelial production of the chemotactic lipid mediator,hepoxilin A3 (HXA3). HXA3 is an eicosanoid derived from arachidonic acid. Although eosinophils are also capable of penetrating mucosal surfaces,eosinophilic infiltration occurs mainly during allergic processes whereas neutrophils dominate mucosal infection. Both neutrophils and eosinophils can respond to chemotactic gradients of certain eicosanoids,however,it is not known whether eosinophils respond to pathogen induced lipid mediators such as HXA3. In this study,neutrophils and eosinophils were isolated from human blood and placed on the basolateral side of polarized epithelial monolayers grown on permeable Transwell filters and challenged by various chemotactic gradients of distinct lipid mediators. We observed that both cell populations migrated across epithelial monolayers in response to a leukotriene B4 (LTB4) gradient,whereas only eosinophils migrated toward a prostaglandin D2 (PGD2) gradient. Interestingly,while pathogen induced neutrophil trans-epithelial migration was substantial,pathogen induced eosinophil trans-epithelial migration was not observed. Further,gradients of chemotactic lipids derived from pathogen infected epithelial cells known to be enriched for HXA3 as well as purified HXA3 drove significant numbers of neutrophils across epithelial barriers,whereas eosinophils failed to respond to these gradients. These data suggest that although the eicosanoid HXA3 serves as an important neutrophil chemo-attractant at mucosal surfaces during pathogenic infection,HXA3 does not appear to exhibit chemotactic activity toward eosinophils. ?? 2013 Elsevier Ltd. All rights reserved. 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

Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here,we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34(+)CD31(+) endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable,and endothelial progenitor differentiation was β-catenin dependent. Furthermore,by clonal analysis,we showed that CD34(+)CD31(+)CD117(+)TIE-2(+) endothelial progenitors were multipotent,capable of differentiating into calponin-expressing smooth muscle cells and CD31(+)CD144(+)vWF(+)I-CAM1(+) endothelial cells. These endothelial cells were capable of 20 population doublings,formed tube-like structures,imported acetylated low-density lipoprotein,and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/β-catenin signaling as a primary regulator for generating vascular cells from hPSCs. View Publication

Manipulation of the CD28/CTLA-4 pathway is at the heart of a number of immunomodulatory approaches used in both autoimmunity and cancer. Although it is clear that CTLA-4 is a critical regulator of T cell responses,the immunological contexts in which CTLA-4 controls immune responses are not well defined. In this study,we show that whereas CD80/CD86-dependent activation of resting human T cells caused extensive T cell proliferation and robust CTLA-4 expression,in this context CTLA-4 blocking Abs had no impact on the response. In contrast,in settings where CTLA-4(+) cells were present as regulators� View Publication

HIV-1 infection leads to numerous B cell abnormalities,including hypergammaglobulinemia,nonspecific B cell activation,nonspecific class switching,increased cell turnover,breakage of tolerance,increased immature/transitional B cells,B cell malignancies,as well as a loss of capacity to generate and maintain memory,all of which contribute to a global impairment of the immune humoral compartment. Several cytokines and soluble factors,which are increased in sera of HIV-1-infected individuals,have been suggested to directly or indirectly contribute to these B cell dysfunctions,and one of these is the B cell-activating factor (BAFF). We report in this study that HIV-1 (X4- and R5-tropic) upregulates BAFF expression and secretion by human monocytes. Moreover,we show that the virus-mediated production of BAFF by monocytes relies on a type I IFN response by a small percentage of plasmacytoid dendritic cells (pDCs) present in the monocyte cultures. HIV-1-induced type I IFN by pDCs triggers BAFF production in both classical and intermediate monocytes,but not in nonclassical monocytes,which nonetheless display a very strong basal BAFF production. We report also that basal BAFF secretion was higher in monocytes obtained from females compared with those from male donors. This study provides a novel mechanistic explanation for the increased BAFF levels observed during HIV-1 infection and highlights the importance of pDC/monocyte crosstalk to drive BAFF secretion. View Publication

Epigenetic modifications refer to a number of biological processes which alter the structure of chromatin and its transcriptional activity such as DNA methylation and histone post-translational processing. Studies have tried to elucidate how the viral genome and its products are affected by epigenetic modifications imposed by cell machinery and how it affects the ability of the virus to either,replicate and produce a viable progeny or be driven to latency. The purpose of this study was to evaluate epigenetic modifications in PBMCs and CD4+ cells after HIV-1 infection analyzing three approaches: (i) global DNA- methylation; (ii) qPCR array and (iii) western blot. HIV-1 infection led to methylation increases in the cellular DNA regardless the activation status of PBMCs. The analysis of H3K9me3 and H3K27me3 suggested a trend towards transcriptional repression in activated cells after HIV-1 infection. Using a qPCR array,we detected genes related to epigenetic processes highly modulated in activated HIV-1 infected cells. SETDB2 and RSK2 transcripts showed highest up-regulation levels. SETDB2 signaling is related to transcriptional silencing while RSK2 is related to either silencing or activation of gene expression depending on the signaling pathway triggered down-stream. In addition,activated cells infected by HIV-1 showed lower CD69 expression and a decrease of IL-2,IFN-γ and metabolism-related factors transcripts indicating a possible functional consequence towards global transcriptional repression found in HIV-1 infected cells. Conversely,based on epigenetic markers studied here,non-stimulated cells infected by HIV-1,showed signs of global transcriptional activation. Our results suggest that HIV-1 infection exerts epigenetic modulations in activated cells that may lead these cells to transcriptional repression with important functional consequences. Moreover,non-stimulated cells seem to increase gene transcription after HIV-1 infection. Based on these observations,it is possible to speculate that the outcome of viral infections may be influenced by the cellular activation status at the moment of infection. View Publication

The extent,regulation and enzymatic basis of RNA editing by cytidine deamination are incompletely understood. Here we show that transcripts of hundreds of genes undergo site-specific CtextgreaterU RNA editing in macrophages during M1 polarization and in monocytes in response to hypoxia and interferons. This editing alters the amino acid sequences for scores of proteins,including many that are involved in pathogenesis of viral diseases. APOBEC3A,which is known to deaminate cytidines of single-stranded DNA and to inhibit viruses and retrotransposons,mediates this RNA editing. Amino acid residues of APOBEC3A that are known to be required for its DNA deamination and anti-retrotransposition activities were also found to affect its RNA deamination activity. Our study demonstrates the cellular RNA editing activity of a member of the APOBEC3 family of innate restriction factors and expands the understanding of CtextgreaterU RNA editing in mammals. View Publication

Targeted nucleases,including zinc-finger nucleases (ZFNs),transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However,realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids,the direct delivery of nuclease proteins to cells provides rapid action and fast turnover,leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression,purification and delivery of ZFN proteins,which are intrinsically cell-permeable; TALEN proteins,which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein,whose nucleofection into cells facilitates rapid induction of multiplexed modifications,are described,along with procedures for evaluating nuclease protein activity. Once they are constructed,nuclease proteins can be expressed and purified within 6 d,and they can be used to induce genomic modifications in human cells within 2 d. View Publication

T cells reactive to β cell Ags are critical players in the development of autoimmune type 1 diabetes. Using a panel of diabetogenic CD4 T cell clones derived from the NOD mouse,we recently identified the β cell secretory granule protein,chromogranin A (ChgA),as a new autoantigen in type 1 diabetes. CD4 T cells reactive to ChgA are pathogenic and rapidly transfer diabetes into young NOD recipients. We report in this article that NOD.ChgA(-/-) mice do not develop diabetes and show little evidence of autoimmunity in the pancreatic islets. Using tetramer analysis,we demonstrate that ChgA-reactive T cells are present in these mice but remain naive. In contrast,in NOD.ChgA(+/+) mice,a majority of the ChgA-reactive T cells are Ag experienced. Our results suggest that the presence of ChgA and subsequent activation of ChgA-reactive T cells are essential for the initiation and development of autoimmune diabetes in NOD mice. View Publication

Studies of repertoires of mouse monoclonal CD4(+) T cells have revealed several mechanisms of self-tolerance; however,which mechanisms operate in normal repertoires is unclear. Here we studied polyclonal CD4(+) T cells specific for green fluorescent protein expressed in various organs,which allowed us to determine the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion,whereas peptides excluded from the thymus were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self antigens. Thus,the immunotolerance of polyclonal CD4(+) T cells was maintained by distinct mechanisms,according to self-peptide expression patterns. View Publication

Myasthenia gravis (MG) is a prototypical autoimmune disease that is among the few for which the target Ag and the pathogenic autoantibodies are clearly defined. The pathology of the disease is affected by autoantibodies directed toward the acetylcholine receptor (AChR). Mature,Ag-experienced B cells rely on the action of Th cells to produce these pathogenic Abs. The phenotype of the MG Ag-reactive T cell compartment is not well defined; thus,we sought to determine whether such cells exhibit both a proinflammatory and a pathogenic phenotype. A novel T cell library assay that affords multiparameter interrogation of rare Ag-reactive CD4(+) T cells was applied. Proliferation and cytokine production in response to both AChR and control Ags were measured from 3120 T cell libraries derived from 11 MG patients and paired healthy control subjects. The frequency of CCR6(+) memory T cells from MG patients proliferating in response to AChR-derived peptides was significantly higher than that of healthy control subjects. Production of both IFN-γ and IL-17,in response to AChR,was also restricted to the CCR6(+) memory T cell compartment in the MG cohort,indicating a proinflammatory phenotype. These T cells also included an elevated expression of GM-CSF and absence of IL-10 expression,indicating a proinflammatory and pathogenic phenotype. This component of the autoimmune response in MG is of particular importance when considering the durability of MG treatment strategies that eliminate B cells,because the autoreactive T cells could renew autoimmunity in the reconstituted B cell compartment with ensuing clinical manifestations. View Publication

Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide-MHC,the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming,low-affinity priming elicited fully differentiated memory T cells with a CD45RB(hi) status. High CD45RB status enabled robust secondary responses in vivo,as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity-primed mice,but not in high affinity-primed mice. Mechanistically,low affinity-primed memory CD8(+) T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RB(hi) status was also a stable marker of priming affinity within polyclonal CD8(+) T cell populations. Following high-affinity rechallenge,low affinity-primed CD45RB(hi) cells became CD45RB(lo),demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8(+) T cells. Thus,these data establish a novel mechanism by which CD45 isoforms tune low affinity-primed memory CD8(+) T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency,low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge. View Publication