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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

Intestinal ischemia-reperfusion (IR)-induced damage requires complement receptor 2 (CR2) for generation of the appropriate natural Ab repertoire. Pathogenic Abs recognize neoantigens on the ischemic tissue,activate complement,and induce intestinal damage. Because C3 cleavage products act as ligands for CR2,we hypothesized that CR2(hi) marginal zone B cells (MZBs) require C3 for generation of the pathogenic Abs. To explore the ability of splenic CR2(+) B cells to generate the damaging Ab repertoire,we adoptively transferred either MZBs or follicular B cells (FOBs) from C57BL/6 or Cr2(-/-) mice into Rag-1(-/-) mice. Adoptive transfer of wild type CR2(hi) MZBs but not CR2(lo) FOBs induced significant damage,C3 deposition,and inflammation in response to IR. In contrast,similarly treated Rag-1(-/-) mice reconstituted with either Cr2(-/-) MZB/B1 B cells (B1Bs) or FOBs lacked significant intestinal damage and displayed limited complement activation. To determine whether C3 cleavage products are critical in CR2-dependent Ab production,we evaluated the ability of the natural Ab repertoire of C3(-/-) mice to induce damage in response to IR. Infusion of C3(-/-) serum into Cr2(-/-) mice restored IR-induced tissue damage. Furthermore,Rag-1(-/-) mice sustained significant damage after infusion of Abs from C3(-/-) but not Cr2(-/-) mice. Finally,adoptive transfer of MZBs from C3(-/-) mice into Rag-1(-/-) mice resulted in significant tissue damage and inflammation. These data indicate that CR2 expression on MZBs is sufficient to induce the appropriate Abs required for IR-induced tissue damage and that C3 is not critical for generation of the pathogenic Abs. View Publication

Pseudo-Pelger-Huët anomaly (PPHA) has been documented in association with transplant medications and other drugs. This iatrogenic neutrophilic dysplasia is reversible with cessation or adjustment of medications but is frequently confused with myelodysplastic syndrome (MDS) based on the conventional concept that PPHA is a marker for dysplasia. We investigated the clinicopathologic features in iatrogenic PPHA and compared them with MDS-related PPHA. The 13 cases studied included 5 bone marrow/stem cell transplantations,3 solid organ transplantations,1 autoimmune disease,3 chronic lymphocytic leukemias,and 1 breast carcinoma. For 12 cases,there was follow-up evaluation,and all demonstrated at least transient normalization of neutrophilic segmentation. All 9 cases of MDS demonstrated at least 2 of the following pathologic abnormalities on bone marrow biopsy: hypercellularity (8/9),morphologic dysplasia (8/9),clonal cytogenetic abnormality (7/9),and increased blasts (3/9),whereas these abnormalities were typically absent in iatrogenic PPHA. Iatrogenic PPHA displayed a higher proportion of circulating PPHA cells than in MDS (mean,47.4%; SD,31.6% vs mean,12.3%; SD,9.8; P textless .01). A diagnostic algorithm is proposed in which isolated PPHA is indicative of transient or benign PPHA unless proven otherwise. View Publication

The antitumor activity of LPS was first described by Dr. William Coley. However,its role in lung cancer remains unclear. The aim of our study was to elucidate the dose-dependent effects of LPS (0.1-10 μg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth increased at 3 and 7 d after the administration of low-dose LPS (0.1 μg/mouse) compared with control mice. This was associated with an influx of plasmacytoid dendritic cells (pDCs),regulatory T cells,myeloid-derived suppressor cells,and CD8(+) regulatory T cells. In contrast,high-dose LPS (10 μg/mouse) reduced lung tumor burden and was associated with a greater influx of pDCs,as well as a stronger Th1 and Th17 polarization. Depletion of pDCs during low-dose LPS administration resulted in a decreased lung tumor burden. Depletion of pDCs during high-dose LPS treatment resulted in an increased tumor burden. The dichotomy in LPS effects was due to the phenotype of pDCs,which were immunosuppressive after the low-dose LPS,and Th1- and T cytotoxic-polarizing cells after the high-dose LPS. Adoptive transfer of T cells into nude mice demonstrated that CD8(+) T cells were responsible for pDC recruitment following low-dose LPS administration,whereas CD4(+) T cells were required for pDC influx after the high-dose LPS. In conclusion,our data suggest differential effects of low-dose versus high-dose LPS on pDC phenotype and tumor progression or regression in the lungs of mice. View Publication

Neutrophils are recruited from the blood to sites of inflammation,where they contribute to immune defense but may also cause tissue damage. During inflammation,neutrophils roll along the microvascular endothelium before arresting and transmigrating. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen 1 (LFA-1),which can be induced by selectin engagement. Here,we demonstrate that a subset of P-selectin glycoprotein ligand-1 (PSGL-1) molecules is constitutively associated with L-selectin. Although this association does not require the known lectin-like interaction between L-selectin and PSGL-1,the signaling output is dependent on this interaction and the cytoplasmic tail of L-selectin. The PSGL-1-L-selectin complex signals through Src family kinases,ITAM domain-containing adaptor proteins,and other kinases to ultimately result in LFA-1 activation. The PSGL-1-L-selectin complex-induced signaling effects on neutrophil slow rolling and recruitment in vivo demonstrate the functional importance of this pathway. We conclude that this is a signaling complex specialized for sensing adhesion under flow. View Publication

Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1,a cell surface glycoprotein expressed on MM cells. In preclinical models,elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein,we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1-CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary,human MM cells. Taken together,these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone. View Publication

Circulating levels of leptin are elevated in type-2 diabetes mellitus (T2DM) and leptin plays a role in immune responses. Elevated circulating IL-18 levels are associated with clinical complications of T2DM. IL-18 regulates cytokine secretion and the function of a number of immune cells including T-cells,neutrophils and macrophages and as such has a key role in immunity and inflammation. Pro-inflammatory monocytes exhibiting elevated cytokine secretion are closely associated with inflammation in T2DM,however,little is known about the role of leptin in modifying monocyte IL-18 secretion. We therefore aimed to investigate the effect of leptin on IL-18 secretion by monocytes. We report herein that leptin increases IL-18 secretion in THP-1 and primary human monocytes but has no effect on IL-18mRNA. Leptin and LPS signalling in monocytes occurs by overlapping but distinct pathways. Thus,in contrast to a strong stimulation by LPS,leptin has no effect on IL-1βmRNA levels or IL-1β secretion. In addition,LPS stimulates the secretion of IL-6 but leptin did not whereas both treatments up regulate IL-8 secretion from the same cells. Although leptin (and LPS) has a synergistic effect with exogenous ATP on IL-18 secretion in both THP-1 and primary monocytes,experiments involving ATP assays and pharmacological inhibition of ATP signalling failed to provide any evidence that endogenous ATP secreted by leptin-stimulated monocytes was responsible for enhancement of monocyte IL-18 secretion by leptin. Analysis of the action of caspase-1 revealed that leptin up regulates caspase-1 activity and the effect of leptin on IL-18 release is prevented by caspase-1 inhibitor (Ac-YVAD-cmk). These data suggest that leptin activates IL-18 processing rather than IL-18 transcription. In conclusion,leptin enhances IL-18 secretion via modulation of the caspase-1 inflammasome function and acts synergistically with ATP in this regard. This process may contribute to aberrant immune responses in T2DM and other conditions of hyperleptinemia. View Publication

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