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

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However,little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional,or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite,thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria. View Publication

Mesenchymal stem cells (MSCs) are capable of down-regulating alloimmune responses and promoting the engraftment of hematopoietic stem cells. MSCs may therefore be suitable for improving donor-specific tolerance induction in solid-organ transplantation. Cells from cadaveric vertebral bone marrow (V-BM),aspirated iliac crest-BM,and peripheral blood progenitor cells were compared. Cells were characterized by flow cytometry and colony assays. MSCs generated from V-BM were assayed for differentiation capacity and immunomodulatory function. A median 5.7 x 10(8) nucleated cells (NCs) were recovered per vertebral body. The mesenchymal progenitor,colony-forming unit-fibroblast,frequency in V-BM (11.6/10(5) NC,range: 6.0-20.0) was considerably higher than in iliac crest-BM (1.4/10(5) NC,range: 0.4-2.6) and peripheral blood progenitor cells (not detectable). MSC generated from V-BM had the typical MSC phenotype (CD105(pos)CD73(pos)CD45(neg)CD34(neg)),displayed multilineage differentiation potential,and suppressed alloreactivity in mixed lymphocyte reactions. V-BM may be an excellent source for MSC cotransplantation approaches. View Publication

Mycoplasma pneumoniae is a human respiratory tract pathogen causing acute and chronic airway disease states that can include long-term carriage and extrapulmonary spread. The mechanisms of persistence and migration beyond the conducting airways,however,remain poorly understood. We previously described an acute exposure model using normal human bronchial epithelium (NHBE) in air-liquid interface culture,showing that M. pneumoniae gliding motility is essential for initial colonisation and subsequent spread,including localisation to epithelial cell junctions. We extended those observations here,characterizing M. pneumoniae infection of NHBE for up to 4 weeks. Colonisation of the apical surface was followed by pericellular invasion of the basolateral compartment and migration across the underlying transwell membrane. Despite fluctuations in transepithelial electrical resistance and increased NHBE cell desquamation,barrier function remained largely intact. Desquamation was accompanied by epithelial remodelling that included cytoskeletal reorganisation and development of deep furrows in the epithelium. Finally,M. pneumoniae strains S1 and M129 differed with respect to invasion and histopathology,consistent with contrasting virulence in experimentally infected mice. In summary,this study reports pericellular invasion,NHBE cytoskeletal reorganisation,and tissue remodelling with persistent infection in a human airway epithelium model,providing clear insight into the likely route for extrapulmonary spread. View Publication

Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death,reactive oxygen species production,mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq,as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally,we show that CRISPR-Cas9-mediated disruption of TOP2B,a gene implicated in DIC in mouse studies,significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response. View Publication

RATIONALE: Cardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis,the key process underlying excitation-contraction coupling. OBJECTIVE: We investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs). METHODS AND RESULTS: We differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes,spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore,in contrast with hESC-derived cardiomyocytes,ryanodine did not reduce the amplitudes,maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition,spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2),sacroplasmic recticulum calcium-ATPase (SERCA),junction (Jun) and triadin (TRDN),were significantly lower in hiPSC than in hESCs. CONCLUSIONS: The results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts. View Publication

Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing,full-length mRNA isoforms are not captured. On the other hand,third-generation sequencing,which yields much longer reads,has current limitations of lower raw accuracy and throughput. Here,we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms,including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover,their reduced expression perturbs the network of pluripotency-associated genes. Results suggest that gene identification,even in well-characterized human cell lines and tissues,is likely far from complete. View Publication

Abs have been shown to be protective in passive immunotherapy of tuberculous infection using mouse experimental models. In this study,we report on the properties of a novel human IgA1,constructed using a single-chain variable fragment clone (2E9),selected from an Ab phage library. The purified Ab monomer revealed high binding affinities for the mycobacterial ?-crystallin Ag and for the human Fc?RI (CD89) IgA receptor. Intranasal inoculations with 2E9IgA1 and recombinant mouse IFN-? significantly inhibited pulmonary H37Rv infection in mice transgenic for human CD89 but not in CD89-negative littermate controls,suggesting that binding to CD89 was necessary for the IgA-imparted passive protection. 2E9IgA1 added to human whole-blood or monocyte cultures inhibited luciferase-tagged H37Rv infection although not for all tested blood donors. Inhibition by 2E9IgA1 was synergistic with human rIFN-? in cultures of purified human monocytes but not in whole-blood cultures. The demonstration of the mandatory role of Fc?RI (CD89) for human IgA-mediated protection is important for understanding of the mechanisms involved and also for translation of this approach toward development of passive immunotherapy of tuberculosis. View Publication

The vascular wall is a source of progenitor cells that are able to induce skeletal repair,primarily by paracrine mechanisms. Here,the paracrine role of extracellular vesicles (EVs) in bone healing was investigated. First,purified human perivascular stem cells (PSCs) were observed to induce mitogenic,pro-migratory,and pro-osteogenic effects on osteoprogenitor cells while in non-contact co-culture via elaboration of EVs. PSC-derived EVs shared mitogenic,pro-migratory,and pro-osteogenic properties of their parent cell. PSC-EV effects were dependent on surface-associated tetraspanins,as demonstrated by EV trypsinization,or neutralizing antibodies for CD9 or CD81. Moreover,shRNA knockdown in recipient cells demonstrated requirement for the CD9/CD81 binding partners IGSF8 and PTGFRN for EV bioactivity. Finally,PSC-EVs stimulated bone repair,and did so via stimulation of skeletal cell proliferation,migration,and osteodifferentiation. In sum,PSC-EVs mediate the same tissue repair effects of perivascular stem cells,and represent an 'off-the-shelf' alternative for bone tissue regeneration. View Publication

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. View Publication

Myeloid Derived Suppressor Cells (MDSCs) play important roles in constituting the immune suppressive environment promoting cancer development and progression. They are consisted of a heterogeneous population of immature myeloid cells including polymorphonuclear MDSC (PMN-MDSC) and monocytes MDSC (M-MDSC) that are found in both the systemic circulation and in the tumor microenvironment (TME). While previous studies had shown that all-trans retinoic acid (ATRA) could induce MDSC differentiation and maturation,the very poor solubility and fast metabolism of the drug limited its applications as an immune-modulator for cancer immunotherapy. We aimed in this study to develop a drug encapsulated liposome formulation L-ATRA with sustained release properties and examined the immuno-modulation effects. We showed that the actively loaded L-ATRA achieved stable encapsulation and enabled controlled drug release and accumulation in the tumor tissues. In vivo administration of L-ATRA promoted the remodeling of the systemic immune homeostasis as well as the tumor microenvironment. They were found to promote MDSCs maturation into DCs and facilitate immune responses against cancer cells. When used as a single agent treatment,L-ATRA deterred tumor growth,but only in immune-competent mice. In mice with impaired immune functions,L-ATRA at the same dose was not effective. When combined with checkpoint inhibitory agents,L-ATRA resulted in greater anti-cancer activities. Thus,L-ATRA may present a new IO strategy targeting the MDSCs that needs be further explored for improving the immunotherapy efficacy in cancer. View Publication

Rationale: IgA can induce activation of neutrophils which are the most abundant cell type in blood,but the development of IgA as therapeutic has been confounded by its short half-life and a weak ability to recruit NK cells as effector cells. Therefore,we generated an X-shaped antibody (X-body) based on the principle of molecular self-assembly that combines the activities of both IgG and IgA,which can effectively recruit and activate NK cells,macrophages,and neutrophils to kill tumor cells. Methods: X-body was generated by using a self-assembly strategy. The affinity of the X-body with the antigen and Fc receptors was tested by surface plasmon resonance. The shape of X-body was examined using negative staining transmission electron microscopy. The tumor cell killing activity of X-body was assessed in vitro and in multiple syngeneic mouse models. To explore the mechanism of X-body,tumor-infiltrating immune cells were analyzed by single-cell RNA-seq and flow cytometry. The dependence of neutrophil,macrophage,and NK cells for the X-body efficacy was confirmed by in vivo depletion of immune cell subsets. Results: The X-body versions of rituximab and trastuzumab combined the full spectrum activity of IgG and IgA and recruited NK cells,macrophages,and neutrophils as effector cells for eradication of tumor cells. Treatment with anti-hCD20 and anti-hHER2 X-bodies leads to a greater reduction in tumor burden in tumor-bearing mice compared with the IgA or IgG counterpart,and no obvious adverse effect is observed upon X-body treatment. Moreover,the X-body has a serum half-life and drug stability comparable to IgG. Conclusions: The X-body,as a myeloid-cell-centered therapeutic strategy,holds promise for the development of more effective cancer-targeting therapies than the current state of the art. View Publication