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

The relevance of blood-based assays to monitor minimal residual disease (MRD) in non-metastatic prostate cancer (PCa) remains unclear. Proving that clinically relevant circulating tumor cells (CTCs) can be detected with available technologies could address this. This study aimed to improve CTC detection in non-metastatic PCa patients by combining three independent CTC assays: the CellSearch system,an in vivo CellCollector and the EPISPOT. Peripheral blood samples from high-risk PCa patients were screened for CTCs before and three months after radical prostatectomy (RP). Combining the results of both time points,CTCs were detected in 37{\%},54.9{\%} and 58.7{\%} of patients using CellSearch,CellCollector and EPISPOT,respectively. The cumulative positivity rate of the three CTC assays was 81.3{\%} (87/107) with 21.5{\%} (23/107) of patients harboring ≥5 CTCs/7.5 ml blood. Matched pair analysis of 30 blood samples taken before and after surgery indicated a significant decrease in CTCs captured by the CellCollector from 66{\%} before RP to 34{\%} after therapy (p = 0.031). CTC detection by EPISPOT before RP significantly correlated with PSA serum values (p {\textless} 0.0001) and clinical tumor stage (p = 0.04),while the other assays showed no significant correlations. In conclusion,CTC-based liquid biopsies have the potential to monitor MRD in patients with non-metastatic prostate cancer. View Publication

Although studies have identified the presence of gut-associated cells in the enthesis of joints affected by spondylarthritis (SpA),a direct link through cellular transit between the gut and joint has yet to be formally demonstrated. Using KikGR transgenic mice to label in situ and track cellular trafficking from the distal colon to the joint under inflammatory conditions of both the gut and joint,we demonstrate bona-fide gut-joint trafficking of T cells from the colon epithelium,also called intraepithelial lymphocytes (IELs),to distal sites including joint enthesis,the pathogenic site of SpA. Similar to patients with SpA,colon IELs from the TNF$\Delta$ARE/+ mouse model of inflammatory bowel disease and SpA display heightened TNF production upon stimulation. Using ex vivo stimulation of photo-labeled gut-joint trafficked T cells from the popliteal lymph nodes of KikGR and KikGR TNF$\Delta$ARE/+ we saw that the CD4+ photo-labeled population was highly enriched for IL-17 competence in healthy as well as arthritic mice,however in the TNF$\Delta$ARE/+ mice these cells were additionally enriched for TNF. Using transfer of magnetically isolated IELs from TNF+/+ and TNF$\Delta$ARE/+ donors into Rag1 -/- hosts,we confirmed that IELs can exacerbate inflammatory processes in the joint. Finally,we blocked IEL recruitment to the colon epithelium using broad spectrum antibiotics in TNF$\Delta$ARE/+ mice. Antibiotic-treated mice had reduced gut-joint IEL migration,contained fewer Il-17A and TNF competent CD4+ T cells,and lessened joint pathology compared to untreated littermate controls. Together these results demonstrate that pro-inflammatory colon-derived IELs can exacerbate inflammatory responses in the joint through systemic trafficking,and that interference with this process through gut-targeted approaches has therapeutic potential in SpA. View Publication

SummaryMotivated by the cellular heterogeneity in complex tissues,particularly in brain and induced pluripotent stem cell (iPSC)-derived brain models,we developed a complete workflow to reproducibly characterize cell types in complex tissues. Our approach combines a flow cytometry (FC) antibody panel with our computational pipeline CelltypeR,enabling dataset aligning,unsupervised clustering optimization,cell type annotating,and statistical comparisons. Applied to human iPSC derived midbrain organoids,it successfully identified the major brain cell types. We performed fluorescence-activated cell sorting of CelltypeR-defined astrocytes,radial glia,and neurons,exploring transcriptional states by single-cell RNA sequencing. Among the sorted neurons,we identified subgroups of dopamine neurons: one reminiscent of substantia nigra cells most vulnerable in Parkinson’s disease. Finally,we used our workflow to track cell types across a time course of organoid differentiation. Overall,our adaptable analysis framework provides a generalizable method for reproducibly identifying cell types across FC datasets in complex tissues. Graphical abstract Highlights•CelltypeR is a flow cytometry and computational pipeline for cell type quantification•Identified brain cell types in midbrain organoids and measured changes in proportions•Enriched selected populations using FACS and characterized by single-cell RNA sequencing•Identified substantia nigra–like dopaminergic neurons sensitive in Parkinson’s disease Neuroscience; Cell biology; Omics View Publication

Supercentenarians (≥110-year-old,SC) are a uniquely informative population not only because they surpass centenarians in age,but because they appear to age more slowly with fewer incidences of chronic age-related disease than centenarians. We reprogramed donor B-lymphoblastoid cell lines (LCL) derived from a 114-year-old (SC),a 43-year-old healthy disease-free control (HDC) and an 8-year-old with a rapid aging disease (Hutchinson-Gilford progeria syndrome (HGPS)) and compared SC-iPSC to HDC-iPSC and HGPS-iPSCs. Reprogramming to pluripotency was confirmed by pluripotency marker expression and differentiation to 3 germ-layers. Each iPSC clone differentiated efficiently to mesenchymal progenitor cells (MPC) as determined by surface marker expression and RNAseq analysis. We identified supercentenarian and HGPS associated gene expression patterns in the differentiated MPC lines that were not evident in the parental iPSC lines. Importantly,telomere length resetting occurred in iPSC from all donors albeit at a lower incidence in supercentenarian iPSCs. These data indicate the potential to use reprogramming to reset both developmental state and cellular age in the oldest of the old." We anticipate that supercentenarian iPSC and their differentiated derivatives will be valuable tools for studying the underlying mechanisms of extreme longevity and disease resistance." View Publication

BACKGROUND Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently,NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here,we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling. RESULTS FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells,Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN),the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hepatocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPK$\alpha$ activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 significantly activated AMPK$\alpha$ and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. CONCLUSION Taken together,these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a potential treatment option for HCC. View Publication

Graphical Abstract Highlights d SMAD activity is active in suprabasal cells but is weaker in basal epithelial cells d SMAD signaling activity correlates with mucociliary differentiation in the airway d Dual TGFb/BMP inhibition prevents spontaneous differentiation in culture d Dual TGFb/BMP inhibition allows prolonged culture of diverse epithelial basal cells Correspondence jrajagopal@partners.org In Brief Mou et al. show that small-molecule-mediated SMAD signaling inhibition allows prolonged feeder-free culture of diverse functional epithelial basal stem cells in a 2D format. This methodology provides a facile patient-specific epithelial disease modeling platform,as shown by the expansion of airway epithelium from non-invasively obtained specimens from cystic fibrosis patients. View Publication

Autosomal recessive osteopetrosis (ARO) is a group of genetic disorders that involve defects that preclude the normal function of osteoclasts,which differentiate from hematopoietic precursors. In half of human cases,ARO is the result of mutations in the TCIRG1 gene,which codes for a subunit of the vacuolar proton pump that plays a fundamental role in the acidification of the cell-bone interface. Functional mutations of this pump severely impair the resorption of bone mineral. Although postnatal hematopoietic stem cell transplantation can partially rescue the hematological phenotype of ARO,other stigmata of the disease,such as secondary neurological and growth defects,are not reversed. For this reason,ARO is a paradigm for genetic diseases that would benefit from effective prenatal treatment. Using the oc/oc mutant mouse,a murine model whose osteopetrotic phenotype closely recapitulates human TCIRG1-dependent ARO,we report that in utero transplantation of adult bone marrow hematopoietic stem cells can correct the ARO phenotype in a limited number of mice. Here we report that in utero injection of allogeneic fetal liver cells,which include hematopoietic stem cells,into oc/oc mouse fetuses at 13.5 days post coitum produces a high level of engraftment,and the oc/oc phenotype is completely rescued in a high percentage of these mice. Therefore,oc/oc pathology appears to be particularly sensitive to this form of early treatment of the ARO genetic disorder. View Publication

HOX genes,notably members of the HOXA cluster,and HOX cofactors have increasingly been linked to human leukemia. Intriguingly,HOXD13,a member of the HOXD cluster not normally expressed in hematopoietic cells,was recently identified as a partner of NUP98 in a t(2;11) translocation associated with t-AML/MDS. We have now tested directly the leukemogenic potential of the NUP98-HOXD13 t(2; 11) fusion gene in the murine hematopoietic model. NUP98-HOXD13 strongly promoted growth and impaired differentiation of early hematopoietic progenitor cells in vitro; this effect was dependent on the NUP98 portion and an intact HOXD13 homeodomain. Expression of the NUP98-HOXD13 fusion gene in vivo resulted in a partial impairment of lymphopoiesis but did not induce evident hematologic disease until late after transplantation (more than 5 months),when some mice developed a myeloproliferative-like disease. In contrast,mice transplanted with bone marrow (BM) cells cotransduced with NUP98-HOXD13 and the HOX cofactor Meis1 rapidly developed lethal and transplantable acute myeloid leukemia (AML),with a median disease onset of 75 days. In summary,this study demonstrates that NUP98-HOXD13 can be directly implicated in the molecular process leading to leukemic transformation,and it supports a model in which the transforming properties of NUP98-HOXD13 are mediated through HOX-dependent pathways. View Publication

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly,this type of immunoglobulin usually pairs with the single germline VL gene,V30 that is typically very conserved in sequence. In this work,we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection,EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly,engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-$\gamma$. Taken together,this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications. View Publication

Despite the fact that glucocorticoids and long acting beta agonists are effective treatments for asthma,their effects on human mast cells (MC) appear to be modest. Although MC are one of the major effector cells in the underlying inflammatory reactions associated with asthma,their regulation by these drugs is not yet fully understood and,in some cases,controversial. Using a human immortalized MC line (LAD2),we studied the effects of fluticasone propionate (FP) and salmeterol (SM),on the release of early and late phase mediators. LAD2 cells were pretreated with FP (100 nM),SM (1 µM),alone and in combination,at various incubation times and subsequently stimulated with agonists substance P,C3a and IgE/anti-IgE. Degranulation was measured by the release of β-hexosaminidase. Cytokine and chemokine expression were measured using quantitative PCR,ELISA and cytometric bead array (CBA) assays. The combination of FP and SM synergistically inhibited degranulation of MC stimulated with substance P (33% inhibition compared to control,n = 3,P>05). Degranulation was inhibited by FP alone,but not SM,when MC were stimulated with C3a (48% inhibition,n = 3,P>05). As previously reported,FP and SM did not inhibit degranulation when MC were stimulated with IgE/anti-IgE. FP and SM in combination inhibited substance P-induced release of tumor necrosis factor (TNF),CCL2,and CXCL8 (98%,99% and 92% inhibition,respectively,n = 4,P>05). Fluticasone and salmeterol synergistically inhibited mediator production by human MC stimulated with the neuropeptide substance P. This synergistic effect on mast cell signaling may be relevant to the therapeutic benefit of combination therapy in asthma. View Publication

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) are a potentially unlimited source of cardiomyocytes (CMs) for cardiac transplantation therapies. The establishment of pure PSC-CM populations is important for this application,but is hampered by a lack of CM-specific surface markers suitable for their identification and sorting. Contemporary purification techniques are either non-specific or require genetic modification. We report a second harmonic generation (SHG) signal detectable in PSC-CMs that is attributable to sarcomeric myosin,dependent on PSC-CM maturity,and retained while PSC-CMs are in suspension. Our study demonstrates the feasibility of developing a SHG-activated flow cytometer for the non-invasive purification of PSC-CMs. View Publication

IL-27,which is produced by activated APCs,bridges innate and adaptive immunity by regulating the development of Th cells. Recent evidence supports a role for IL-27 in the activation of monocytic cells in terms of inflammatory responses. Indeed,proinflammatory and anti-inflammatory activities are attributed to IL-27,and IL-27 production itself is modulated by inflammatory agents such as LPS. IL-27 primes LPS responses in monocytes; however,the molecular mechanism behind this phenomenon is not understood. In this study,we demonstrate that IL-27 priming results in enhanced LPS-induced IL-6,TNF-α,MIP-1α,and MIP-1β expression in human primary monocytes. To elucidate the molecular mechanisms responsible for IL-27 priming,we measured levels of CD14 and TLR4 required for LPS binding. We determined that IL-27 upregulates TLR4 in a STAT3- and NF-κB-dependent manner. Immunofluorescence microscopy revealed enhanced membrane expression of TLR4 and more distinct colocalization of CD14 and TLR4 upon IL-27 priming. Furthermore,IL-27 priming enhanced LPS-induced activation of NF-κB family members. To our knowledge,this study is the first to show a role for IL-27 in regulating TLR4 expression and function. This work is significant as it reveals new mechanisms by which IL-27 can enhance proinflammatory responses that can occur during bacterial infections. View Publication