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

Background/Objectives: The limited translatability of preclinical experimental findings to patients remains an obstacle for successful treatment of brain diseases. Relevant models to elucidate mechanisms behind brain pathogenesis,including cell-specific contributions and cell-cell interactions,and support successful targeting and prediction of drug responses in humans are urgently needed,given the species differences in brain and blood-brain barrier (BBB) functions. Human microphysiological systems (MPS),such as Organ-Chips,are emerging as a promising approach to address these challenges. Here,we examined and advanced a Brain-Chip that recapitulates aspects of the human cortical parenchyma and the BBB in one model. Methods: We utilized human primary astrocytes and pericytes,human induced pluripotent stem cell (hiPSC)-derived cortical neurons,and hiPSC-derived brain microvascular endothelial-like cells and included for the first time on-chip hiPSC-derived microglia. Results: Using Tumor necrosis factor alpha (TNF?) to emulate neuroinflammation,we demonstrate that our model recapitulates in vivo-relevant responses. Importantly,we show microglia-derived responses,highlighting the Brain-Chip’s sensitivity to capture cell-specific contributions in human disease-associated pathology. We then tested BBB crossing of human transferrin receptor antibodies and conjugated adeno-associated viruses. We demonstrate successful in vitro/in vivo correlation in identifying crossing differences,underscoring the model’s capacity as a screening platform for BBB crossing therapeutic strategies and ability to predict in vivo responses. Conclusions: These findings highlight the potential of the Brain-Chip as a reliable and time-efficient model to support therapeutic development and provide mechanistic insights into brain diseases,adding to the growing evidence supporting the value of MPS in translational research and drug discovery. View Publication

Siglec-6 is a lectin receptor with restricted expression in the placenta,mast cells and memory B-cells. Although Siglec-6 is expressed in patients with chronic lymphocytic leukemia (CLL),its pathophysiological role has not been elucidated. We describe here a role for Siglec-6 in migration and adhesion of CLL B cells to CLL- bone marrow stromal cells (BMSCs) in vitro and compromised migration to bone marrow and spleen in vivo. Mass spectrometry analysis revealed interaction of Siglec-6 with DOCK8,a guanine nucleotide exchange factor. Stimulation of MEC1-002 CLL cells with a Siglec-6 ligand,sTn,results in Cdc42 activation,WASP protein recruitment and F-actin polymerization,which are all associated with cell migration. Therapeutically,a Siglec-6/CD3-bispecific T-cell-recruiting antibody (T-biAb) improves overall survival in an immunocompetent mouse model and eliminates CLL cells in a patient derived xenograft model. Our findings thus reveal a migratory role for Siglec-6 in CLL,which can be therapeutically targeted using a Siglec-6 specific T-biAb. Siglec-6 is often overexpressed in chronic lymphocytic leukaemia (CLL),but its role is unclear. Here,the author report that Siglec-6 regulates the migration and adhesion of CLL B cells via interaction with sialyl Tn on bone marrow stromal cells driving invasion which could be therapeutically targeted using a Siglec-6/CD3-bispecfiic antibody. View Publication

BackgroundFANCA mutations have been detected in a variety of cancers and found to be pro-carcinogenic. However,no functional studies have been identified regarding the involvement of FANCA in the occurrence and the immune response of LUAD.MethodsThe mRNA expression and overall survival rates of FANCA were evaluated by the TIMER,PrognoScan and TCGA database in LUAD tissues,and FANCA expression was further validated by clinical serum samples using ELISA. The correlation between FANCA and immune infiltration level was investigated via TISIDB database and CIBERSORT algorithm. The Kaplan–Meier plotter was used to further evaluate the prognostic value based on the expression levels of FANCA in related immune cells. Then,the influence of FANCA knockout on the proliferation,migration,and invasion of A549 and H1299 cells was validated using CCK8,cloning formation,and Transwell assays. Subsequently,HLA-A2-restricted FANCA antigenic peptides were predicted and synthesized by NetMHC4.0 and SYFPEITHI,and DCs were induced and cultured in vitro. Finally,DCs loaded with HLA-A2-restricted FANCA antigenic peptides were co-cultured with autologous peripheral blood lymphocyte to generate specific CTLs. The killing effects of different CTLs on LUAD cells were studied.ResultsThe results showed that high levels of FANCA in patients with LUAD were significantly correlated with worse OS survival,which was correlated with age,clinical stage,pathological T stage,M stage,and N stage in LUAD. Knockdown of FANCA in A549 and H1299 cells significantly inhibited proliferation,metastasis,and invasion in vitro. In addition,FANCA was significantly related to immune infiltrate,genomic alterations and TMB. FANCA expression infuenced the prognosis of LUAD patients by directly affecting immune cell infltration. Finally,HLA-A2-restricted FANCA antigenic peptides were synthesized. And FANCA 146–154 (SLLEFAQYL) antigenic peptide exhibit a stronger affinity for DCs,and induce CTLs to produce stronger targeted killing ability for LUAD cells at an effector-to-target ratio of 40:1.ConclusionThese results demonstrated that the elevation of FANCA promotes malignant phenotype of LUAD,and the potential peptide P2 (SLLEFAQYL) derived from FANCA may be used as an epitope vaccine for the treatment of LUAD. View Publication

Neoscytalidium dimidiatum is a non-dermatophyte mold that commonly causes skin and nail infections in tropical regions and often resists conventional antifungal therapies. Because its clinical and laboratory features often resemble dermatophyte infections,diagnosis is frequently delayed and treatment is sometimes inappropriate. We therefore developed a dot-immunobinding assay (Dot-Iba) to detect N. dimidiatum antigens. We generated a highly specific monoclonal antibody,3E6F7 (MAb 3E6F7),for antigen capture,and used goat anti-mouse Ig conjugated with alkaline phosphatase (AP) as the signal generator. The test pad comprised a test hole,a nitrocellulose membrane (NC),and water-absorbent pads in a vertical flow-through format to allow a rapid antigen–antibody reaction. The assembled system detected N. dimidiatum antigens in vitro with high specificity and yielded visible results within 2 h; its detection limit was 0.9 µg without cross-reactivity to dermatophyte or non-dermatophyte fungi. This rapid,specific,and easy-to-use assay shows strong potential as a diagnostic tool,particularly in settings with limited access to fungal culture or advanced molecular diagnostics,where early,accurate identification is crucial. View Publication

Several recent reports suggest that there is far more plasticity that previously believed in the developmental potential of bone-marrow-derived cells (BMCs) that can be induced by extracellular developmental signals of other lineages whose nature is still largely unknown. In this study,we demonstrate that bone-marrow-derived mesenchymal stem cells (MSCs) co-cultured with mouse proliferating or fixed (by paraformaldehyde or methanol) neural stem cells (NSCs) generate neural stem cell-like cells with a higher expression of Sox-2 and nestin when grown in NS-A medium supplemented with N2,NSC conditioned medium (NSCcm) and bFGF. These neurally induced MSCs eventually differentiate into beta-III-tubulin and GFAP expressing cells with neuronal and glial morphology when grown an additional week in Neurobasal/B27 without bFGF. We conclude that juxtacrine interaction between NSCs and MSCs combined with soluble factors released from NSCs are important for generation of neural-like cells from bone-marrow-derived adherent MSCs. View Publication

Interleukin-15 (IL-15) is essential for natural killer (NK) cell differentiation. In this study,we assessed whether the receptor tyrosine kinase Axl and its ligand,Gas6,are involved in IL-15-mediated human NK differentiation from CD34(+) hematopoietic progenitor cells (HPCs). Blocking the Axl-Gas6 interaction with a soluble Axl fusion protein (Axl-Fc) or the vitamin K inhibitor warfarin significantly diminished the absolute number and percentage of CD3(-)CD56(+) NK cells derived from human CD34(+) HPCs cultured in the presence of IL-15,probably resulting in part from reduced phosphorylation of STAT5. In addition,CD3(-)CD56(+) NK cells derived from culture of CD34(+) HPCs with IL-15 and Axl-Fc had a significantly diminished capacity to express interferon-gamma or its master regulator,T-BET. Culture of CD34(+) HPCs in the presence of c-Kit ligand and Axl-Fc resulted in a significant decrease in the frequency of NK precursor cells responding to IL-15,probably the result of reduced c-Kit phosphorylation. Collectively,our data suggest that the Axl/Gas6 pathway contributes to normal human NK-cell development,at least in part via its regulatory effects on both the IL-15 and c-Kit signaling pathways in CD34(+) HPCs,and to functional NK-cell maturation via an effect on the master regulatory transcription factor T-BET. View Publication

There is an enormous gap between the antiproliferative and in vivo antitumor efficacy of gemcitabine in cell line-based models and its clinical efficacy. This may be due to insensitiveness of the precursor,cancer stem cell (CSC) compartment to cytotoxic agents. The hedgehog pathway is associated with CSC signaling and control. We used a direct xenograft model of pancreatic cancer and a two-stage approach was used to test the hypotheses that targeting CSC could increase the efficacy of gemcitabine. Tumors from a gemcitabine-sensitive xenograft were treated with gemcitabine first,and randomized,after tumor regression to continuing treatment with gemcitabine,a hedgehog inhibitor alone or in combination with gemcitabine. We tested markers described as associated with CSC such as CD24,CD44,ALDH,nestin,and the hedgehog pathway. After induction with gemcitabine,treated tumor showed an enrichment in CSC markers such as ALDH and CD24. Subsequently,a release from gemcitabine prompted a repopulation of proliferating cells and a decrease in such markers to equilibrate from pretreatment levels. Combined treatment with gemcitabine and cyclopamine induced tumor regression and decrease in CSC markers and hedgehog signaling. Cytoplasmic CD24 and ALDH were inversely and strongly associated with growth and were expressed in a minority of cells that we propose constitute the CSC compartment. Hedgehog inhibitors as part of a dual compartment therapeutic approach were able to further reduce tumor growth and decreased both static and dynamic markers of CSC. Direct tumor xenografts are a valid platform to test multicompartment therapeutic approaches in pancreatic cancer. View Publication

PURPOSE: To evaluate the expression of stem cell-related markers at the cellular level in human breast tumors of different subtypes and histologic stage. EXPERIMENTAL DESIGN: We performed immunohistochemical analyses of 12 proteins [CD44,CD24,ALDH1,vimentin,osteonectin,EPCR,caveolin 1,connexin 43,cytokeratin 18 (CK18),MUC1,claudin 7,and GATA3] selected based on their differential expression in breast cancer cells with more differentiated and stem cell-like characteristics in 47 cases of invasive ductal carcinoma (IDC) only,135 cases of IDC with ductal carcinoma in situ (DCIS),35 cases of DCIS with microinvasion,and 58 cases of pure DCIS. We also analyzed 73 IDCs with adjacent DCIS to determine the differences in the expression of markers by histology within individual tumors. CD44+/CD24- and CD24-/CD24+ cells were detected using double immunohistochemistry. RESULTS: CD44 and EPCR expression was different among the four histologic groups and was lower in invasive compared with in situ tumors,especially in luminal A subtype. The expression of vimentin,osteonectin,connexin 43,ALDH1,CK18,GATA3,and MUC1 differed by tumor subtype in some histologic groups. ALDH1-positive cells were more frequent in basal-like and HER2+ than in luminal tumors. CD44+/CD24- cells were detected in 69% of all tumors with 100% of the basal-like and 52% of HER2+ tumors having some of these cells. CONCLUSIONS: Our findings suggest that in breast cancer,the frequency of tumor cells positive for stem cell-like and more differentiated cell markers varies according to tumor subtype and histologic stage. View Publication

Raman spectra often contain undesirable,randomly positioned,intense,narrow-bandwidth,positive,unidirectional spectral features generated when cosmic rays strike charge-coupled device cameras. These must be removed prior to analysis,but doing so manually is not feasible for large data sets. We developed a quick,simple,effective,semi-automated procedure to remove cosmic ray spikes from spectral data sets that contain large numbers of relatively homogenous spectra. Although some inhomogeneous spectral data sets can be accommodated—it requires replacing excessively modified spectra with the originals and removing their spikes with a median filter instead—caution is advised when processing such data sets. In addition,the technique is suitable for interpolating missing spectra or replacing aberrant spectra with good spectral estimates. The method is applied to baseline-flattened spectra and relies on fitting a third-order (or higher) polynomial through all the spectra at every wavenumber. Pixel intensities in excess of a threshold of 3× the noise standard deviation above the fit are reduced to the threshold level. Because only two parameters (with readily specified default values) might require further adjustment,the method is easily implemented for semi-automated processing of large spectral sets. View Publication

The neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) is caused by a CAG-repeat expansion in the ATXN3 gene. In this study,induced pluripotent stem cell (iPSC) lines were established from two SCA3 patients. Dermal fibroblasts were reprogrammed using an integration-free method and the resulting SCA3 iPSCs were differentiated into neurons. These neuronal lines harbored the disease causing mutation,expressed comparable levels of several neuronal markers and responded to the neurotransmitters,glutamate/glycine,GABA and acetylcholine. Additionally,all neuronal cultures formed networks displaying synchronized spontaneous calcium oscillations within 28days of maturation,and expressed the mature neuronal markers NeuN and Synapsin 1 implying a relatively advanced state of maturity,although not comparable to that of the adult human brain. Interestingly,we were not able to recapitulate the glutamate-induced ataxin-3 aggregation shown in a previously published iPSC-derived SCA3 model. In conclusion,we have generated a panel of SCA3 patient iPSCs and a robust protocol to derive neurons of relatively advanced maturity,which could potentially be valuable for the study of SCA3 disease mechanisms. View Publication

Thiazolidinedione derivatives are antidiabetic agents that increase the insulin sensitivity of target tissues in animal models of non-insulin-dependent diabetes mellitus. In vitro,thiazolidinediones promote adipocyte differentiation of preadipocyte and mesenchymal stem cell lines; however,the molecular basis for this adipogenic effect has remained unclear. Here,we report that thiazolidinediones are potent and selective activators of peroxisome proliferator-activated receptor gamma (PPAR gamma),a member of the nuclear receptor superfamily recently shown to function in adipogenesis. The most potent of these agents,BRL49653,binds to PPAR gamma with a Kd of approximately 40 nM. Treatment of pluripotent C3H10T1/2 stem cells with BRL49653 results in efficient differentiation to adipocytes. These data are the first demonstration of a high affinity PPAR ligand and provide strong evidence that PPAR gamma is a molecular target for the adipogenic effects of thiazolidinediones. Furthermore,these data raise the intriguing possibility that PPAR gamma is a target for the therapeutic actions of this class of compounds. View Publication