搜索结果: 'gmp t cell activator'

Background/Objectives: The SARS-CoV-2’s high mutations and replication rates contribute to its high infectivity and resistance to current vaccinations and treatments. The primary cause of resistance to most current treatments aligns within the coding regions for the spike S protein of SARS-CoV-2 that has mutated. As a potential novel immunotherapy,we generated a novel fusion protein composed of a soluble ACE2 (sACE2) linked to llama-derived anti-CD16 that targets different variants of spike proteins and enhances natural killer cells to target infected cells. Methods: Here,we generated a novel sACE2-AntiCD16VHH fusion protein using a Gly4Ser linker,synthesized and cloned into the pLVX-EF1alpha-IRES-Puro vector,and further expressed in ExpiCHO-S cells and purified using Ni+NTA chromatography. Results: The fusion protein significantly blocked SARS-CoV-2 alpha,beta,delta,gamma,and omicron S-proteins binding and activating angiotensin-converting enzyme receptor-2 (ACE2) on ACE2-expressing RAW-Blue macrophage cells and the secretion of several key inflammatory cytokines,G-CSF,MIP-1A,and MCP-1,implicated in the cytokine release storm (CRS). The sACE2-Anti-CD16VHH fusion protein also bridged NK cells to ACE2-expressing human lung carcinoma A549 cells and significantly activated NK-dependent cytotoxicity. Conclusions: The findings show that a VHH directed against CD16 could be an excellent candidate to be linked to soluble ACE2 to generate a bi-specific molecule (sACE2-AntiCD16VHH) suitable for bridging effector cells and infected target cells to inhibit SARS-CoV-2 variant spike proteins binding to the ACE2 receptor in the RAW-Blue cell line and pro-inflammatory cytokines and to activate natural killer cell cytotoxicity. View Publication

Diffuse large B cell lymphoma (DLBCL) is the commonest disorder derived from the B-lymphocytes. Inhibiting the immune checkpoint through naturalizing programmed death-1 (PD-1) and programmed death ligand 1 (PD-L1) is proved to be a successful therapeutic regime for lymphoma. Long non-coding RNAs (lncRNAs) are unceasingly reported to be promising biological targets for the cancer therapies. This study planned to explore the regulation of small nucleolar RNA host gene 14 (SNHG14) on DLBCL. SNHG14 level in DLBCL samples and cell lines was analyzed by GEPIA bioinformatics tool and RT-qPCR. Biological functions of SNHG14 in DLBCL were detected by CCK-8,colony formation,and transwell invasion assays. Molecular interaction was determined by RNA immunoprecipitation (RIP) and luciferase reporter assays. MiR-5590-3p-related pathway was identified through KEGG pathway analysis applying DAVID6.8 online bioinformatics tool. Effect of SNHG14 on CD8+ T cells was detected by flow cytometry. Results depicted that SNHG14 was upregulated in DLBCL and its depletion retarded proliferation,migration and epithelial-to-mesenchymal transition (EMT). Mechanistically,SNHG14 sponged miR-5590-3p to upregulate Zinc finger E-box binding homeobox 1 (ZEB1),and ZEB1 transcriptionally activated SNHG14 and PD-L1 to promote the immune evasion of DLBCL cells. In conclusion,we firstly showed that SNHG14/miR-5590-3p/ZEB1 positive feedback loop promoted diffuse large B cell lymphoma progression and immune evasion through regulating PD-1/PD-L1 checkpoint,indicating that targeting SNHG14 was a potential approach to improve the efficacy of immunotherapy in DLBCL. View Publication

NAD(P)-dependent steroid dehydrogenase-like protein (NSDHL),which is involved in breast tumor growth and metastasis,has been implicated in the maintenance of cancer stem cells. However,its role in regulating breast cancer stem-like cells (BCSCs) remains unclear. We have previously reported the clinical significance of NSDHL in patients with estrogen receptor-positive (ER +) breast cancer. This study aimed to elucidate the molecular mechanisms by which NSDHL regulates the capacity of BCSCs in the ER + human breast cancer cell line,MCF-7. NSDHL knockdown suppressed tumor spheroid formation in MCF-7 human breast cancer cells grown on ultralow-attachment plates. RNA sequencing revealed that NSDHL knockdown induced widespread transcriptional changes in the MCF-7 spheroids. TGF-β signaling pathway was the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (fold change ≥ 2,P ≤ 0.05) identified in NSDHL-knockdown MCF-7 spheroids compared with the control. In orthotopic tumor models injected with NSDHL-knockdown MCF-7 spheroids,tumor initiation and growth were strongly suppressed compared with those in the control. BCSC populations with CD44+/CD24- and CD49f+/EpCAM + phenotypes and high ALDH activity were decreased in NSDHL-knockdown MCF-7 spheroids and xenograft tumors relative to controls,along with decreased secretion of TGF-β1 and 3,phosphorylation of Smad2/3,and expression of SOX2. In RNA-sequencing data from The (TCGA) database,a positive correlation between the expression of NSDHL and SOX2 was found in luminal-type breast cancer specimens ( n = 998). Our findings revealed that NSDHL plays an important role in maintaining the BCSC population and tumor-initiating capacity of ER-positive MCF-7 spheroids,suggesting that NSDHL is an attractive therapeutic target for eliminating BCSCs,thus preventing breast cancer initiation and progression. Our findings suggest that NSDHL regulates the BCSC/tumor-initiating cell population in MCF-7 spheroids and xenograft tumors. The online version contains supplementary material available at 10.1186/s12885-024-13143-3. View Publication

BACKGROUND The epidermal growth factor receptor (EGFR) and Notch signaling pathways have been implicated in self-renewal of normal breast stem cells. We investigated the involvement of these signaling pathways in ductal carcinoma in situ (DCIS) of the breast. METHODS Samples of normal breast tissue (n = 15),pure DCIS tissue of varying grades (n = 35),and DCIS tissue surrounding an invasive cancer (n = 7) were used for nonadherent (i.e.,mammosphere) culture. Mammosphere cultures were treated at day 0 with gefitinib (an EGFR inhibitor),DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) (a gamma-secretase inhibitor),or Notch 4-neutralizing antibody. Mammosphere-forming efficiency (MFE) was calculated by dividing the number of mammospheres of 60 microm or more formed by the number of single cells seeded and is expressed as a percentage. The Notch 1 intracellular domain (NICD) was detected immunohistochemically in paraffin-embedded DCIS tissue from 50 patients with at least 60 months of follow-up. All statistical tests were two-sided. RESULTS DCIS had a greater MFE than normal breast tissue (1.5% versus 0.5%,difference = 1%,95% confidence interval [CI] = 0.62% to 1.25%,Ptextless.001). High-grade DCIS had a greater MFE than low-grade DCIS (1.6% versus 1.09%,difference = 0.51%,95% CI = 0.07% to 0.94%,P = .01). The MFE of high-grade DCIS treated with gefitinib in the absence of exogenous EGF was lower than that of high-grade DCIS treated with mammosphere medium lacking gefitinib and exogenous EGF (0.56% versus 1.36%,difference 0.8%,95% CI = 0.33% to 1.4%,P = .004). Increased Notch signaling as detected by NICD staining was associated with recurrence at 5 years (P = .012). DCIS MFE was reduced when Notch signaling was inhibited using either DAPT (0.89% versus 0.51%,difference = 0.38%,95% CI = 0.2% to 0.6%,Ptextless.001) or a Notch 4-neutralizing antibody (0.97% versus 0.2%,difference = 0.77%,95% CI = 0.52% to 1.0%,Ptextless.001). CONCLUSION We describe a novel primary culture technique for DCIS. Inhibition of the EGFR or Notch signaling pathways reduced DCIS MFE. View Publication

CD24 is a signal-transducing molecule on the surfaces of most human B cells that can modulate their response to activation signals by antagonizing IL-induced differentiation into antibody-forming cells and inducing proliferation in combination with signals generated by Ag receptors. A cDNA that directs the expression of CD24 on the surfaces of transfected COS cells was cloned by its homology to a cDNA encoding the murine M1/69-J11d heat stable Ag. The CD24 cDNA encodes a mature peptide of only 31 to 35 amino acids that is extensively glycosylated and is attached to the outer surface of the plasma membrane by a glycosyl phosphatidylinositol lipid anchor. Although CD24 is structurally similar to M1/69-J11d,and the two Ag appear to have a common genetic ancestry,the homology of CD24 to the M1/69-J11d Ag is confined to a small cluster of amino acids comprising potential N-linked glycosylation sites. Combined with the differences in expression patterns of the human and murine Ag,this indicates that CD24 and M1/69-J11d may not have equivalent functional roles in lymphoid development. The novel structure of CD24 suggests that signaling could be triggered by the binding of a lectin-like ligand to the carbohydrates projecting from the CD24 peptide,and transduced through the release of second messengers derived from the glycosyl phosphatidylinositol membrane anchor of CD24. View Publication

Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro,which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these,glutamic acid/alanine-rich protein (GARP),is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms,the variant surface glycoprotein (VSG) that protects the parasite membrane,and is involved in antigenic variation. Unlike VSG,however,the function of GARP is not known,which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP,we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG,the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively,these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal. View Publication

Human embryonic stem cells (hESCs) require specific niches for adhesion,expansion,and lineage-specific differentiation. In this study,we showed that a membrane substrate offers better tissue niches for hESC attachment,spreading,proliferation,and differentiation. The cell doubling time was shortened from 46.3±5.7 h for hESCs grown on solid substrates to 25.6±2.6 h for those on polyester (PE) membrane substrates with pore size of 0.4 μm. In addition,we observed an increase of approximately five- to ninefold of definitive endoderm marker gene expression in hESCs differentiated on PE or polyethylene terephthalate membrane substrates. Global gene expression analysis revealed upregulated expressions of a number of extracellular matrix and cell adhesion molecules in hESCs grown on membrane substrates. Further,an enhanced nuclear translocation of β-catenin was detected in these cells. These observations suggested the augmentation of Wnt signaling in hESCs grown on membrane substrates. These results also demonstrated that a membrane substrate can offer better physicochemical cues for enhancing in vitro hESC attachment,proliferation,and differentiation. View Publication

Sickle cell disease (SCD) is the most common human genetic disease which is caused by a single mutation of human β-globin (HBB) gene. The lack of long-term treatment makes the development of reliable cell and gene therapies highly desirable. Disease-specific patient-derived human induced pluripotent stem cells (hiPSCs) have great potential for developing novel cell and gene therapies. With the disease-causing mutations corrected in situ,patient-derived hiPSCs can restore normal cell functions and serve as a renewable autologous cell source for the treatment of genetic disorders. Here we successfully utilized transcription activator-like effector nucleases (TALENs),a recently emerged novel genome editing tool,to correct the SCD mutation in patient-derived hiPSCs. The TALENs we have engineered are highly specific and generate minimal off-target effects. In combination with piggyBac transposon,TALEN-mediated gene targeting leaves no residual ectopic sequences at the site of correction and the corrected hiPSCs retain full pluripotency and a normal karyotype. Our study demonstrates an important first step of using TALENs for the treatment of genetic diseases such as SCD,which represents a significant advance toward hiPSC-based cell and gene therapies. View Publication

BACKGROUND: Epithelial-mesenchymal transition (EMT) is involved in important malignant features of cancer cells,like invasion,metastatic potential,anti-apoptotic and stem-cell like phenotypes. Among several transcription factors,SNAI2/SLUG is supposed to play an essential role for EMT. METHODS: Paraffin embedded tumor samples from 63 patients with metastatic non-small cell lung cancer,enrolled in a randomized phase II trial,were prospectively collected,53 samples qualified for further analysis. Automated RNA extraction from paraffin and RT-quantitative PCR was used for evaluation of SNAI2/SLUG,estrogen receptor 1 (ESR1) and matrix-metalloproteinases (MMP) mRNA expression. RESULTS: Clinical features like age,gender,performance status,histological subtype and stage were similarly distributed among SNAI2/SLUG positive and negative patients. SNAI2/SLUG was significantly,inversely correlated with ESR1 mRNA expression (p textless 0.0001). In contrast,MMP2 (p = 0.387),MMP7 (p = 0.396) and MMP9 mRNA expression (p = 0.366) did not correlate with SNAI2/SLUG. Patients with high SNAI2/SLUG expression (grouped by median expression) had a worse outcome. Median overall survival in patients with high SNAI2/SLUG expression was 5.7 months versus 11.6 months with low SNAI2/SLUG expression (p = .038). Inversely,patients with high ESR1 expression (grouped by median expression) had an improved median OS with 10.9 months vs. 5.0 months in the low expression group (p = .032). In multivariate analysis,SNAI2/SLUG2 (p = .022) and ESR1 (p = .017) separately were independent prognostic factors for survival. CONCLUSION: SNAI2/SLUG is prognostic of patients' outcome. The strong inverse correlation with ESR1 indicates a significant impact of estrogen receptor pathway regarding these malignant features. View Publication

Olfactory epithelium (OE) has a lifelong capacity for neurogenesis due to the presence of basal stem cells. Despite the ability to generate short-term cultures,the successful in vitro expansion of purified stem cells from adult OE has not been reported. We sought to establish expansion-competent OE stem cell cultures to facilitate further study of the mechanisms and cell populations important in OE renewal. Successful cultures were prepared using adult mouse basal cells selected for expression of c-KIT. We show that c-KIT signaling regulates self-renewal capacity and prevents neurodifferentiation in culture. Inhibition of TGFβ family signaling,a known negative regulator of embryonic basal cells,is also necessary for maintenance of the proliferative,undifferentiated state in vitro Characterizing successful cultures,we identified expression of BMI1 and other Polycomb proteins not previously identified in olfactory basal cells but known to be essential for self-renewal in other stem cell populations. Inducible fate mapping demonstrates that BMI1 is expressed in vivo by multipotent OE progenitors,validating our culture model. These findings provide mechanistic insights into the renewal and potency of olfactory stem cells. View Publication

Cellular damage by reactive oxygen species and altered neurogenesis are implicated in the etiology of AD and the pathogenic actions of amyloid β-peptide (Aβ); the underlying mechanisms and the early oxidative intracellular events triggered by Aβ are not established. In the present study,we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). The incidence of mitochondria SO flash activity in neural progenitor cells (NPCs) increased during the first 6-24 hours of exposure to aggregating amyloid β-peptide (Aβ1-42),indicating an increase in transient mPTP opening. Subsequently,the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Aβ1-42,during which time global cellular reactive oxygen species increased,mitochondrial membrane potential decreased,cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Aβ1-42 on NPC proliferation and survival. Our findings suggest that mPTP-mediated bursts of mitochondrial SO production is a relatively early and pivotal event in the adverse effects of Aβ1-42 on NPCs. If Aβ inhibits NPC proliferation in the brains of AD patients by a similar mechanism,then interventions that inhibit mPTP-mediated superoxide flashes would be expected to protect NPCs against the adverse effects of Aβ. View Publication

Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast,lung,colon and prostate. Using CR,we have established matched normal and tumor cultures,GUMC-29 and GUMC-30 respectively,from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly,only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice,demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry,both normal and tumor CR cells expressed basal,luminal,and stem cell markers,with the majority of the normal and tumor CR cells expressing prostate basal cell markers,CD44 and Trop2,as well as luminal marker,CD13,suggesting a transit-amplifying phenotype. Consistent with this phenotype,real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5,KRT14 and p63),and low levels of luminal markers. When the CR tumor cells were injected into SCID mice,the expression of luminal markers (AR,NKX3.1) increased significantly,while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor,but undergo differentiation once injected into SCID mice. Genomic analyses,including SNP and INDEL,identified genes mutated in tumor cells,including components of apoptosis,cell attachment,and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer. View Publication