参考文献

Regulatory mechanisms governing adhesion of hematopoietic progenitor cells to the stromal nische are poorly understood. Growth factors such as stem cell factor (SCF),granulocyte-macrophage colony-stimulating factor,and thrombopoietin were reported to upregulate the adhesion of hematopoietic progenitors to immobilized fibronectin through activation of integrin alpha4beta1 and alpha5beta1. Macrophage inflammatory protein (MIP)-1alpha is a C-C chemokine that suppresses colony formation by stem/progenitor cells in vitro. We asked if MIP-1alpha would modulate the adhesive phenotype of colony-forming cells (CFCs) obtained from healthy donor bone marrow (BM),cord blood (CB),and mobilized peripheral blood (mPB) CD34+ cells,in comparison with SCF,using immobilized fibronectin. SCF significantly increased the level of adhesion of CFCs from BM,CB,and mPB. On the other hand,MIP-1alpha significantly increased the level of adhesion of CFCs from BM and CB,but less so from mPB. The effects of MIP-1alpha were inhibited by blocking antibodies to integrin alpha4,alpha5,or beta1,and polymerization plus rearrangement of F-actin were observed in affected cells by labeling with rhodamine-conjugated phalloidine. These data indicate that the effect of MIP-1alpha on the adhesive phenotype of CFCs is mediated by modulation of the organization of integrin. The amount of MIP-1alpha receptor on mPB was less than for BM or CB,which may explain the distinct characteristics in the adhesive response induced by MIP-1alpha. We suggest that hematopoietic progenitor cells from different sources may be heterogeneous with respect to maturation,integrin affinity,MIP-1alpha receptor expression,and regulation of MIP-1alpha signaling. Our data indicate that MIP-1alpha may affect migration,homing,and mobilization of hematopoietic progenitors by modulating the adhesive phenotype of these cells. View Publication

Basophils (Ba) and mast cells (MC) are important effector cells of inflammatory reactions. Both cell types derive from CD34(+) hematopoietic progenitors. However,little is known about the cell subsets that become committed to and give rise to Ba and/or MC. We have generated a monoclonal antibody (MoAb),97A6,that specifically detects human Ba,MC (lung,skin),and their CD34(+) progenitors. Other mature hematopoietic cells (neutrophils,eosinophils,monocytes,lymphocytes,platelets) did not react with MoAb 97A6,and sorting of 97A6(+) peripheral blood (PB) and bone marrow (BM) cells resulted in an almost pure population (textgreater98%) of Ba. Approximately 1% of CD34(+) BM and PB cells was found to be 97A6(+). Culture of sorted CD34(+)97A6(+) BM cells in semisolid medium containing phytohemagglutinin-stimulated leukocyte supernatant for 16 days (multilineage assay) resulted in the formation of pure Ba colonies (10 of 40),Ba-eosinophil colonies (7 of 40),Ba-macrophage colonies (3 of 40),and multilineage Ba-eosinophil-macrophage and/or neutrophil colonies (12 of 40). In contrast,no Ba could be cultured from CD34(+)97A6(-) cells. Liquid culture of CD34(+) PB cells in the presence of 100 ng/mL interleukin (IL)-3 (Ba progenitor assay) resulted in an increase of 97A6(+) cells,starting from 1% of day-0 cells to almost 70% (basophils) after day 7. Culture of sorted BM CD34(+)97A6(+) cells in the presence of 100 ng/mL stem cell factor (SCF) for 35 days (mast cell progenitor assay) resulted in the growth of MC (textgreater30% on day 35). Anti-IgE-induced IgE receptor cross-linking on Ba for 15 minutes resulted in a 4-fold to 5-fold upregulation of 97A6 antigen expression. These data show that the 97A6-reactive antigen plays a role in basophil activation and is expressed on multipotent CD34(+) progenitors,MC progenitors,Ba progenitors,as well as on mature Ba and tissue MC. The lineage-specificity of MoAb 97A6 suggests that this novel marker may be a useful tool to isolate and analyze Ba/MC and their progenitors. View Publication

Huntington’s disease (HD) is the best-known example of a neurodegenerative disorder caused by the expansion of a glutamine-encoding CAG repeat in the causative gene. Growing evidence indicates that somatic CAG expansions play a key role in disease progression,providing a strong rationale for therapeutic strategies directly targeting the repeat tract. However,achieving sufficient efficacy while maintaining allele selectivity and minimizing off-target effects remains a major challenge. Here,we developed allele-selective,CAG-targeting artificial microRNA (amiRNA) molecules that exhibit significantly reduced off-target risk. This was achieved by introducing specific substitutions at selected positions within the guide strand. These molecules effectively downregulated polyglutamine (polyQ) proteins in cellular models of HD,spinocerebellar ataxias types 1 and 3,and dentatorubral pallidoluysian atrophy. The most promising candidate,amiR136-13A,reduced mutant huntingtin levels in different brain regions of the HD mouse model and did not induce toxicity up to 28 weeks following a single administration of an AAV5 vector. Transcriptomic profiling of human HD neural stem cells treated with amiR136-13A revealed minor changes in gene expression. Moreover,amiR136-13A reduced the level of HTT1a,a short pathogenic isoform of huntingtin. Collectively,these findings identify amiR136-13A as a potent,selective,and safe therapeutic candidate for HD and potentially other polyQ disorders. View Publication

Widespread uses of nuclear materials increase the risk of accidental or intentional radiation exposure,which can result in acute radiation syndrome (ARS). Hematopoietic ARS (H-ARS) occurs at relatively low doses and is potentially lethal without intervention. While several FDA-approved cytokine-based radiomitigators exist,many require repeated dosing,complicating deployment in mass-casualty scenarios. This study evaluated a novel long-acting,murine-reactive granulocyte–macrophage colony-stimulating factor (LA-GM-CSF; mPDM608) as a prophylactic and mitigative countermeasure for H-ARS. Male and female C57BL/6 mice were exposed to lethal or sublethal total body irradiation (TBI) and treated with LA-GM-CSF using single- or multi-dose regimens administered before or after TBI. Safety,30-day survival,hematologic recovery,bone marrow cellularity,serum GM-CSF pharmacokinetics,endothelial injury markers,and cytokine profiles were assessed using standard hematology,histopathology,ELISA,and multiplex assays. LA-GM-CSF was well tolerated at doses up to 30 mg/kg. Single or limited dosing conferred significant survival benefits compared with vehicle controls,with optimal efficacy observed at lower doses (3 mg/kg). Post-TBI administration as a single dose 24 h after exposure markedly improved survival in both sexes,with stronger hematopoietic recovery in males. LA-GM-CSF accelerated recovery of neutrophils,red blood cells,platelets,hematocrit,and sternal megakaryocytes,prolonged circulating GM-CSF levels,and favorably modulated endothelial injury markers and select cytokines. LA-GM-CSF demonstrates strong potential as a next-generation radiation countermeasure,providing robust survival benefit and hematopoietic recovery with minimal dosing. The results shown here support further development for H-ARS management under the FDA Animal Rule. View Publication

Mesenchymal stromal cells (MSCs) have shown immunomodulatory effects and great promise in many inflammatory diseases such as acute respiratory distress syndrome (ARDS). However,several barriers to translation remain such as cell availability and potency. This study evaluates the therapeutic potentials of three types of MSCs,bone marrow-derived MSCs (BM-MSC),the human induced pluripotent stem cell-derived MSC wild type (iMSC WT) and β2 microglobulin-knockout iMSCs (iMSC B2M KO) with or without proinflammatory cytokine preconditioning. BM-MSC,iMSC WT and iMSC B2M KO were preconditioned with a proinflammatory cytokine cocktail (Cytomix: IL-1β,IFN-γ and TNF-α). Immunoregulatory biomarkers were analysed by flow cytometry and cytokines released by ELISA. MSC antimicrobial properties were analysed via CFU assays while the MSCs’ immunomodulatory effects were evaluated using macrophage activation and T cell proliferation assays. Proinflammatory cytokine preconditioning enhanced the therapeutic potency of all three types of MSCs by increasing immunomodulatory marker expression,enhancing the antimicrobial effects and improving MSC-mediated inhibition of T cell proliferation. These findings provided new insights into the therapeutic potencies of MSCs in inflammation. Further studies are required for in vitro characterisation of the MSCs and in vivo efficacy verification of these MSCs prior to their clinical application. View Publication

Background/Objectives: Overexpression of transferrin receptor (TFR1) is common in cancer and may be associated with inferior treatment outcomes. Due to these patterns and TFR1’s essential role in iron metabolism,the protein has been targeted for cytotoxic drug delivery. More recently,increased TFR1 expression has been linked to tumor microenvironment (TME) infiltration by immune effectors in selected tumors,but a comprehensive assessment of the genomic landscape associated TFRC (the gene encoding TFR1) expression has not been conducted. Methods: By utilizing a pan-cancer database of 93,248 patients with whole-exome and whole-transcriptome sequencing,we assessed TFRC-associated multiomic patterns. Results: We found that high TFRC expression correlates with significantly worse overall survival in multiple common solid tumor types,a higher tumor mutational burden (TMB),an increase in infiltrating effector cells with upregulated immune checkpoint markers within the TME,and increased frequency of specific high-risk genomic alterations. Further assessment in cell line models revealed increased susceptibility to cytotoxic T cells when iron metabolism is elevated,despite upregulation of the checkpoint ligand PD-L1. Conclusions: High TFRC expression,therefore,indicates worse clinical risk across multiple common tumor types but potentially increased susceptibility to cytotoxic immune effectors,informing the development of TFR1 biomarker-driven therapeutic strategies. View Publication

Background: Acute erythroid leukemia (AEL) or AML-M6 predominantly affects older adults and is rare in childhood. Compared with other AML subtypes,AEL remains relatively understudied because of its rarity. We established LS-CHM,a novel AEL cell line derived from the ascitic fluid of a patient with congenital leukemia. Interestingly,leukemic cells persisted in the ascitic fluid even after successful eradication from the bone marrow and extramedullary sites. Method: Leukemia cells from the ascites fluid exhibited robust proliferation in culture independent of cytokine requirement and were further characterized by flow cytometric immunophenotyping,cytogenetics,cell cycle and doubling time analysis,colony formation,genome and RNA sequencing,myeloid gene next generation sequencing,and cytotoxicity analysis. Results: LS-CHM displayed CD36,partial CD235a,CD31,CD43,and CD71 expression and demonstrated in vitro robust growth and high sensitivity to chemotherapeutic agents. A PDX mouse model showed development of leukemia. Genomic analysis revealed a frameshift BCOR mutation in the absence of additional mutations and downregulated TP53 expression with an exonic non-deleterious mutation. RNA sequencing of LS-CHM cells revealed upregulation of two cohesin complex genes,RAD21 and SMC3,whose high levels are associated with hematopoietic stem cell differentiation into erythroid lineage. Conclusions: LS-CHM represents the first congenital AEL-derived cell line,in contrast to the predominantly adult-origin and often secondary erythroid leukemia cell lines available currently. Thus,LS-CHM provides a unique pediatric and extramedullary AEL model,expanding the existing spectrum of AEL cell lines and offering valuable opportunities for biologic and therapeutic investigations. View Publication

Dpep is a cell-penetrating peptide that targets transcription factors ATF5,CEBPB and CEBPD to selectively suppress growth and survival of diverse tumor cell types in vitro and in vivo. Due to these actions and its apparent safety,the peptide has potential as a cancer therapeutic. How Dpep might be combined with other anti-cancer agents to achieve synergistic efficacy and to overcome possible peptide resistance has not been assessed in depth. Based on prior work indicating that Dpep promotes apoptotic cancer cell death and up-regulates multiple pro-apoptotic and tumor suppressor genes,we studied combinations of Dpep with ABT-263,a pro-apoptotic BCL2 family inhibitor,and decitabine,a hypomethylating drug. Combining Dpep with each agent alone or together synergistically suppressed the growth of a range of solid and liquid tumor cell types. Moreover,the combinations synergistically inhibited the growth of cells lines that were selected either in vivo or in vitro for Dpep resistance. Finally,we tested the combination of Dpep with ABT-263 in a mouse melanoma xenograft model. The combination more effectively inhibited tumor growth than either agent alone and,in contrast to vehicle or ABT-263,produced a 40% durable survival rate. Taken together,these observations highlight potential drug partners for the therapeutic development of Dpep. View Publication

Progressive supranuclear palsy-Richardson’s syndrome (PSP-RS) is a primary 4R tauopathy in which early axonal dysfunction may precede overt neurodegeneration; however,the mechanisms linking Tau dysregulation to cytoskeletal vulnerability remain poorly defined. Here,we generated induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic neurons from individuals with sporadic PSP-RS and matched healthy controls and performed integrated transcriptomic and proteomic analyses. PSP-RS neurons exhibited coordinated suppression of dopaminergic and synaptic programs alongside activation of cytoskeletal remodeling and stress-related pathways. These changes were accompanied by increased Tau phosphorylation,neurofilament accumulation,and structural alterations of the axonal compartment,consistent with an early axonopathic phenotype. Notably,mechanistic target of rapamycin (mTOR) signaling significantly increased. Pharmacological inhibition of mTOR reduced Tau phosphorylation and neurofilament levels,indicating that mTOR activity contributes to the maintenance of cytoskeletal imbalance. In conclusion,our findings support a model in which early cytoskeletal dysfunction in PSP-RS arises from the convergence of Tau dysregulation,impaired structural homeostasis,and altered signaling pathways. Rather than acting as a primary driver,mTOR appears to function as a pathogenic amplifier that sustains axonal stress. This study provides a human cellular framework to investigate early axonopathic mechanisms in sporadic PSP-RS. View Publication

Background: Crocodilians rarely develop cancer despite long lifespans and continuous exposure to environmental carcinogens,suggesting robust natural anti-tumor defense mechanisms. Methods: We investigated the anti-cancer activity of sera derived from the phylogenetically related species—alligators,crocodiles,and chickens,and studied their underlying immune mechanisms. The anti-tumor activity of alligator serum was tested in murine models of melanoma and lymphoma. Results: Alligator serum (AS) and its (NH4)2SO4-precipitated fraction (ASa) showed rapid and potent cytotoxicity toward multiple murine and human cancer cell lines while sparing non-malignant human cells. Importantly,ASa attenuated melanoma and lymphoma tumor growth in mice. Electrophysiological analyses in PN71 cancer cells treated with ASa revealed rapid membrane depolarization and formation of high-conductance pores consistent with Complement-mediated membrane attack complex (MAC) activity. Proteomic analyses identified the Complement component C5 as a major protein enriched in active fractions,implicating the Complement system in cancer cell killing. Based on phylogenetic similarity of C5,crocodile and chicken sera exhibit alligator-like comparable anti-cancer activity. Mechanistic studies in chicken serum showed that the anti-cancer activity depends on Ca2+ and Mg2+ ions,terminal Complement components (C5–C8),and IgM antibodies that initiate Complement activation. Immunodepletion of IgM from CSa significantly reduced cytotoxicity,whereas purified chicken IgM activated human Complement to induce cancer cell death. Conclusions: These findings identify a conserved IgM–Complement immune mechanism capable of selectively targeting malignant cells. The evolutionary conservation and cross-species functionality of this pathway highlight its potential as a bio-inspired strategy for developing novel Complement-based cancer immunotherapies. View Publication

Triple-negative breast cancer (TNBC) remains a significant challenge in oncology,contributing to a significant portion of cancer-related deaths among women. Current therapeutic options,including chemotherapy,surgery,radiation,and hormonal targeting therapies,exhibit limited efficacy,necessitating the exploration of innovative treatment modalities. The emergence of drug resistance and the persistence of cancer stem cells (CSCs) further emphasize the urgent need for novel therapeutic strategies. In this context,natural killer cell-derived extracellular vesicles (NK-EVs) have emerged as a promising cell-free therapeutic approach that exhibits high tumor infiltration and cytotoxicity against cancer cells and CSCs. This study aims to investigate the efficacy of NK-EVs as a therapeutic strategy for TNBC using various clinically relevant models,including patient-derived xenografts. Pathway analysis suggests strong activation of apoptosis via canonical caspase activation,as well as necrosis,thereby confirming the important cytotoxic effect of NK-EVs. Interestingly,NK-EVs were also found to suppress TNBC CSCs by disrupting their functionality and viability,and NK-EV treatment increased the expression of apoptosis markers in both CSCs and non-CSCs. By elucidating the therapeutic efficacy and translational potential of NK-EV-based interventions in TNBC,these findings offer critical insights for the development of future immunotherapeutic strategies against this aggressive subtype of breast cancer. View Publication

Microvascular dysfunction due to hypoxia is a key contributor in the pathogenesis of many disorders including cancer and retinal and cardiovascular diseases,but relevant human models are missing. Here,we present a robust 3D in vitro method with the use of human induced pluripotent stem cell–derived blood vessel organoids to analyze in vitro microvascular remodeling. We present a detailed practical pipeline combining optical tissue clearing,high-resolution immunofluorescence,and surface marker analysis to quantitatively assess hypoxia-driven changes in endothelial cells,pericytes,and the basal lamina. Exposure of these blood vessel organoids to chronic hypoxia (1% O2) for 1 week recapitulated key pathological features,including structural remodeling and a dysregulated secretome with altered vascular endothelial growth factor signaling. This approach establishes a versatile and human-relevant platform to study microvascular remodeling induced by chronic hypoxia and other pathological stimuli and their contribution to microvascular-related diseases. View Publication