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

Type I IFNs are key mediators of immune defense against viruses and bacteria. Type I IFNs were also previously implicated in protection against fungal infection,but their roles in antifungal immunity have not been thoroughly investigated. A recent study demonstrated that bacterial and fungal β-glucans stimulate IFN-β production by dendritic cells (DCs) following detection by the Dectin-1 receptor,but the effects of β-glucan-induced type I IFNs have not been defined. We investigated whether type I IFNs regulate CD8 T cell activation by fungal β-glucan particle-stimulated DCs. We demonstrate that β-glucan-stimulated DCs induce CD8 T cell proliferation,activation marker (CD44 and CD69) expression,and production of IFN-γ,IL-2,and granzyme B. Moreover,we show that type I IFNs support robust CD8 T cell activation (proliferation and IFN-γ and granzyme B production) by β-glucan-stimulated DCs in vitro and in vivo due to autocrine effects on the DCs. Specifically,type I IFNs promote Ag presentation on MHC I molecules,CD86 and CD40 expression,and the production of IL-12 p70,IL-2,IL-6,and TNF-α by β-glucan-stimulated DCs. We also demonstrate a role for autocrine type I IFN signaling in bacterial LPS-induced DC maturation,although,in the context of LPS stimulation,this mechanism is not so critical for CD8 T cell activation (promotes IFN-γ production but not proliferation or granzyme B production). This study provides insight into the mechanisms underlying CD8 T cell activation during infection,which may be useful in the rational design of vaccines directed against pathogens and tumors. View Publication

BACKGROUND: Critical limb ischemia due to peripheral arterial occlusive disease is associated with a severely increased morbidity and mortality. There is no effective pharmacological therapy available. Injection of autologous bone marrow-derived mononuclear cells (BM-MNC) is a promising therapeutic option in patients with critical limb ischemia,but double-blind,randomized trials are lacking. METHODS AND RESULTS: Forty patients with critical limb ischemia were included in a multicenter,phase II,double-blind,randomized-start trial to receive either intraarterial administration of BM-MNC or placebo followed by active treatment with BM-MNC (open label) after 3 months. Intraarterial administration of BM-MNC did not significantly increase ankle-brachial index and,thus,the trial missed its primary end point. However,cell therapy was associated with significantly improved ulcer healing (ulcer area,3.2±4.7 cm(2) to 1.89±3.5 cm(2) [P=0.014] versus placebo,2.92±3.5 cm(2) to 2.89±4.1 cm(2) [P=0.5]) and reduced rest pain (5.2±1.8 to 2.2±1.3 [P=0.009] versus placebo,4.5±2.4 to 3.9±2.6 [P=0.3]) within 3 months. Limb salvage and amputation-free survival rates did not differ between the groups. Repeated BM-MNC administration and higher BM-MNC numbers and functionality were the only independent predictors of improved ulcer healing. Ulcer healing induced by repeated BM-MNC administration significantly correlated with limb salvage (r=0.8; Ptextless0.001). CONCLUSIONS: Intraarterial administration of BM-MNC is safe and feasible and accelerates wound healing in patients without extensive gangrene and impending amputation. These exploratory findings of this pilot trial need to be confirmed in a larger randomized trial in patients with critical limb ischemia and stable ulcers. View Publication

BACKGROUND: Stem cell-based therapy for repair and replacement of lost neural cells is a promising treatment for central nervous system (CNS) diseases. Bone marrow (BM)-derived mesenchymal stem cells (MSCs) can differentiate into neural phenotypes and be isolated and expanded for autotransplantation with no risk of rejection. OBJECTIVE: The authors examined whether transplanted neurally induced human MSCs (NI hMSCs),developed by a new procedure,can survive,differentiate,and promote tissue protection and functional recovery in injured spinal cord (ISC) rats. METHODS: Neural induction was achieved by exposing cells simultaneously to inhibitors of DNA methylation,histone deacetylation,and pharmacological agents that increased cAMP levels. Three groups of adult female Sprague-Dawley rats were injected immediately rostral and caudal to the midline lesion with phosphate-buffered saline,MSCs,or NI hMSCs,1 week after a spinal cord impact injury at T-8. Functional outcome was measured using the Basso Beattie Bresnahan (BBB) locomotor rating scale and thermal sensitivity test on a weekly basis up to 12 weeks postinjury. Graft integration and anatomy of spinal cord was assessed by stereological,histochemical,and immunohistochemical techniques. RESULTS: The transplanted NI hMSCs survived,differentiated,and significantly improved locomotor recovery of ISC rats. Transplantation also reduced the volume of lesion cavity and white matter loss. CONCLUSION: This method of hMSC modification may provide an alternative source of autologous adult stem cells for CNS repair. View Publication

Fibroblasts from patients with Type I bipolar disorder (BPD) and their unaffected siblings were obtained from an Old Order Amish pedigree with a high incidence of BPD and reprogrammed to induced pluripotent stem cells (iPSCs). Established iPSCs were subsequently differentiated into neuroprogenitors (NPs) and then to neurons. Transcriptomic microarray analysis was conducted on RNA samples from iPSCs,NPs and neurons matured in culture for either 2 weeks (termed early neurons,E) or 4 weeks (termed late neurons,L). Global RNA profiling indicated that BPD and control iPSCs differentiated into NPs and neurons at a similar rate,enabling studies of differentially expressed genes in neurons from controls and BPD cases. Significant disease-associated differences in gene expression were observed only in L neurons. Specifically,328 genes were differentially expressed between BPD and control L neurons including GAD1,glutamate decarboxylase 1 (2.5 fold) and SCN4B,the voltage gated type IV sodium channel beta subunit (-14.6 fold). Quantitative RT-PCR confirmed the up-regulation of GAD1 in BPD compared to control L neurons. Gene Ontology,GeneGo and Ingenuity Pathway Analysis of differentially regulated genes in L neurons suggest that alterations in RNA biosynthesis and metabolism,protein trafficking as well as receptor signaling pathways may play an important role in the pathophysiology of BPD. View Publication

CD4+ T cells,especially T-helper (Th) cells (Th1,Th2 and Th17) and regulatory T cells (Treg) play pivotal role in the pathogenesis of multiple sclerosis (MS),a demyelinating autoimmune disease occurring in central nervous system (CNS). Astragaloside IV (ASI,CAS: 84687-43-4) is one of the saponins isolated from Astragalus membranceus,a traditional Chinese medicine with immunomodulatory effect. So far,whether ASI has curative effect on experimental autoimmune encephalomyelitis (EAE),an animal model of MS,and how it affects the subsets of CD4+ T cells,as well as the underlying mechanism have not been clearly elucidated. In the present study,ASI was found to ameliorate the progression and hamper the recurrence of EAE effectively in the treatment regimens. It significantly reduced the demyelination and inflammatory infiltration of CNS in EAE mice by suppressing the percentage of Th1 and Th17 cells,which was closely associated with the inhibition of JAK/STAT and NF-$\kappa$B signaling pathways. ASI also increased the percentage of Treg cells in spleen and CNS,which was accompanied by elevated Foxp3. However,in vitro experiments disclosed that ASI could regulate the differentiation of Th17 and Treg cells but not Th1 cells. In addition,it induced the apoptosis of MOG-stimulated CD4+ T cells probably through modulating STAT3/Bcl-2/Bax signaling pathways. Together,our findings suggested that ASI can modulate the differentiation of autoreactive CD4+ T cells and is a potential prodrug or drug for the treatment of MS and other similar autoimmune diseases. View Publication

Dendritic cells (DCs) are pivotal in regulating allergic asthma. Our research has shown that the absence of Sema3E worsens asthma symptoms in acute and chronic asthma models. However,the specific role of PlexinD1 in these processes,particularly in DCs,remains unclear. This study investigates the role of PlexinD1 in CD11c+ DCs using a house dust mite (HDM) model of asthma. We generated CD11c+ DC-specific PlexinD1 knockout (CD11cPLXND1 KO) mice and subjected them,alongside wild-type controls (PLXND1fl/fl),to an HDM allergen protocol. Airway hyperresponsiveness (AHR) was measured using FlexiVent,and immune cell populations were analyzed via flow cytometry. Cytokine levels and immunoglobulin concentrations were assessed using mesoscale and ELISA,while collagen deposition and mucus production were examined through Sirius-red and periodic acid Schiff (PAS) staining respectively. Our results indicate that CD11cPLXND1 KO mice exhibit significantly exacerbated AHR,characterized by increased airway resistance and tissue elastance. Enhanced mucus production and collagen gene expression were observed in these mice compared to wild-type counterparts. Flow cytometry revealed higher CD11c+ MHCIIhigh CD11b+ cell recruitment into the lungs,and elevated total and HDM-specific serum IgE levels in CD11cPLXND1 KO mice. Mechanistically,co-cultures of B cells with DCs from CD11cPLXND1 KO mice showed significantly increased IgE production compared to wild-type mice.These findings highlight the critical regulatory role of the plexinD1 signaling pathway in CD11c+ DCs in modulating asthma features. View Publication

Aging is associated with immune dysfunction,but long-term endurance training may confer protective effects on immune cell function. This study investigates how natural killer (NK) cell phenotypes,functional markers,and metabolism differ between endurance-trained and untrained older adults. Ex vivo expanded NK cells from endurance-trained (63.6 ± 2.1 years) and untrained (64.3 ± 3.3 years) males were exposed to adrenergic blockade (propranolol; 0–200 ng/mL) or mTOR inhibition (rapamycin; 10–100 ng/mL),both with or without PMA-induced inflammatory stimulation. Flow cytometry assessed NK subsets,activation (CD38,CD57,CD107a,NKG2D),senescence (KLRG1),and inhibitory markers (PD-1,LAG-3,TIM-3,NKG2A). Seahorse analysis measured metabolic parameters. Trained participants displayed healthier immune profiles (lower NLR,SII) and higher effector NK cells with lower cytotoxic subsets. Propranolol at 100 ng/mL blunted PMA-driven increases in CD57,CD107a,and NKG2D,while potentiating regulatory markers KLRG1,LAG-3,and PD-1 in the trained group,indicating stronger immunoregulation. With rapamycin,trained NK cells preserved NKG2D and CD107a at 10 ng/mL,maintaining cytotoxicity and degranulation. In contrast,at 100 ng/mL rapamycin plus PMA,trained NK cells shifted toward an effector phenotype with higher CD57 and CD107a,yet a blunted PMA-increased LAG-3 and TIM-3,suggesting resistance to exhaustion. PD-1 and KLRG1 remained elevated,reflecting balanced immune control. Mitochondrial analysis revealed that trained NK cells exhibited higher basal and maximal OCR,greater spare respiratory capacity,and OCR/ECAR ratio,reflecting superior metabolic fitness. These findings indicate that endurance-trained older adults have NK cells with greater functional adaptability,reduced senescence,and enhanced metabolism under inflammatory and pharmacological stress.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-06057-y. View Publication

Venetoclax plus azacitidine treatment is clinically beneficial for elderly and unfit acute myeloid leukemia (AML) patients. However,the treatment is rarely curative,and relapse due to resistant disease eventually emerges. Since no current clinically feasible treatments are known to be effective at the state of acquired venetoclax resistance,this is becoming a major challenge in AML treatment. Studying venetoclax-resistant AML cell lines,we observed that venetoclax induced sublethal apoptotic signaling and DNA damage even though cell survival and growth were unaffected. This effect could be due to venetoclax inducing a sublethal degree of mitochondrial outer membrane permeabilization. Based on these results,we hypothesized that the sublethal apoptotic signaling induced by venetoclax could constitute a vulnerability in venetoclax-resistant AML cells. This was supported by screens with a broad collection of drugs,where we observed a synergistic effect between venetoclax and PARP inhibition in venetoclax-resistant cells. Additionally,the venetoclax-PARP inhibitor combination prevented the acquisition of venetoclax resistance in treatment naïve AML cell lines. Furthermore,the addition of azacitidine to the venetoclax-PARP inhibitor combination enhanced venetoclax induced DNA damage and exhibited exceptional sensitivity and long-term responses in the venetoclax-resistant AML cell lines and samples from AML patients that had clinically relapsed under venetoclax-azacitidine therapy. In conclusion,we mechanistically identify a new vulnerability in acquired venetoclax-resistant AML cells and identify PARP inhibition as a potential therapeutic approach to overcome acquired venetoclax resistance in AML. Subject terms: Acute myeloid leukaemia,Acute myeloid leukaemia View Publication

Atezolizumab plus bevacizumab and lenvatinib are standard treatments for unresectable hepatocellular carcinoma; however,tumor markers such as alpha-fetoprotein and des-gamma-carboxy prothrombin have a limited ability to reflect treatment responses. Circulating tumor cells are non-invasive biomarkers associated with cancer stemness and treatment resistance. We assessed circulating tumor cell subsets expressing cancer stem cell markers (CD90,epithelial cell adhesion molecule,CD133,vimentin) using multiparametric flow cytometry at early and maximal response phases in patients receiving atezolizumab plus bevacizumab or lenvatinib. Early decreases in CD90-positive circulating tumor cells after therapy initiation were associated with tumor shrinkage and longer progression-free survival in both groups,as well as prolonged overall survival in the atezolizumab plus bevacizumab group. At maximal response,changes in CD90-positive circulating tumor cells reflected tumor burden more accurately than alpha-fetoprotein or des-gamma-carboxy prothrombin. These findings highlight the potential of CD90-positive circulating tumor cells to become dynamic biomarkers in systemic therapy for unresectable hepatocellular carcinoma. View Publication

Ataxia with oculomotor apraxia type 1 (AOA1) is a rare,autosomal recessive,early-onset,progressive cerebellar ataxia caused by mutations in the APTX gene,which encodes aprataxin,a DNA-adenylate hydrolase involved in DNA damage repair. The pathogenesis of AOA1 remains unclear. The purpose of this study was to investigate the pathogenesis of a novel mutation,p.H201P/H201R,carried by our AOA1 patient and the mechanism of AOA1 in an induced pluripotent stem cells (iPSCs) model. We edited iPSCs derived from a healthy individual to carry the APTX homozygous mutation p.H201P (H201P-iPSCs) or p.H201R (H201R-iPSCs) via CRISPR/Cas9. We found that aprataxin expression was absent in both H201P- and H201R-iPSCs. The capacity of these APTX-mutant iPSCs to differentiate into neural progenitor cells (NPCs) and mature neurons was diminished. We observed an increase in DNA single-strand breaks (SSB) via a comet assay and poly(ADP-ribose) staining,and an increase in the ratio of cleaved PARP-1/total PARP-1 in APTX-mutant NPCs and early immature neurons (EiNs),in addition of a heightened sensitivity to tert-butyl hydroperoxide in APTX-mutant EiNs. Moreover,a decrease of APE1 expression was observed in APTX-mutant NPCs and H201R-EiNs during neural differentiation. Our study established a practical iPSCs model to investigate AOA1 disease. We found that mutant aprataxin leads to defective neural differentiation,accompanied by the accumulation of DNA SSBs with increased cleaved PARP-1 and reduced APE1 expression of the base excision repair pathway. View Publication

The beta-globin locus control region (LCR) is a large DNA element that is required for high-level expression of beta-like globin genes from the endogenous mouse locus or in transgenic mice carrying the human beta-globin locus. The LCR encompasses 6 DNaseI hypersensitive sites (HSs) that bind transcription factors. These HSs each contain a core of a few hundred base pairs (bp) that has most of the functional activity and exhibits high interspecies sequence homology. Adjoining the cores are 500- to 1000-bp flanks" with weaker functional activity and lower interspecies homology. Studies of human beta-globin transgenes and of the endogenous murine locus show that deletion of an entire HS (core plus flanks) moderately suppresses expression. However View Publication

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes,respectively. Using patient-derived induced pluripotent stem cells (iPSC),we corrected the dysferlin nonsense mutation c.5713CtextgreaterT; p.R1905X and the most common alpha-sarcoglycan mutation,missense c.229CtextgreaterT; p.R77C,by single-stranded oligonucleotide-mediated gene editing,using the CRISPR/Cas9 gene editing system to enhance the frequency of homology-directed repair. We demonstrated seamless,allele-specific correction at efficiencies of 0.7-1.5%. As an alternative,we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22,using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination,and DICE also utilized site-specific recombinases. With DICE and THRIP,we obtained targeting efficiencies after selection of ˜20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization,as shown by immunoblot and immunocytochemistry. In summary,we demonstrate for the first time precise correction of LGMD iPSC and validation of expression,opening the possibility of cell therapy utilizing these corrected iPSC.Molecular Therapy (2016); doi:10.1038/mt.2016.40. View Publication