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As a malignant tumour of the central nervous system, glioma exhibits high incidence and poor prognosis. Although TNIP1 and the TNF-α/NF-κB axis play key roles in immune diseases and inflammatory responses, their relationship and role in glioma remain unknown. Here, we revealed high levels of TNIP1 and TNF-α/NF-κB in glioma tissue. Glioma cell proliferation was activated with TNF-α treatment and showed extreme sensitivity to the TNF receptor antagonist. Furthermore, loss of TNIP1 disbanded the A20 complex responsible for IκB degradation and NF-κB nucleus translocation, and consequently erased TNFα-induced glioma cell proliferation. Thus, our investigation uncovered a vital function of the TNIP1-mediated TNF-α/NF-κB axis in glioma cell proliferation and provides novel insight into glioma pathology and diagnosis.
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Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteínas de Ligação a DNA/metabolismo , Glioma/metabolismo , Glioma/patologia , NF-kappa B/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Animais , Neoplasias Encefálicas/genética , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Humanos , Masculino , Camundongos Nus , Modelos Biológicos , Análise de SobrevidaRESUMO
Secondary failure of platelet recovery (SFPR) can occur after allogeneic hematopoietic stem cell transplantation (alloHSCT), and 20% of cases are related to acute graft-versus-host disease (aGVHD). The underlying mechanisms of this association are unclear, however. The aim of the present study was to investigate the potential mechanisms of SFPR secondary to aGVHD, which may provide a new therapeutic strategy for these patients. A total of 468 patients with malignant hematologic disease who underwent alloHSCT were included. Sixty-six patients developed SFPR after alloHSCT, and in 45 of these 66 patients (68.2%), SFPR was secondary to grade II-IV aGVHD (SFPR/aGVHD). Compared with patients with good graft function (GGF), patients with SFPR had poor overall survival (20.72% versus 88.01%; P < .0001). Grade II-IV aGVHD was identified as an independent risk factor for SFPR in multivariate analysis (hazard ratio, 9.512; P < .0001). We observed reduced erythroid and megakaryocyte colony formation in bone marrow (BM) samples isolated from SFPR/aGVHD patients, consistent with the lower frequency of megakaryocyte and erythrocyte progenitors in BM. Levels of the inflammatory cytokines IL-2R and TNF-R1 were significantly higher in the SFPR/aGVHD group compared with the GGF group (P = .002 and .001, respectively), as were the frequencies of proinflammatory T helper subsets. Furthermore, the pathways that regulate hematopoiesis and immune responses were universally underexpressed in CD34+ cells isolated from SFPR/aGVHD patients. Differentially expressed genes were significantly enriched in the hematopoietic cell lineage pathway and other pathways involved in both immune responses and megakaryopoiesis. In summary, we found that both the immune microenvironment and compromised proliferation of hematopoietic primitive cells contribute to the development of SFPR secondary to aGVHD, and our data provide new insight into the mechanisms of SFPR in the context of aGVHD.
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Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Doença Aguda , Plaquetas , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Megacariócitos , Transplante HomólogoRESUMO
BACKGROUND/AIMS: The serum inhibited gene (Si1) was named according to its inhibited expression in response to serum exposure. Si1 has an important relationship with tumors. Autophagy and apoptosis are two types of cell death. However, there are few studies regarding the association between Si1 and autophagy, or apoptosis in tumors. In this, we investigated the effect of Si1 on the proliferation and cell cycle progression of MCF-7 cells and its influence on autophagy and apoptosis in MCF-7 cells. METHODS: To investigate these functions of Si1 in tumor cells, we firstly constructed a pEGFP-Si1 overexpression vector and a pSilencer-Si1 interference vector, and we subsequently tested the proliferation and cell cycle progression of MCF-7 cells using the MTT assay and flow cytometry, and we then detected autophagy by western blotting and MDC (Monodansylcadaverine) staining as well as apoptosis by western blotting and Hoechst 33258 staining. RESULTS: We found that the Si1 gene can significantly inhibit the viability of MCF-7 cells and arrest the cell cycle at the G2/M phase. Si1 can induce autophagy through upregulation of LC3-II and Beclin1, it can induce apoptosis through cleavage of PARP in MCF-7 cells. CONCLUSION: Altogether, our study indicated that Si1 can inhibit cell proliferation of MCF-7, and also induces autophagy and apoptosis. This study firstly investigated the effect of Si1 on autophagy and apoptosis in MCF-7 cells. Moreover, it also improves the current understanding of the mechanisms related to the effect of Si1 on tumor cells and also provides a foundation for gene-targeted therapy.
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Apoptose , Autofagia , Proteínas/genética , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Células MCF-7 , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Regulação para CimaRESUMO
Cancer stem cells (CSCs) contribute to the relapse and development of new neoplasm lesions. While most available clinical approaches, such as chemical and radiation therapies, will kill the majority of cancer cells, they do not kill them all. Some resisting cells, like CSCs, are able to survive due to their excellent self-maintaining capabilities, even in challenging environments. In the present study, we investigated the mRNA level of DNA repair genes of colon CSCs from the HT29 cell line in response to single-strand damage and double-strand breaks, as well as the evident upregulation of key genes in base excision repair, mismatch repair, non-homologous end-joining, and homologous recombination pathways in these cells. Digital gene expression analysis identified upregulated genes in CD44+ HT29 cells that may play important roles in DNA repair. Our results reveal that colon CSCs bear efficient DNA repair abilities, which might explain the survival of colon CSCs after repeated chemical and radiation therapy.
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Neoplasias do Colo/genética , Reparo do DNA/genética , Perfilação da Expressão Gênica , Células-Tronco Neoplásicas/metabolismo , Neoplasias do Colo/patologia , Células HT29 , HumanosRESUMO
Although the Warburg effect is a dominant metabolic phenotype observed in cancers, the metabolic changes and adaptation occurring in tumors have been demonstrated to extend beyond the Warburg effect and thus considered a secondary effect to the transformation process of carcinogenesis, including nutritional deficiencies. However, the role of nutritional deficiencies in this metabolic reprogramming (e. g., oxidative phosphorylation (OXPHOS)/glycolysis interconversion) is not completely known yet. Here, we showed that under regular culture condition, the proliferation of U251 cells, but not other tumor cell lines, preferentially performed the Warburg effect and was remarkably inhibited by oxamic acid which can inhibit the activity of lactate dehydrogenase (LDH); whereas under serum starvation, glycolysis was depressed, tricarboxylic acid cycle (TCA) was enhanced, and the activity of OXPHOS was reinforced to maintain cellular ATP content in a high level, but interestingly, we observed a decreased expression of reactive oxygen species (ROS). Moreover, the upregulated activity of mitochondrial complex I was confirmed by Western blots and showed that the mitochondrial-related protein, NDUFA9, NDUFB8, ND1, and VDAC1 were remarkably increased after serum starved. Mechanistically, nutritional deficiencies could reduce hypoxia-inducible factor α (HIF-1α) protein expression to increase C-MYC protein level, which in turn increased nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) transcription to enhance the activity of OXPHOS, suggesting that metabolic reprogramming by the changes of microenvironment during the carcinogenesis can provide some novel therapeutic clues to traditional cancer treatments.
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Metabolismo Energético , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/biossíntese , Linhagem Celular Tumoral , Células Cultivadas , Ciclo do Ácido Cítrico , Glicólise , Humanos , Redes e Vias Metabólicas , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Fenótipo , Inanição/metabolismoRESUMO
BACKGROUND: The development of rapid and safe antidepressants for the treatment of major depression is in urgent demand. Converging evidence suggests that glutamatergic signaling seems to play important roles in the pathophysiology of depression. METHODS: We studied the antidepressant effects of 3(')-deoxyadenosine (3'-dA, Cordycepin) and the critical role of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor in male CD-1 mice via behavioral and biochemical experiments. After 3'-dA treatment, the phosphorylation and synaptic localization of the AMPA receptors GluR1 and GluR2 were determined in the prefrontal cortex (PFC) and hippocampus (HIP). The traditional antidepressant imipramine was applied as a positive control. RESULTS: We found that an injection of 3'-dA led to a rapid and robust antidepressant effect, which was significantly faster and stronger than imipramine, after 45min in tail suspension and forced swim tests. This antidepressant effect remained after 5 days of treatment with 3'-dA. Unlike the psycho-stimulants, 3'-dA did not show a hyperactive effect in the open field test. After 45min or 5 days of treatment, 3'-dA enhanced GluR1 S845 phosphorylation in both the PFC and HIP. In addition, after 45min of treatment, 3'-dA significantly up-regulated GluR1 S845 phosphorylation and GluR1, but not GluR2 levels, at the synapses in the PFC. After 5 days of treatment, 3'-dA significantly enhanced GluR1 S845 phosphorylation and GluR1, but not GluR2, at the synapses in the PFC and HIP. Moreover, the AMPA-specific antagonist GYKI 52466 was able to block the rapid antidepressant effects of 3'-dA. CONCLUSION: This study identified 3'-dA as a novel rapid antidepressant with clinical potential and multiple beneficial mechanisms, particularly in regulating the prefrontal AMPA receptor signaling pathway.
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Antidepressivos/farmacologia , Desoxiadenosinas/farmacologia , Transtorno Depressivo/tratamento farmacológico , Córtex Pré-Frontal/efeitos dos fármacos , Receptores de AMPA/metabolismo , Animais , Benzodiazepinas/farmacologia , Transtorno Depressivo/metabolismo , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Imipramina/farmacologia , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Fosforilação/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Distribuição Aleatória , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Fatores de Tempo , Resultado do TratamentoRESUMO
The mitochondrial transcription termination factor (MTERF) family is a group of highly conserved DNA-binding proteins composed of four key members, MTERF1-4. To date, several studies have investigated the binding sites of MTERF1 on mitochondrial genome and the regulation of mitochondrial gene transcription, but the more intricate connection between mitochondrial genes transcription regulation, mitochondrial oxidative phosphorylation (OXPHOS), and cell proliferation is still poorly understood. In this study, we constructed over-expression and knockdown vectors of MTERF1 that were transfected into HeLa cells to investigate the functions of MTERF1. Results showed that although MTERF1 is a positive regulatory factor of mitochondrial genes transcription, it had no significant effect on the replication of mitochondrial DNA. Over-expression of MTERF1 increased mitochondrial oxidative phosphorylation activity and promoted ATP synthesis, cyclin D1 expression, and cell proliferation, while its knockdown inhibited ATP synthesis, decreased cyclin D1 expression, and slowed the cell growth. These results suggested that MTERF1 may promote cell proliferation by regulating oxidative phosphorylation activity in HeLa cells. Ultimately, these findings create a foundation for further and more conclusive studies on the physiological functions of MTERF family by providing novel insights into the potential mechanisms underlying cell proliferation regulation.
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Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Proliferação de Células , Proteínas Mitocondriais/fisiologia , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Primers do DNA , Replicação do DNA , DNA Mitocondrial/metabolismo , DNA Mitocondrial/fisiologia , Citometria de Fluxo , Células HeLa , Humanos , Fosforilação Oxidativa , Reação em Cadeia da PolimeraseRESUMO
Type 2 CXC chemokine receptor CXCR2 plays roles in development, tumorigenesis, and inflammation. CXCR2 also promotes demyelination and decreases remyelination by actions toward hematopoietic cells and nonhematopoietic cells. Germline CXCR2 deficient (Cxcr2(-/-) ) mice reported in 1994 revealed the complexity of CXCR2 function and its differential expression in varied cell-types. Here, we describe Cxcr2(fl/fl) mice for which the targeting construct was generated by recombineering based on homologous recombination in E. coli. Without recombination Cxcr2(fl/fl) mice have CXCR2 expression on neutrophils in peripheral blood, bone marrow and spleen. Cxcr2(fl/fl) mice were crossed to Mx-Cre mice in which Cre recombinase is induced by Type I interferons, elicited by injection with polyinosinic-polycytidylic acid (poly(I:C)). CXCR2-deficient neutrophils were observed in poly(I:C) treated Cxcr2(fl/fl) ::Mx-Cre(+) (Cxcr2-CKO) mice, but not in poly(I:C) treated Cxcr2(f//+) ::Mx-Cre(+) mice. CXCR2 deletion was mainly observed peripherally but not in the CNS. Cxcr2-CKO mice showed impaired neutrophil migration in sterile peritonitis. Cxcr2-CKO mice reported here will provide a genetic reagent to dissect roles of CXCR2 in the neutrophil granulocyte lineage. Furthermore Cxcr2(fl/fl) mice will provide useful genetic models to evaluate CXCR2 function in varied cell populations.
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Deleção de Genes , Neutrófilos/metabolismo , Receptores de Interleucina-8B/genética , Animais , Movimento Celular/genética , Camundongos , Camundongos Knockout , Neutrófilos/fisiologia , Receptores de Interleucina-8B/metabolismoRESUMO
OBJECTIVE: Glioblastomas (GBMs) are lethal cancers that display cellular hierarchies parallel to normal brain. At the apex are GBM stem cells (GSCs), which are relatively resistant to conventional therapy. Interactions with the adjacent perivascular niche are an important driver of malignancy and self-renewal in GSCs. Extracellular matrix (ECM) cues instruct neural stem/progenitor cell-niche interactions, and the objective of our study was to elucidate its composition and contribution to GSC maintenance in the perivascular niche. METHODS: We interrogated human tumor tissue for immunofluorescence analysis and derived GSCs from tumor tissues for functional studies. Bioinformatics analyses were conducted by mining publicly available databases. RESULTS: We find that laminin ECM proteins are localized to the perivascular GBM niche and inform negative patient prognosis. To identify the source of laminins, we characterized cellular elements within the niche and found that laminin α chains were expressed by nonstem tumor cells and tumor-associated endothelial cells (ECs). RNA interference targeting laminin α2 inhibited GSC growth and self-renewal. In co-culture studies of GSCs and ECs, laminin α2 knockdown in ECs resulted in decreased tumor growth. INTERPRETATION: Our studies highlight the contribution of nonstem tumor cell-derived laminin juxtracrine signaling. As laminin α2 has recently been identified as a molecular marker of aggressive ependymoma, we propose that the brain vascular ECM promotes tumor malignancy through maintenance of the GSC compartment, providing not only a molecular fingerprint but also a possible therapeutic target.
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Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioblastoma/patologia , Laminina/metabolismo , Células-Tronco Neoplásicas/fisiologia , Antígeno AC133 , Análise de Variância , Antígenos CD/metabolismo , Neoplasias Encefálicas/mortalidade , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Biologia Computacional , Relação Dose-Resposta à Radiação , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Glioblastoma/mortalidade , Glicoproteínas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Laminina/genética , Imageamento por Ressonância Magnética , Masculino , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos da radiação , Peptídeos/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , Radiação , Análise de Regressão , Fatores de Tempo , Análise Serial de Tecidos , Células Tumorais Cultivadas , Microambiente Tumoral/fisiologiaRESUMO
Hepatocellular carcinoma is a common tumor with a high fatality rate worldwide, and exploring its pathogenesis and deterioration mechanism is a focus for many researchers. Increasing evidence has shown that miRNAs are involved in the occurrence and progression of a variety of cancers, including hepatocellular carcinoma. Therefore, this study mainly aimed identify key miRNAs related to hepatocellular carcinoma and explore their potential functions and clinical significance. In this study, we performed miRNA sequencing on three pairs of hepatocellular carcinoma tissue samples and screened 26 differentially expressed miRNAs. Then 2 key miRNAs (miR-139-5p and miR-582-3p) were screened by Kaplan-Meier curve analysis, Cox multivariate analysis and qPCR methods. The expression of miR-582-3p was positively correlated with clinicopathological parameters in patients with hepatocellular carcinoma. Subsequently, miRwalk and starbase were used to predict the target genes of key miRNAs, and then the key pairs miR-582-3p/SMAD2 identified by WGCNA, PPI, qPCR and Pearson correlation analysis. Finally, a dual luciferase experiment, the rescue-of-function experiment and qPCR confirmed that miR-582-3p directly targets SMAD2 and regulates the proliferation, migration and invasion of HepG2 cells by targeting SMAD2. At the same time, interference with SMAD2 can influence the effect of miR-582-3p on HepG2 cells. In conclusion, our findings confirm that miR-582-3p is an independent factor for the prognosis of hepatocellular carcinoma patients, and can regulate the progression of hepatocellular carcinoma cells by targeting SMAD2.
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CXC chemokine receptor CXCR4 is expressed in vitro in both human and rodent adult neural progenitor cells (NPCs). It has been suggested that the CXCL12-CXCR4 axis potentially enhances the proliferation of NPCs. However, whether CXCR4 is expressed in the neural stem cells (NSCs), a subset of self-renewing and multipotent NPCs, and whether CXCR4 signaling is directly required for their proliferation are not clear. In this study, we report that CXCR4 is expressed in a subpopulation of NPCs in the early embryonic ventricular zone. In studies of a CXCR4(eGFP) bacterial artificial chromosomal (BAC) transgenic mouse line, we further isolated NPCs from E12.5 transgenic telencephalon and GFP(+) cells demonstrated self-renewal and multipotency in neurosphere assays in vitro. Consistent with these observations, we enriched GFP(+)/CXCR4(+) cells by fluorescence activated cell sorting (FACS) with either CXCR4 antibody 12G5 or GFP. Furthermore, we observed that CXCL12 alone did not activate the self-renewal of NPCs or increase the proliferation of NPCs that are induced by bFGF/EGF. However, we found that blocking CXCR4 receptor with antagonist AMD3100 impaired the bFGF/EGF-induced expansion of GFP(+) NPCs through modulating their cell cycling. In addition, AMD3100 treatment of pregnant mice reduced the generation of neurospheres from E12.5 embryos. Our data suggest that CXCR4 is a potential cell surface marker for early embryonic NSCs and modulates growth-factor signaling.
Assuntos
Ciclo Celular/fisiologia , Células-Tronco Multipotentes/metabolismo , Células-Tronco Neurais/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Citometria de Fluxo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
The formation of blood vessels in embryos and tumors are different processes but under the control of common molecular mechanisms. Chemokine CXCL12 involved in both embryonic and tumor angiogenesis. In this review, we summarize recent advances in understanding the roles of CXCL12 in brain tumor angiogenesis/vasculogenesis. CXCL12 and its cognate receptors are abnormally induced in brain tumors, in particular in tumor cells and endothelium. Pathologically enhanced CXCL12 signaling may promote the formation of new vessels through recruiting circulating endothelial progenitor cells or directly enhancing the migration/growth of endothelial cells. Therefore, CXCL12 signaling represents an important mechanism that regulates brain tumor angiogenesis/vasculogenesis and may provide potential targets for anti-angiogenic therapy in malignant gliomas.
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Neoplasias Encefálicas/patologia , Quimiocina CXCL12/fisiologia , Neovascularização Patológica/patologia , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Animais , Neoplasias Encefálicas/metabolismo , Humanos , Neovascularização Patológica/metabolismoRESUMO
Background: Gut microbiome is proved to affect the activity of immunotherapy in certain tumors. However, little is known if there is universal impact on both the treatment response and adverse effects (AEs) of immune checkpoint inhibitors (ICIs) across multiple solid tumors, and whether such impact can be modulated by common gut microbiome modifiers, such as antibiotics and diet. Methods: A systematic search in PubMed followed by stringent manual review were performed to identify clinical cohort studies that evaluated the relevance of gut microbiome to ICIs (response and/or AEs, 12 studies), or association of antibiotics with ICIs (17 studies), or impact of diet on gut microbiome (16 studies). Only original studies published in English before April 1st, 2020 were used. Qualified studies identified in the reference were also included. Results: At the phylum level, patients who had enriched abundance in Firmicutes and Verrucomicrobia almost universally had better response from ICIs, whereas those who were enriched in Proteobacteria universally presented with unfavorable outcome. Mixed correlations were observed for Bacteroidetes in relating to treatment response. Regarding the AEs, Firmicutes correlated to higher incidence whereas Bacteroidetes were clearly associated with less occurrence. Interestingly, across various solid tumors, majority of the studies suggested a negative association of antibiotic use with clinical response from ICIs, especially within 1-2 month prior to the initiation of ICIs. Finally, we observed a significant correlation of plant-based diet in relating to the enrichment of "ICI-favoring" gut microbiome (P = 0.0476). Conclusions: Gut microbiome may serve as a novel modifiable biomarker for both the treatment response and AEs of ICIs across various solid tumors. Further study is needed to understand the underlying mechanism, minimize the negative impact of antibiotics on ICIs, and gain insight regarding the role of diet so that this important lifestyle factor can be harnessed to improve the therapeutic outcomes of cancer immunotherapy partly through its impact on gut microbiome.
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Both mesenchymal stem cells (MSCs) and their corresponding small extracellular vesicles (sEVs, commonly referred to as exosomes) share similar immunomodulatory properties that are potentially beneficial for the treatment of acute graft versus host disease (aGvHD). We report that clinical grade Wharton's Jelly-derived MSCs (WJMSCs) secrete sEVs enriched in programmed death-ligand 1 (PD-L1), an essential ligand for an inhibitory immune checkpoint. A rapid increase in circulating sEV-associated PD-L1 was observed in patients with aGvHD and was directly associated with the infusion time of clinical grade WJMSCs. In addition, in vitro inhibitory antibody mediated blocking of sEV-associated PD-L1 restored T cell activation (TCA), suggesting a functional inhibitory role of sEVs-PD-L1. PD-L1-deficient sEVs isolated from WJMSCs following CRISPR-Cas9 gene editing fail to inhibit TCA. Furthermore, we found that PD-L1 is essential for WJMSC-derived sEVs to modulate T cell receptors (TCRs). Our study reveals an important mechanism by which therapeutic WJMSCs modulate TCR-mediated TCA through sEVs or sEV-carried immune checkpoints. In addition, our clinical data suggest that sEV-associated PD-L1 may be not only useful in predicting the outcomes from WJMSC clinical administration, but also in developing cell-independent therapy for aGvHD patients.
Assuntos
Antígeno B7-H1/imunologia , Vesículas Extracelulares/imunologia , Doença Enxerto-Hospedeiro/imunologia , Células-Tronco Mesenquimais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Geleia de Wharton/imunologia , Adolescente , Adulto , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Exossomos/imunologia , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Feminino , Técnicas de Inativação de Genes , Humanos , Ativação Linfocitária , Células-Tronco Mesenquimais/metabolismo , Gravidez , Adulto JovemRESUMO
Chemotactic cytokines (chemokines) have been traditionally defined as small (10-14kDa) secreted leukocyte chemoattractants. However, chemokines and their cognate receptors are constitutively expressed in the central nervous system (CNS) where immune activities are under stringent control. Why and how the CNS uses the chemokine system to carry out its complex physiological functions has intrigued neurobiologists. Here, we focus on chemokine CXCL12 and its receptor CXCR4 that have been widely characterized in peripheral tissues and delineate their main functions in the CNS. Extensive evidence supports CXCL12 as a key regulator for early development of the CNS. CXCR4 signaling is required for the migration of neuronal precursors, axon guidance/pathfinding and maintenance of neural progenitor cells (NPCs). In the mature CNS, CXCL12 modulates neurotransmission, neurotoxicity and neuroglial interactions. Thus, chemokines represent an inherent system that helps establish and maintain CNS homeostasis. In addition, growing evidence implicates altered expression of CXCL12 and CXCR4 in the pathogenesis of CNS disorders such as HIV-associated encephalopathy, brain tumor, stroke and multiple sclerosis (MS), making them the plausible targets for future pharmacological intervention.
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Sistema Nervoso Central/fisiologia , Quimiocina CXCL12/fisiologia , Transdução de Sinais/fisiologia , Animais , Sistema Nervoso Central/embriologia , Doenças do Sistema Nervoso Central/fisiopatologia , Humanos , Receptores CXCR4/fisiologiaRESUMO
BACKGROUND: Tumor-infiltrating lymphocyte (TIL) therapy is a personalized cancer treatment which involves generating ex vivo cultures of tumor-reactive T cells from surgically resected tumors and administering the expanded TILs as a therapeutic infusion. Phase 1 of many TIL production protocols use aldesleukin (IL-2) alone to establish TIL cultures (termed "PreREP" (Pre-Rapid Expansion Protocol)); however, this fails to consistently produce TIL cultures from renal cell carcinoma (RCC) in a timely manner. Adding mitogenic stimulation via anti-CD3/anti-CD28 beads along with IL-2 to the fresh tumor digest (FTD) during TIL generation (termed "FTD+ beads") increases successful TIL culture rates; however, T cells produced by this method may be suboptimal for adoptive transfer. We hypothesize that adherent cell depletion (ACD) before TIL expansion will produce a superior TIL product by removing the immunosuppressive signals originating from adherent tumor and stromal cells. Here we investigate if "panning," a technique for ACD prior to TIL expansion, will impact the phenotype, functionality and/or clonality of ex vivo expanded RCC TILs. METHODS: Tumor specimens from 55 patients who underwent radical or partial nephrectomy at the University of Kansas Medical Center (KUMC) were used to develop the panning method and an additional 19 specimens were used to validate the protocol. Next-generation sequencing, immunohistochemistry/immunocytochemistry and flow cytometry were used during method development. The phenotype, functionality and clonality of autologous TILs generated in parallel by panning, PreREP, and FTD+ beads were assessed by flow cytometry, in vitro co-culture assays, and TCRB CDR3 sequencing. RESULTS: TIL cultures were successfully generated using the panning protocol from 15/16 clear cell, 0/1 chromophobe, and 0/2 papillary RCC samples. Significantly fewer regulatory (CD4+/CD25+/FOXP3+) (p=0.049, p=0.005), tissue-resident memory (CD8+/CD103+) (p=0.027, p=0.009), PD-1+/TIM-3+ double-positive (p=0.009, p=0.011) and TIGIT+ T cells (p=0.049, p=0.026) are generated by panning relative to PreREP and FTD+ beads respectively. Critically, a subset of TILs generated by panning were able to degranulate and/or produce interferon gamma in response to autologous tumor cells and the average tumor-reactive TIL yield was greatest when using the panning protocol. CONCLUSIONS: Removing immunosuppressive adherent cells within an RCC digest prior to TIL expansion allow for the rapid production of tumor-reactive T cells with optimal characteristics for adoptive transfer.
Assuntos
Carcinoma de Células Renais/imunologia , Imunoterapia Adotiva/métodos , Imunoterapia/métodos , Neoplasias Renais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T/imunologia , HumanosRESUMO
BACKGROUND: Because of their well-described immunosuppressive properties, allogeneic adult human mesenchymal stromal cells (MSC) derived from bone marrow have demonstrated safety and efficacy in steroid refractory acute graft versus host disease (SR aGVHD). Clinical trials have resulted in variable success and an optimal source of MSC has yet to be defined. Based on the importance of maternal-fetal interface immune tolerance, extraembryonic fetal tissues, such as the umbilical cord, may provide an superior tissue source of MSC to mediate immunomodulation in aGVHD. METHODS: A two-dose cohort trial allogeneic Wharton's Jelly-derived mesenchymal stromal cells (WJMSC, referred to as MSCTC-0010, here) were tested in 10 patients with de novo high risk (HR) or SR aGVHD post allogeneic hematopoietic stem cell transplantation (allo-HCT). Following Good Manufacturing Practices isolation, expansion and cryostorage, WJMSC were thawed and administered via intravenous infusions on days 0 and 7 at one of two doses (low dose cohort, 2 × 106/kg, n = 5; high dose cohort, 10 × 106/kg, n = 5). To evaluate safety, patients were monitored for infusion related toxicity, Treatment Related Adverse Events (TRAE) til day 42, or ectopic tissue formation at day 90. Clinical responses were monitored at time points up to 180 days post infusion. Serum biomarkers ST2 and REG3α were acquired 1 day prior to first MSCTC-0010 infusion and on day 14. RESULTS: Safety was indicated, e.g., no infusion-related toxicity, no development of TRAE, nor ectopic tissue formation in either low or high dose cohort was observed. Clinical response was suggested at day 28: the overall response rate (ORR) was 70%, 4 of 10 patients had a complete response (CR) and 3 had a partial response (PR). By study day 90, the addition of escalated immunosuppressive therapy was necessary in 2 of 9 surviving patients. Day 100 and 180 post infusion survival was 90% and 60%, respectively. Serum biomarker REG3α decreased, particularly in the high dose cohort, and with REG3α decrease correlated with clinical response. CONCLUSIONS: Treatment of patients with de novo HR or SR aGVHD with low or high dose MSCTC-0010 was safe: the infusion was well-tolerated, and no TRAEs or ectopic tissue formation was observed. A clinical improvement was seen in about 70% patients, with 4 of 10 showing a complete response that may have been attributable to MSCTC-0010 infusions. These observations indicate safety of two different doses of MSCTC-0010, and suggest that the 10 × 106 cells/ kg dose be tested in an expanded randomized, controlled Phase 2 trial. Graphical abstract.
Assuntos
Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Geleia de Wharton/citologia , Doença Aguda , Adulto , Idoso , Biomarcadores/metabolismo , Estudos de Viabilidade , Feminino , Doença Enxerto-Hospedeiro/patologia , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Pessoa de Meia-Idade , Proteínas Associadas a Pancreatite/metabolismo , Recidiva , Fatores de Risco , Análise de SobrevidaRESUMO
Actions of chemokines and the interaction with specific receptors go beyond their original, defined role of recruiting leukocytes to inflamed tissues. Chemokine receptor expression in peripheral elements and resident cells of the central nervous system (CNS) represents a relevant communication system during neuroinflammatory conditions. The following examples are described in this review: Chemokine receptors play important homeostatic properties by regulating levels of specific ligands in blood and tissues during healthy and pathological conditions; chemokines and their receptors are clearly involved in leukocyte extravasation and recruitment to the CNS, and current studies are directed toward understanding the interaction between chemokine receptors and matrix metalloproteinases in the process of blood brain barrier breakdown. We also propose novel functions of chemokine receptors during demyelination/remyelination, and developmental processes.
Assuntos
Encéfalo/metabolismo , Quimiocinas/fisiologia , Quimiotaxia/fisiologia , Encefalomielite Autoimune Experimental/metabolismo , Animais , Barreira Hematoencefálica , Humanos , Receptores de Quimiocinas/metabolismoRESUMO
Chronic pulmonary inflammation (CPI) gives rise to serious lung injuries in rheumatoid arthritis (RA) patients. However, the molecular mechanism underlying the pathogenesis of RA-associated CPI remains little understood. Here we established a novel tree shrew-based collagen-induced arthritis (TsCIA) model to study RA-associated CPI. Our results showed that typical CPI but not fibrosis developed pathologically in the TsCIA model. Furthermore, abnormal up-regulation of pulmonary chemokine CXCL10 was directly associated with lung damage. Specific blockage of CXCR3 (a CXCL10 receptor) significantly decreased the severity of CPI by decreasing the recruitment of inflammatory cells. Therefore, CXCL10 is proposed as a key player responsible for the development of TsCIA-associated CPI. Our findings also suggest that CXCR3 could be developed as a potential diagnosis biomarker for RA-associated CPI.
Assuntos
Quimiocina CXCL10/metabolismo , Pneumonia/etiologia , Pneumonia/metabolismo , Animais , Artrite Experimental/metabolismo , Artrite Reumatoide/complicações , Artrite Reumatoide/metabolismo , Quimiocina CXCL10/genética , Quimiocinas/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Modelos Animais , Pneumonia/genética , Receptores CXCR3/fisiologia , Musaranhos , Ativação Transcricional , Tupaiidae/metabolismo , Regulação para CimaRESUMO
Chemokines represent a superfamily of small secretion proteins that functionally mediate immune cell transmigration in normal or inflammatory conditions. Although anatomic and polygenetic evidence suggests that tree shrews are primate-like species, understanding of the structure and function of tree shrew chemokines has only just commenced. In this study, we cloned tree shrew chemokine CXCL8 and its cognate receptors. Predicted three-dimensional (3D) structures showed that binding domains in CXCL8 and CXCR1/2 were highly conserved between tree shrews and humans. We found that the human CXCL8 (hCXCL8) protein induced migration of tree shrew peripheral blood mononuclear cells (PBMCs) expressed by CXCR1/2 (tsCXCR1/2). Blocking interaction between hCXCL8 and tsCXCR1/2 with allosteric antagonists (reparixin and SB265610) significantly decreased tree shrew PBMC transmigration. Over-expressing tree shrew CXCR1 in human HEK 293 T cells further enhanced cellular in vitro transmigration. Similar to primate species, our findings suggest that CXCL8 and CXCR1/2 constitute a structurally- and functionally-conserved chemotaxis responsible for tree shrew immune activities.