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OBJECTIVES: To explore the possibility of kidney organoids generated using patient derived human induced pluripotent stem cells (hiPSC) for modeling of Fabry disease nephropathy (FDN). METHODS: First, we generated hiPSC line using peripheral blood mononuclear cells (PBMCs) from two male FD-patients with different types of GLA mutation: a classic type mutation (CMC-Fb-001) and a non-classic type (CMC-Fb-003) mutation. Second, we generated kidney organoids using wild-type (WT) hiPSC (WTC-11) and mutant hiPSCs (CMC-Fb-001 and CMC-Fb-003). We then compared alpha-galactosidase A (α-GalA) activity, deposition of globotriaosylceremide (Gb-3), and zebra body formation under electromicroscopy (EM). RESULTS: Both FD patients derived hiPSCs had the same mutations as those detected in PBMCs of patients, showing typical pluripotency markers, normal karyotyping, and successful tri-lineage differentiation. Kidney organoids generated using WT-hiPSC and both FD patients derived hiPSCs expressed typical nephron markers without structural deformity. Activity of α-GalA was decreased and deposition of Gb-3 was increased in FD patients derived hiPSCs and kidney organoids in comparison with WT, with such changes being far more significant in CMC-Fb-001 than in CMC-Fb-003. In EM finding, multi-lammelated inclusion body was detected in both CMC-Fb-001 and CMC-Fb-003 kidney organoids, but not in WT. CONCLUSIONS: Kidney organoids generated using hiPSCs from male FD patients might recapitulate the disease phenotype and represent the severity of FD according to the GLA mutation type.
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Doença de Fabry , Células-Tronco Pluripotentes Induzidas , Nefropatias , Humanos , Masculino , Doença de Fabry/genética , Leucócitos Mononucleares , Rim , Diferenciação Celular , OrganoidesRESUMO
The objective of this study was to investigate whether CRISPR/Cas9-mediated suppression of A4GALT could rescue phenotype of Fabry disease nephropathy (FDN) using human induced pluripotent stem cells (hiPSCs) derived kidney organoid system. We generated FDN patient-derived hiPSC (CMC-Fb-002) and FD-specific hiPSCs (GLA-KO) by knock-out (KO) of GLA in wild-type (WT) hiPSCs using CRISPR/Cas9. We then performed A4GALT KO in both CMC-Fb-002 and GLA-KO to make Fb-002-A4GALT-KO and GLA/A4GALT-KO, respectively. Using these hiPSCs, we generated kidney organoids and compared alpha-galactosidase-A enzyme (α-GalA) activity, globotriaosylceramide (Gb-3) deposition, and zebra body formation under electron microscopy (EM). We also compared mRNA expression levels using RNA-seq and qPCR. Generated hiPSCs showed typical pluripotency markers without chromosomal disruption. Expression levels of GLA in CMC-Fb-002 and GLA-KO and expression levels of A4GALT in Fb-002-A4GALT-KO and GLA/A4GALT-KO were successfully decreased compared to those in WT-hiPSCs, respectively. Generated kidney organoids using these hiPSCs expressed typical nephron markers. In CMC-Fb-002 and GLA-KO organoids, α-GalA activity was significantly decreased along with increased deposition of Gb-3 in comparison with WT organoids. Intralysosomal inclusion body was also detected under EM. However, these disease phenotypes were rescued by KO of A4GALT in both GLA/A4GALT-KO and Fb-002-A4GALT-KO kidney organoids. RNA-seq showed increased expression levels of genes related to FDN progression in both GLA-mutant organoids compared to those in WT. Such increases were rescued in GLA/A4GALT-KO or Fb-002-A4GALT-KO organoids. CRISPR/Cas9 mediated suppression of A4GALT could rescue FDN phenotype. Hence, it can be proposed as a therapeutic approach to treat FDN.
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Doença de Fabry , Células-Tronco Pluripotentes Induzidas , Nefropatias , Humanos , Doença de Fabry/genética , Doença de Fabry/metabolismo , Sistemas CRISPR-Cas/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/metabolismo , Nefropatias/genética , Fenótipo , OrganoidesRESUMO
Necroptosis is a form of programmed cell death with features of necrosis and apoptosis that occurs in the intestinal epithelium of patients with inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease. In addition, necroptosis has also been observed in enterocytes in animal models of dextran sulfate sodium (DSS)-induced colitis. Thus, the discovery of natural products for regulating necroptosis may represent an important therapeutic strategy for improving IBD. We found that Magnolia officinalis bark extract (MBE) prevented weight loss and suppressed the activation of the proinflammatory cytokine IL6 in DSS-induced colitis. Furthermore, MBE restored the length of the damaged colon and decreased the expression of necroptosis markers in mice with DSS-induced colitis. In vitro, necroptosis-induced reactive oxygen species (ROS) production was reduced by MBE, and the expression of COX2, a target protein of ROS, was simultaneously suppressed. Both magnolol and honokiol, the two major bioactive compounds in MBE, inhibited necroptosis in human primary intestinal epithelial cells and colorectal adenocarcinoma cells. Our findings highlight the effectiveness of MBE in modulating enterocyte necroptosis and suggest that MBE may be developed as a natural, disease-targeting drug for the treatment of colitis.
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Plasma-treated media (PTM) serve as an adjuvant therapy to postoperatively remove residual cancerous lesions. We speculated that PTM could selectively kill cells infected with Mycobacterium tuberculosis (Mtb) and remove postoperative residual tuberculous lesions. We therefore investigated the effects of a medium exposed to a non-thermal plasma jet on the suppression of intracellular Mtb replication, cell death, signaling, and selectivity. We propose that PTM elevates the levels of the detoxifying enzymes, glutathione peroxidase, catalase, and ataxia-telangiectasia mutated serine/threonine kinase and increases intracellular reactive oxygen species production in Mtb-infected cells. The bacterial load was significantly decreased in spleen and lung tissues and single-cell suspensions from mice intraperitoneally injected with PTM compared with saline and untreated medium. Therefore, PTM has the potential as a novel treatment that can eliminate residual Mtb-infected cells after infected tissues are surgically resected.
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Muscle atrophy in postmenopausal women is caused by estrogen deficiency and a variety of inflammatory factors, including tumor necrosis factor alpha (TNFα). Paeoniflorin (PNF), a natural compound with anti-inflammatory properties, improves estradiol synthesis. Here, we demonstrate that PNF inhibits the progression of TNFα-induced skeletal muscle atrophy after menopause by restoring mitochondrial biosynthesis. Differentiated myoblasts damaged by TNFα were restored by PNF, as evident by the increase in the expression of myogenin (MyoG) and myosin heavy chain 3 (Myh3)-the markers of muscle differentiation. Moreover, diameter of atrophied myotubes was restored by PNF treatment. TNFα-repressed nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) (a major regulator of mitochondrial biosynthesis) were restored by PNF, via regulation by estrogen receptor alpha (ERα), an upregulator of NRF1. This mechanism was confirmed in ovariectomized (OVX) mice with a ~40% reduction in the cross-sectional area of the anterior tibialis muscle. OVX mice administered PNF (100, 300 mg/kg/day) for 12 weeks recovered more than ~20%. Behavioral, rotarod, and inverted screen tests showed that PNF enhances reduced muscle function in OVX mice. ERα restored expression of mitofusin 1 (MFN1) and mitofusin 2 (MFN2) (mitochondrial fusion markers) and dynamin-related protein (DRP1) and fission 1 (FIS1) (mitochondrial fission markers). Therefore, PNF can prevent muscle atrophy in postmenopausal women by inhibiting dysfunctional mitochondrial biogenesis.
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Huntington's disease (HD) is a severe inherited neurological disorder caused by a CAG repeat expansion in the huntingtin gene (HTT), leading to the accumulation of mutant huntingtin with polyglutamine repeats. Despite its severity, there is no cure for this debilitating disease. HTT lowering strategies, including antisense oligonucleotides (ASO) showed promising results very recently. Attempts to develop stem cell-based therapeutics have shown efficacy in preclinical HD models. Using an HD patient's autologous cells, which have genetic defects, may hamper therapeutic efficacy due to mutant HTT. Pretreating these cells to reduce mutant HTT expression and transcription may improve the transplanted cells' therapeutic efficacy. To investigate this, we targeted the SUPT4H1 gene that selectively supports the transcription of long trinucleotide repeats. Transplanting SUPT4H1-edited HD-induced pluripotent stem cell-derived neural precursor cells (iPSC-NPCs) into the YAC128 HD transgenic mouse model improved motor function compared to unedited HD iPSC-NPCs. Immunohistochemical analysis revealed reduced mutant HTT expression without compensating wild-type HTT expression. Further, SUPT4H1 editing increased neuronal and decreased reactive astrocyte differentiation in HD iPSC-NPCs compared to the unedited HD iPSC-NPCs. This suggests that ex vivo editing of SUPT4H1 can reduce mutant HTT expression and provide a therapeutic gene editing strategy for autologous stem cell transplantation in HD.
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Although Mycobacterium tuberculosis (Mtb) is an intracellular pathogen in phagocytic cells, the factors and mechanisms by which they invade and persist in host cells are still not well understood. Characterization of the bacterial proteins modulating macrophage function is essential for understanding tuberculosis pathogenesis and bacterial virulence. Here we investigated the pathogenic role of the Rv2145c protein in stimulating IL-10 production. We first found that recombinant Rv2145c stimulated bone marrow-derived macrophages (BMDMs) to secrete IL-10, IL-6 and TNF-α but not IL-12p70 and to increase the expression of surface molecules through the MAPK, NF-κB, and TLR4 pathways and enhanced STAT3 activation and the expression of IL-10 receptor in Mtb-infected BMDMs. Rv2145c significantly enhanced intracellular Mtb growth in BMDMs compared with that in untreated cells, which was abrogated by STAT3 inhibition and IL-10 receptor (IL-10R) blockade. Expression of Rv2145c in Mycobacterium smegmatis (M. smegmatis) led to STAT3-dependent IL-10 production and enhancement of intracellular growth in BMDMs. Furthermore, the clearance of Rv2145c-expressing M. smegmatis in the lungs and spleens of mice was delayed, and these effects were abrogated by administration of anti-IL-10R antibodies. Finally, all mice infected with Rv2145c-expressing M. smegmatis died, but those infected with the vector control strain did not. Our data suggest that Rv2145c plays a role in creating a favorable environment for bacterial survival by modulating host signals.
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Proteínas de Bactérias/imunologia , Mycobacterium tuberculosis/patogenicidade , Receptores de Interleucina-10/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Proteínas de Bactérias/genética , Interleucina-10/metabolismo , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana/genética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/imunologia , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Receptores de Interleucina-10/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , VirulênciaRESUMO
Mycobacterium tuberculosis contains diverse immunologically active components. This study investigated the biological function of a newly identified component, Rv1654, with the potential to induce apoptosis in macrophages. Recombinant Rv1654 induced macrophage apoptosis in a caspase-9/3-dependent manner through the production of reactive oxygen species (ROS) and interaction with Toll-like receptor 4. In addition, Rv1654 induced the production of tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 through the mitogen-activated protein kinase pathway. Furthermore, Rv1654-induced c-Jun N-terminal kinase (JNK) activation was inhibited by the ROS scavenger and Rv1654-induced apoptosis was inhibited by the JNK inhibitor. Moreover, it was found that treatment of macrophages with Rv1654 led to the loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, and translocation of Bax into the mitochondria. Finally, Rv1654-mediated apoptosis was inhibited in macrophages transfected with Bax siRNA. These results suggest that Rv1654 induces macrophage apoptosis through a mitochondrial-dependent pathway and ROS-mediated JNK activation.
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Apoptose , Proteínas de Bactérias/imunologia , Macrófagos/microbiologia , Mitocôndrias , Mycobacterium tuberculosis , Caspases , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/imunologia , Receptores Toll-LikeRESUMO
Human-induced pluripotent stem cell lines (hiPSCs) derived from the peripheral blood mononuclear cells (PBMCs) of a woman (CMCi007-A) and her son (CMCi006-A) diagnosed with Fabry disease (FD) caused by the frameshift deletion mutation c.969delC in the alpha-galactosidase A (GLA) gene were generated. These hiPSCs showed typical human embryonic stem cell-like morphology and expressed pluripotency-associated markers, and directly differentiated into all three germ-layers. Karyotyping showed normal 46, XY (CMCi006-A) and 46, XX (CMCi007-A). In summary, we generated novel patient-specific hiPSC lines from both a female and male containing the same mutation, which may provide additional insight into the pathophysiology of FD.
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Doença de Fabry , Células-Tronco Pluripotentes Induzidas , Doença de Fabry/genética , Feminino , Humanos , Leucócitos Mononucleares , Masculino , Mutação , Deleção de Sequência , alfa-Galactosidase/genéticaRESUMO
The human-induced pluripotent stem cell lines (hiPSCs) (CMCi009), derived from peripheral blood mononuclear cells (PBMCs) of a 42-year-old woman who were diagnosed as Birt-Hogg-Dubé syndrome (BHD) caused by the frameshift deletion mutation c.1285delC in FCLN gene, was generated using synthetic mRNA. Generated hiPSCs showed a typical human embryonic stem cell like morphology and expressed all pluripotency-associated markers, and directly differentiated into all three germ layers. Karyotyping of generated iPSCs showed normal 46, XY (CMCi009-A) respectively. In summary, we generated a novel patient-specific hiPSCs line containing the same mutation of FLCN gene and it can be used to provide additional insights for BHD pathophysiology.
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Síndrome de Birt-Hogg-Dubé , Células-Tronco Pluripotentes Induzidas , Adulto , Síndrome de Birt-Hogg-Dubé/genética , Feminino , Humanos , Leucócitos Mononucleares , Mutação , Proteínas Proto-Oncogênicas/genética , Deleção de Sequência , Proteínas Supressoras de Tumor/genéticaRESUMO
Human embryonic stem cells (hESCs) hold promise in regenerative medicine but allogeneic immune rejections caused by highly polymorphic human leukocyte antigens (HLAs) remain a barrier to their clinical applications. Here, we used a CRISPR/Cas9-mediated HLA-editing strategy to generate a variety of HLA homozygous-like hESC lines from pre-established hESC lines. We edited four pre-established HLA-heterozygous hESC lines and created a mini library of 14 HLA-edited hESC lines in which single HLA-A and HLA-B alleles and both HLA-DR alleles are disrupted. The HLA-edited hESC derivatives elicited both low T cell- and low NK cell-mediated immune responses. Our library would cover about 40% of the Asian-Pacific population. We estimate that HLA-editing of only 19 pre-established hESC lines would give rise to 46 different hESC lines to cover 90% of the Asian-Pacific population. This study offers an opportunity to generate an off-the-shelf HLA-compatible hESC bank, available for immune-compatible cell transplantation, without embryo destruction. Graphical Abstract.
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Edição de Genes , Células-Tronco Embrionárias Humanas , Embrião de Mamíferos , Transplante de Células-Tronco Hematopoéticas , Humanos , Medicina RegenerativaRESUMO
Mycobacterium tuberculosis (Mtb) is an intracellular pathogen known to persist in host cells. The apoptotic response of macrophages serves as a defense mechanism to inhibit the growth of intracellular bacteria, the failure of which can favor the spread of the pathogen to new cells. However, the mycobacterial components that regulate cell death and the related underlying mechanisms remain poorly understood. In this study, we investigated protein Rv3261, isolated from an Mtb culture filtrate, for its apoptotic potential using multidimensional fractionation. Rv3261 was found to induce macrophage apoptosis through the caspase-3/-9-dependent pathway. Furthermore, the ROS-dependent JNK activation pathway was found to be critical in Rv3261-mediated apoptosis. Rv3261 inhibited the growth of intracellular Mtb, which was significantly abrogated by pre-treatment with the ROS scavenger N-acetylcysteine (NAC), suggesting that Rv3261-mediated apoptosis may act as a host defense response. These findings suggest that Rv3261 is involved in the apoptotic modulation of Mtb-infected macrophages.
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Proteínas de Bactérias/metabolismo , Macrófagos/microbiologia , Mitocôndrias/metabolismo , Mycobacterium tuberculosis/fisiologia , Acetilcisteína/farmacologia , Animais , Apoptose , Caspase 3/metabolismo , Caspase 9/metabolismo , Processos de Crescimento Celular , Evasão da Resposta Imune , Imunidade Inata , Espaço Intracelular , MAP Quinase Quinase 4/metabolismo , Macrófagos/imunologia , Camundongos , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Macrophages are responsible for innate and adaptive immune response activation necessary for eliminating infections. Optimal activation of macrophages to phagocytize Mycobacterium tuberculosis is critical in anti-mycobacterial defense. Here, we identified a novel Rv3463 hypothetical protein that induces macrophage activation in Mtb culture filtrate. Recombinant Rv3463 activated mouse bone marrow-derived macrophages to induce the expression of surface molecules and secrete pro-inflammatory cytokines via the TLR2 and TLR4 pathways. Mitogen activated protein kinase, phospatidylinositol-4,5-bisphosphate 3-kinases, and the NF-κB signaling pathways are involved in Rv3463-mediated macrophage activation. Furthermore, Rv3463 induced bactericidal effects in Mtb-infected macrophages through phagosome maturation and phagolysosomal fusion enhanced by phospatidylinositol-4,5-bisphosphate 3-kinases and Ca2+ signaling pathways and exhibited therapeutic effects in a short-term Mtb-infection mouse model. Overexpression of Rv3463 in M. smegmatis caused rapid clearance of bacteria in macrophages and mice. Our study suggests that Rv3463 is a promising target for the development of post-exposure tuberculosis vaccines or adjunct immune-therapy.
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Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/uso terapêutico , Tuberculose/prevenção & controle , Animais , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Lisossomos/imunologia , Lisossomos/microbiologia , Ativação de Macrófagos , Macrófagos/microbiologia , Camundongos , Fagocitose/imunologia , Profilaxia Pós-Exposição/métodos , Transdução de Sinais/imunologia , Células THP-1 , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Tuberculose/imunologia , Tuberculose/microbiologia , Vacinas contra a Tuberculose/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
Mycobacterial acyl carrier protein (AcpM; Rv2244) is a meromycolate extension acyl carrier protein of Mycobacterium tuberculosis (Mtb), which participates in multistep mycolic acid biosynthesis. However, the function of AcpM in host-mycobacterium interactions during infection remains largely uncharacterized. Here we show that AcpM inhibits host cell apoptosis during mycobacterial infection. To examine the function of AcpM during infection, we generated a recombinant Mycobacterium smegmatis (M. smegmatis) strain overexpressing AcpM (Ms_AcpM) and a strain transformed with an empty vector (Ms_Vec). Ms_AcpM promoted intracellular survival of M. smegmatis and led to a significant decrease in the death rate of primary bone marrow-derived macrophages (BMDMs). Importantly, Ms_AcpM showed significantly decreased reactive oxygen species (ROS) generation and activation of c-Jun N-terminal kinase (JNK) signaling compared with Ms_Vec. In addition, treatment of BMDMs with recombinant AcpM significantly inhibited the apoptosis and ROS/JNK signaling induced by M. smegmatis. Moreover, recombinant AcpM enhanced intracellular survival of Mtb H37Rv. Taken together, these results indicate that AcpM plays a role as a virulence factor by modulating host cell apoptosis during mycobacterial infection.
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Apoptose/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/patologia , Mycobacterium tuberculosis/química , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Células Cultivadas , Feminino , Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Viabilidade Microbiana/efeitos dos fármacos , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/metabolismo , Infecções por Mycobacterium/microbiologia , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/fisiologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacosRESUMO
Mycobacterium abscessus (MAB) is a species of nontuberculous mycobacteria (NTM) and a major causative pathogen of pulmonary diseases especially in patients with cystic fibrosis. MAB infection is notoriously difficult to treat because of its intrinsic or inducible resistance to most antibiotics. The rough (R) morphotype of MAB, lacking cell surface glycopeptidolipids (GPLs), is associated with more severe and persistent infection than the smooth (S) type; however, the mechanisms underlying the R type's virulence and the relation with GPLs remain unclear. In this study, we found that R-type MAB is much more proapoptotic than the S type, as a result of GPL-mediated inhibition of macrophage apoptosis. Polar GPLs inhibited an apoptotic response (induced by proapoptotic stimuli) by suppressing ROS production and the cytochrome c release and by preserving mitochondrial transmembrane potential. Furthermore, GPLs were found to be targeted to mitochondria and interacted with cyclophilin D; their acetylation was essential for this interaction. Finally, GPLs inhibited the intracellular growth and bacterial spreading of R-type MAB among macrophages via apoptosis inhibition. These findings suggest that GPLs limit MAB virulence by inhibiting apoptosis and the spread of bacteria and therefore provide a novel insight into the mechanism underlying virulence of MAB.
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Ciclofilinas/metabolismo , Macrófagos/metabolismo , Mitocôndrias/metabolismo , Mycobacterium abscessus/patogenicidade , Micobactérias não Tuberculosas/patogenicidade , Apoptose , Peptidil-Prolil Isomerase F , HumanosRESUMO
Mycobacterium avium complex induces macrophage apoptosis. However, the M. avium components that inhibit or trigger apoptosis and their regulating mechanisms remain unclear. We recently identified the immunodominant MAV2054 protein by fractionating M. avium culture filtrate protein by multistep chromatography; this protein showed strong immuno-reactivity in M. avium complex pulmonary disease and in patients with tuberculosis. Here, we investigated the biological effects of MAV2054 on murine macrophages. Recombinant MAV2054 induced caspase-dependent macrophage apoptosis. Enhanced reactive oxygen species production and JNK activation were essential for MAV2054-mediated apoptosis and MAV2054-induced interleukin-6, tumour necrosis factor, and monocyte chemoattractant protein-1 production. MAV2054 was targeted to the mitochondrial compartment of macrophages treated with MAV2054 and infected with M. avium. Dissipation of the mitochondrial transmembrane potential (ΔΨm) and depletion of cytochrome c also occurred in MAV2054-treated macrophages. Apoptotic response, reactive oxygen species production, and ΔΨm collapse were significantly increased in bone marrow-derived macrophages infected with Mycobacterium smegmatis expressing MAV2054, compared to that in M. smegmatis control. Furthermore, MAV2054 expression suppressed intracellular growth of M. smegmatis and increased the survival rate of M. smegmatis-infected mice. Thus, MAV2054 induces apoptosis via a mitochondrial pathway in macrophages, which may be an innate cellular response to limit intracellular M. avium multiplication.
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Apoptose/fisiologia , Proteínas de Bactérias/metabolismo , Macrófagos/metabolismo , Mitocôndrias/metabolismo , Mycobacterium avium/metabolismo , Mycobacterium avium/fisiologia , Animais , Citocromos c/metabolismo , Feminino , Interleucina-6/metabolismo , Macrófagos/fisiologia , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/fisiologia , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Taxa de Sobrevida , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.
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Apoptose/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Macrófagos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Linhagem Celular , Citocromos c/metabolismo , Feminino , Interleucina-6/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium avium/metabolismo , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Skin diseases, including hair-related diseases and neoplasia, are a major public health problem. While their prevalence is increasing, their treatment options are limited. Researchers have tried to investigate the genes and signal pathways underlying hair follicles (HFs) to develop genetically targeted therapies through microarrays, which represent an appropriate modality for the analysis of small genomes. To enable the comprehensive transcriptome analysis of large and/or complex transcriptomes, we performed RNA-seq using next-generation sequencing (NGS). We isolated interfollicular keratinocytes (IFKs), HFs, and dermal fibroblasts including dermal papilla cells (DFs-DPCs) from normal C57BL/6 murine skin, transplanted combinations of these samples into nude mice, and followed the mice over time. Sustained hair growth was supported by HFs and DFs-DPCs. We then investigated the pathways and the relevant gene ontology associated with any identified differentially expressed genes (DEGs). In addition, in the culture and flow cytometry (FCM), the HFs had a more quiescent cell cycle pattern than did the IFKs and DFs-DPCs. Therefore, the representative cell cycle-related gene expression of IFKs, HFs, and DFs-DPCs was analyzed by NGS. Our study will allow researchers to further investigate the potential interactions and signaling pathways that are active in HF-related diseases and cancer and may aid in future bioengineering applications.
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Derme/citologia , Folículo Piloso/citologia , Cabelo/metabolismo , Queratinócitos/citologia , Dermatopatias/genética , Animais , Proliferação de Células , Células Cultivadas , Fibroblastos/citologia , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
INTRODUCTION: Induced pluripotent stem cells (iPSCs) have emerged as a promising cell source for immune-compatible cell therapy. Although a variety of somatic cells have been tried for iPSC generation, it is still of great interest to test new cell types, especially those which are hardly obtainable in a normal situation. METHODS: In this study, we generated iPSCs by using the cells originated from intervertebral disc which were removed during a spinal operation after spinal cord injury. We investigated the pluripotency of disc cell-derived iPSCs (diPSCs) and neural differentiation capability as well as therapeutic effect in spinal cord injury. RESULTS: The diPSCs displayed similar characteristics to human embryonic stem cells and were efficiently differentiated into neural precursor cells (NPCs) with the capability of differentiation into mature neurons in vitro. When the diPSC-derived NPCs were transplanted into mice 9 days after spinal cord injury, we detected a significant amelioration of hindlimb dysfunction during follow-up recovery periods. Histological analysis at 5 weeks after transplantation identified undifferentiated human NPCs (Nestin(+)) as well as early (Tuj1(+)) and mature (MAP2(+)) neurons derived from the transplanted NPCs. Furthermore, NPC transplantation demonstrated a preventive effect on spinal cord degeneration resulting from the secondary injury. CONCLUSION: This study revealed that intervertebral discs removed during surgery for spinal stabilization after spinal cord injury, previously considered a "waste" tissue, may provide a unique opportunity to study iPSCs derived from difficult-to-access somatic cells and a useful therapeutic resource for autologous cell replacement therapy in spinal cord injury.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Disco Intervertebral/citologia , Células-Tronco Neurais/transplante , Neurônios/patologia , Traumatismos da Medula Espinal/terapia , Animais , Comportamento Animal , Diferenciação Celular , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Células-Tronco Neurais/citologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase em Tempo Real , Recuperação de Função Fisiológica , Canais de Sódio/metabolismo , Medula Espinal/patologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transplante HeterólogoRESUMO
Mycobacterium kansasii (Mk) is an emerging pathogen that causes a pulmonary disease similar to tuberculosis. Macrophage apoptosis contributes to innate host defense against mycobacterial infection. Recent studies have suggested that lithium significantly enhances the cytotoxic activity of death stimuli in many cell types. We examined the effect of lithium on the viability of host cells and intracellular Mk in infected macrophages. Lithium treatment resulted in a substantial reduction in the viability of intracellular Mk in macrophages. Macrophage cell death was significantly enhanced after adding lithium to Mk-infected cells but not after adding to uninfected macrophages. Lithium-enhanced cell death was due to an apoptotic response, as evidenced by augmented DNA fragmentation and caspase activation. Reactive oxygen species were essential for lithium-induced apoptosis. Intracellular scavenging by N-acetylcysteine abrogated the lithium-mediated decrease in intracellular Mk growth as well as apoptosis. These data suggest that lithium is associated with control of intracellular Mk growth through modulation of the apoptotic response in infected macrophages.