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1.
PLoS One ; 19(4): e0301822, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38603764

RESUMO

The human gut is a complex environment where the microbiota and its metabolites play a crucial role in the maintenance of a healthy state. The aim of the present work is the reconstruction of a new in vitro minimal human gut microbiota resembling the microbe-microbe networking comprising the principal phyla (Bacillota, Bacteroidota, Pseudomonadota, and Actinomycetota), to comprehend the intestinal ecosystem complexity. In the reductionist model, we mimicked the administration of Maitake extract as prebiotic and a probiotic formulation (three strains belonging to Lactobacillus and Bifidobacterium genera), evaluating the modulation of strain levels, the release of beneficial metabolites, and their health-promoting effects on human cell lines of the intestinal environment. The administration of Maitake and the selected probiotic strains generated a positive modulation of the in vitro bacterial community by qPCR analyses, evidencing the prominence of beneficial strains (Lactiplantibacillus plantarum and Bifidobacterium animalis subsp. lactis) after 48 hours. The bacterial community growths were associated with the production of metabolites over time through GC-MSD analyses such as lactate, butyrate, and propionate. Their effects on the host were evaluated on cell lines of the intestinal epithelium and the immune system, evidencing positive antioxidant (upregulation of SOD1 and NQO1 genes in HT-29 cell line) and anti-inflammatory effects (production of IL-10 from all the PBMCs). Therefore, the results highlighted a positive modulation induced by the synergic activities of probiotics and Maitake, inducing a tolerogenic microenvironment.


Assuntos
Bifidobacterium animalis , Microbioma Gastrointestinal , Grifola , Probióticos , Humanos , Ecossistema , Mucosa Intestinal/microbiologia , Lactobacillus/fisiologia , Probióticos/farmacologia
2.
Mucosal Immunol ; 16(3): 326-340, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37004750

RESUMO

iNKT cells account for a relevant fraction of effector T-cells in the intestine and are considered an attractive platform for cancer immunotherapy. Although iNKT cells are cytotoxic lymphocytes, their functional role in colorectal cancer (CRC) is still controversial, limiting their therapeutic use. Thus, we examined the immune cell composition and iNKT cell phenotype of CRC lesions in patients (n = 118) and different murine models. High-dimensional single-cell flow-cytometry, metagenomics, and RNA sequencing experiments revealed that iNKT cells are enriched in tumor lesions. The tumor-associated pathobiont Fusobacterium nucleatum induces IL-17 and Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in iNKT cells without affecting their cytotoxic capability but promoting iNKT-mediated recruitment of neutrophils with polymorphonuclear myeloid-derived suppressor cells-like phenotype and functions. The lack of iNKT cells reduced the tumor burden and recruitment of immune suppressive neutrophils. iNKT cells in-vivo activation with α-galactosylceramide restored their anti-tumor function, suggesting that iNKT cells can be modulated to overcome CRC-associated immune evasion. Tumor co-infiltration by iNKT cells and neutrophils correlates with negative clinical outcomes, highlighting the importance of iNKT cells in the pathophysiology of CRC. Our results reveal a functional plasticity of iNKT cells in CRC, suggesting a pivotal role of iNKT cells in shaping the tumor microenvironment, with relevant implications for treatment.


Assuntos
Antineoplásicos , Neoplasias Colorretais , Células T Matadoras Naturais , Camundongos , Animais , Neutrófilos , Antineoplásicos/farmacologia , Imunoterapia , Neoplasias Colorretais/patologia , Microambiente Tumoral
3.
Biomed Pharmacother ; 150: 112930, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35427821

RESUMO

Dupuytren disease (DD) is a hand-localized fibrotic disorder characterized by a scar-like, collagen-rich cord. Treatment usually comprises surgical removal of the cord, but is associated with a high relapse rate, in some cases requiring finger amputation. There is currently no consensual medical approach for treating DD. Numerous preclinical studies have highlighted antifibrotic properties of metformin, and the aim of this study was to assess a potential antifibrotic role of metformin in DD. Fibroblasts from DD cords (DF) and phenotypically normal palmar fascia (PF) were extracted from surgical specimens and cultured. The fibrotic status of DF and PF was compared at baseline, and under profibrotic (TGF-ß stimulation) and antifibrotic (metformin stimulation) conditions, using quantitative RT-PCR, western blot, immunocytochemistry, and a functional fibroblast contraction assay. At baseline, DF showed higher levels of fibrotic markers and contraction capacity compared with PF. Both types of fibroblasts responded to TGF-ß stimulation. Treatment of DF and PF with metformin did not affect basal levels of fibrotic markers and contraction but largely prevented their induction by TGF-ß. In conclusion, our data show that metformin inhibits TGF-ß-induced expression of fibrotic markers and contraction in hand-derived fibroblasts. This supports the case for a clinical trial to assess the repurposing of metformin as an adjuvant to surgery, to prevent, reduce, or delay recurrence in at-risk DD patients.


Assuntos
Contratura de Dupuytren , Metformina , Células Cultivadas , Contratura de Dupuytren/tratamento farmacológico , Contratura de Dupuytren/metabolismo , Fibroblastos/metabolismo , Fibrose , Humanos , Metformina/metabolismo , Metformina/farmacologia , Metformina/uso terapêutico , Recidiva Local de Neoplasia/metabolismo , Fator de Crescimento Transformador beta/metabolismo
4.
EMBO Mol Med ; 14(3): e15295, 2022 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-35156321

RESUMO

Lineage dedifferentiation toward a mesenchymal-like state displaying myofibroblast and fibrotic features is a common mechanism of adaptive and acquired resistance to targeted therapy in melanoma. Here, we show that the anti-fibrotic drug nintedanib is active to normalize the fibrous ECM network, enhance the efficacy of MAPK-targeted therapy, and delay tumor relapse in a preclinical model of melanoma. Acquisition of this resistant phenotype and its reversion by nintedanib pointed to miR-143/-145 pro-fibrotic cluster as a driver of this mesenchymal-like phenotype. Upregulation of the miR-143/-145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile. The 2 mature miRNAs generated from this cluster, miR-143-3p and miR-145-5p, collaborated to mediate transition toward a drug-resistant undifferentiated mesenchymal-like state by targeting Fascin actin-bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics and mechanotransduction pathways. Our study brings insights into a novel miRNA-mediated regulatory network that contributes to non-genetic adaptive drug resistance and provides proof of principle that preventing MAPKi-induced pro-fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy.


Assuntos
Indóis/farmacologia , Melanoma , MicroRNAs , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Humanos , Mecanotransdução Celular , Melanoma/tratamento farmacológico , Melanoma/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas dos Microfilamentos/metabolismo , Recidiva Local de Neoplasia
5.
PLoS Pathog ; 16(10): e1008660, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33075093

RESUMO

Mammary carcinoma, including triple-negative breast carcinomas (TNBC) are tumor-types for which human and canine pathologies are closely related at the molecular level. The efficacy of an oncolytic vaccinia virus (VV) was compared in low-passage primary carcinoma cells from TNBC versus non-TNBC. Non-TNBC cells were 28 fold more sensitive to VV than TNBC cells in which VV replication is impaired. Single-cell RNA-seq performed on two different TNBC cell samples, infected or not with VV, highlighted three distinct populations: naïve cells, bystander cells, defined as cells exposed to the virus but not infected and infected cells. The transcriptomes of these three populations showed striking variations in the modulation of pathways regulated by cytokines and growth factors. We hypothesized that the pool of genes expressed in the bystander populations was enriched in antiviral genes. Bioinformatic analysis suggested that the reduced activity of the virus was associated with a higher mesenchymal status of the cells. In addition, we demonstrated experimentally that high expression of one gene, DDIT4, is detrimental to VV production. Considering that DDIT4 is associated with a poor prognosis in various cancers including TNBC, our data highlight DDIT4 as a candidate resistance marker for oncolytic poxvirus therapy. This information could be used to design new generations of oncolytic poxviruses. Beyond the field of gene therapy, this study demonstrates that single-cell transcriptomics can be used to identify cellular factors influencing viral replication.


Assuntos
Neoplasias Mamárias Animais/metabolismo , Terapia Viral Oncolítica/métodos , Fatores de Transcrição/metabolismo , Transcriptoma , Vaccinia virus/genética , Vacínia/metabolismo , Replicação Viral , Animais , Biologia Computacional , Cães , Feminino , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/terapia , Neoplasias Mamárias Animais/virologia , Análise de Célula Única , Fatores de Transcrição/genética , Vacínia/genética , Vacínia/virologia
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