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1.
Nat Commun ; 15(1): 4862, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862464

RESUMO

As spaceflight becomes more common with commercial crews, blood-based measures of crew health can guide both astronaut biomedicine and countermeasures. By profiling plasma proteins, metabolites, and extracellular vesicles/particles (EVPs) from the SpaceX Inspiration4 crew, we generated "spaceflight secretome profiles," which showed significant differences in coagulation, oxidative stress, and brain-enriched proteins. While >93% of differentially abundant proteins (DAPs) in vesicles and metabolites recovered within six months, the majority (73%) of plasma DAPs were still perturbed post-flight. Moreover, these proteomic alterations correlated better with peripheral blood mononuclear cells than whole blood, suggesting that immune cells contribute more DAPs than erythrocytes. Finally, to discern possible mechanisms leading to brain-enriched protein detection and blood-brain barrier (BBB) disruption, we examined protein changes in dissected brains of spaceflight mice, which showed increases in PECAM-1, a marker of BBB integrity. These data highlight how even short-duration spaceflight can disrupt human and murine physiology and identify spaceflight biomarkers that can guide countermeasure development.


Assuntos
Coagulação Sanguínea , Barreira Hematoencefálica , Encéfalo , Homeostase , Estresse Oxidativo , Voo Espacial , Animais , Humanos , Encéfalo/metabolismo , Barreira Hematoencefálica/metabolismo , Camundongos , Coagulação Sanguínea/fisiologia , Masculino , Secretoma/metabolismo , Camundongos Endogâmicos C57BL , Vesículas Extracelulares/metabolismo , Proteômica/métodos , Biomarcadores/metabolismo , Biomarcadores/sangue , Feminino , Adulto , Proteínas Sanguíneas/metabolismo , Pessoa de Meia-Idade , Leucócitos Mononucleares/metabolismo , Proteoma/metabolismo
2.
Cells ; 13(11)2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38891113

RESUMO

Tigilanol tiglate (TT, also known as EBC-46) is a novel, plant-derived diterpene ester possessing anticancer and wound-healing properties. Here, we show that TT-evoked PKC-dependent S985 phosphorylation of the tyrosine kinase MET leads to subsequent degradation of tyrosine phosphorylated p-Y1003 and p-Y1234/5 MET species. PKC inhibition with BIM-1 blocked S985 phosphorylation of MET and led to MET cell surface accumulation. Treatment with metalloproteinase inhibitors prevented MET-ECD release into cell culture media, which was also blocked by PKC inhibitors. Furthermore, unbiased secretome analysis, performed using TMT-technology, identified additional targets of TT-dependent release of cell surface proteins from H357 head and neck cancer cells. We confirm that the MET co-signalling receptor syndecan-1 was cleaved from the cell surface in response to TT treatment. This was accompanied by rapid cleavage of the cellular junction adhesion protein Nectin-1 and the nerve growth factor receptor NGFRp75/TNFR16. These findings, that TT is a novel negative regulator of protumorigenic c-MET and NGFRp75/TNFR16 signalling, as well as regulating Nectin-1-mediated cell adhesion, further contribute to our understanding of the mode of action and efficacy of TT in the treatment of solid tumours.


Assuntos
Neoplasias de Cabeça e Pescoço , Proteínas Proto-Oncogênicas c-met , Humanos , Proteínas Proto-Oncogênicas c-met/metabolismo , Fosforilação/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Linhagem Celular Tumoral , Secretoma/metabolismo , Diterpenos/farmacologia , Proteínas de Membrana/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sindecana-1/metabolismo , Nectinas/metabolismo , Proteína Quinase C/metabolismo
3.
J Med Microbiol ; 73(6)2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38836745

RESUMO

Introduction. The fungal pathogen Aspergillus fumigatus can induce prolonged colonization of the lungs of susceptible patients, resulting in conditions such as allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis.Hypothesis. Analysis of the A. fumigatus secretome released during sub-lethal infection of G. mellonella larvae may give an insight into products released during prolonged human colonisation.Methodology. Galleria mellonella larvae were infected with A. fumigatus, and the metabolism of host carbohydrate and proteins and production of fungal virulence factors were analysed. Label-free qualitative proteomic analysis was performed to identify fungal proteins in larvae at 96 hours post-infection and also to identify changes in the Galleria proteome as a result of infection.Results. Infected larvae demonstrated increasing concentrations of gliotoxin and siderophore and displayed reduced amounts of haemolymph carbohydrate and protein. Fungal proteins (399) were detected by qualitative proteomic analysis in cell-free haemolymph at 96 hours and could be categorized into seven groups, including virulence (n = 25), stress response (n = 34), DNA repair and replication (n = 39), translation (n = 22), metabolism (n = 42), released intracellular (n = 28) and cellular development and cell cycle (n = 53). Analysis of the Gallerial proteome at 96 hours post-infection revealed changes in the abundance of proteins associated with immune function, metabolism, cellular structure, insect development, transcription/translation and detoxification.Conclusion. Characterizing the impact of the fungal secretome on the host may provide an insight into how A. fumigatus damages tissue and suppresses the immune response during long-term pulmonary colonization.


Assuntos
Aspergillus fumigatus , Proteínas Fúngicas , Larva , Mariposas , Animais , Aspergillus fumigatus/metabolismo , Larva/microbiologia , Mariposas/microbiologia , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Secretoma/metabolismo , Proteômica , Fatores de Virulência/metabolismo , Proteoma/análise , Hemolinfa/microbiologia , Hemolinfa/metabolismo , Virulência , Aspergilose/microbiologia , Aspergilose/metabolismo
4.
Talanta ; 276: 126216, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38761653

RESUMO

Human amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties making them attractive candidates for regenerative applications in inflammatory diseases. Most of their beneficial properties are mediated through their secretome. The bioactive factors concurring to its therapeutic activity are still unknown. Evidence suggests synergy between the two main components of the secretome, soluble factors and vesicular fractions, pivotal in shifting inflammation and promoting self-healing. Biological variability and the absence of quality control (QC) protocols hinder secretome-based therapy translation to clinical applications. Moreover, vesicular secretome contains a multitude of particles with varying size, cargos and functions whose complexity hinders full characterization and comprehension. This study achieved a significant advancement in secretome characterization by utilizing native, FFF-based separation and characterizing extracellular vesicles derived from hAMSCs. This was accomplished by obtaining dimensionally homogeneous fractions then characterized based on their protein content, potentially enabling the identification of subpopulations with diverse functionalities. This method proved to be successful as an independent technique for secretome profiling, with the potential to contribute to the standardization of a qualitative method. Additionally, it served as a preparative separation tool, streamlining populations before ELISA and LC-MS characterization. This approach facilitated the categorization of distinctive and recurring proteins, along with the identification of clusters associated with vesicle activity and functions. However, the presence of proteins unique to each fraction obtained through the FFF separation tool presents a challenge for further analysis of the protein content within these cargoes.


Assuntos
Âmnio , Vesículas Extracelulares , Células-Tronco Mesenquimais , Secretoma , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Secretoma/metabolismo , Âmnio/química , Âmnio/citologia , Âmnio/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Controle de Qualidade , Células Cultivadas
5.
Int J Mol Sci ; 25(9)2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38731966

RESUMO

Leukemias are among the most prevalent types of cancer worldwide. Bone marrow mesenchymal stem cells (MSCs) participate in the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases such as leukemias, to a yet unknown extent. Here we described the effect of secretome of bone marrow MSCs obtained from healthy donors and from patients with acute myeloid leukemia (AML) on leukemic cell lineages, sensitive (K562) or resistant (K562-Lucena) to chemotherapy drugs. Cell proliferation, viability and death were evaluated, together with cell cycle, cytokine production and gene expression of ABC transporters and cyclins. The secretome of healthy MSCs decreased proliferation and viability of both K562 and K562-Lucena cells; moreover, an increase in apoptosis and necrosis rates was observed, together with the activation of caspase 3/7, cell cycle arrest in G0/G1 phase and changes in expression of several ABC proteins and cyclins D1 and D2. These effects were not observed using the secretome of MSCs derived from AML patients. In conclusion, the secretome of healthy MSCs have the capacity to inhibit the development of leukemia cells, at least in the studied conditions. However, MSCs from AML patients seem to have lost this capacity, and could therefore contribute to the development of leukemia.


Assuntos
Proliferação de Células , Leucemia Mieloide Aguda , Células-Tronco Mesenquimais , Humanos , Células-Tronco Mesenquimais/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/genética , Células K562 , Apoptose , Secretoma/metabolismo , Pessoa de Meia-Idade , Feminino , Masculino , Células da Medula Óssea/metabolismo , Linhagem da Célula/genética , Sobrevivência Celular , Adulto
6.
Asian Pac J Cancer Prev ; 25(5): 1803-1813, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38809653

RESUMO

BACKGROUND: Breast cancer stem cells (BCSCs) play a role in the high rates of resistance, recurrence, and metastasis. The precise biomarkers of BCSCs can assist effectively in identifying cancer, assessing prognosis, diagnosing, and monitoring therapy. The aim of this study was to give a complete analysis for predicting specific biomarkers of BCSCs. METHODS: We aggregated profile datasets in this work to shed light on the underlying critical genes and pathways of BCSCs. We obtained two expression profiling by array datasets (GSE7513 and GSE7515) from the Gene Expression Omnibus (GEO) database to identify biomarkers in BCSCs. Enrichr was used to do functional analysis, including gene ontology (GO) and reactome pathway. Furthermore, the protein-protein interaction (PPI) of these differential expression genes (DEGs) was visualized using Cytoscape with the search tool for the retrieval of interacting genes (STRING). The hub genes in the PPI network were chosen for further investigation. RESULTS: We identified 65 up-regulated and 190 down- regulated DEGs and the GO enrichment analysis revealed that these DEGs were enriched in biological process related to tumorigenesis and stemness, including alter the extracellular matrix's physicochemical properties, cytoskeletal reorganisation, adhesion, motility, migration, growth, and survival. The Reactome analysis indicated that these DEGs were also involved in modulating function of ECM, regulation cancer metabolism and angiogenesis, tumor growth, proliferation, and metastasis. CONCLUSION: Our bioinformatic study revealed that FYN, INADL, OCLN, F11R, and TOP2A were potential biomarker panel of BCSCs from secretome.


Assuntos
Biomarcadores Tumorais , Neoplasias da Mama , Células-Tronco Neoplásicas , Mapas de Interação de Proteínas , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Feminino , Secretoma/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Biologia Computacional/métodos , Prognóstico
7.
Stem Cell Res Ther ; 15(1): 137, 2024 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-38735979

RESUMO

Scar tissue is the inevitable result of repairing human skin after it has been subjected to external destructive stimuli. It leads to localized damage to the appearance of the skin, accompanied by symptoms such as itching and pain, which reduces the quality of life of the patient and causes serious medical burdens. With the continuous development of economy and society, there is an increasing demand for beauty. People are looking forward to a safer and more effective method to eliminate pathological scarring. In recent years, adipose-derived stem cells (ADSCs) have received increasing attention from researchers. It can effectively improve pathological scarring by mediating inflammation, regulating fibroblast proliferation and activation, and vascular reconstruction. This review focuses on the pathophysiological mechanisms of hypertrophic scarring, summarizing the therapeutic effects of in vitro, in vivo, and clinical studies on the therapeutic effects of ADSCs in the field of hypertrophic scarring prevention and treatment, the latest application techniques, such as cell-free therapies utilizing ADSCs, and discussing the advantages and limitations of ADSCs. Through this review, we hope to further understand the characterization of ADSC and clarify the effectiveness of its application in hypertrophic scarring treatment, so as to provide clinical guidance.


Assuntos
Tecido Adiposo , Cicatriz Hipertrófica , Humanos , Cicatriz Hipertrófica/terapia , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/patologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Células-Tronco/metabolismo , Células-Tronco/citologia , Secretoma/metabolismo , Animais , Transplante de Células-Tronco/métodos
8.
Sci Rep ; 14(1): 10243, 2024 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-38702388

RESUMO

The widespread use of multipotent mesenchymal stromal cell-derived secretome (MSC-sec) requires optimal preservation methods. Lyophilization offers benefits like concentrating the secretome, reducing the storage volume, and making storage conditions more flexible. This study evaluated the influence of storage duration and temperature on lyophilized MSC-sec. The conditioned medium from Wharton's jelly MSCs was stored at - 80 °C or lyophilized with or without trehalose. Lyophilized formulations were kept at - 80 °C, - 20 °C, 4 °C, or room temperature (RT) for 3 and 30 months. After storage and reconstitution, the levels of growth factors and cytokines were assessed using multiplex assay. The storage of lyophilized MSC-sec at - 80 °C ensured biomolecule preservation for 3 and 30 months. Following 3 month storage at 4 °C and RT, a notable decrease occurred in BDNF, bNGF, and sVCAM-1 levels. Prolonged 30 month storage at the same temperatures significantly reduced BDNF, bNGF, VEGF-A, IL-6, and sVCAM-1, while storage at - 20 °C decreased BDNF, bNGF, and VEGF- A levels. Trehalose supplementation of MSC-sec improved the outcome during storage at 4 °C and RT. Proper storage conditions were crucial for the preservation of lyophilized MSC-sec composition. Short-term storage at various temperatures maintained over 60% of the studied growth factors and cytokines; long-term preservation was only adequate at -80 °C.


Assuntos
Liofilização , Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Humanos , Secretoma/metabolismo , Trealose/metabolismo , Trealose/farmacologia , Citocinas/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/química , Criopreservação/métodos , Temperatura
9.
EBioMedicine ; 103: 105145, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38713924

RESUMO

BACKGROUND: There is increased evidence that the effects of stem cells can mostly be duplicated by administration of their secretome which might streamline the translation towards the clinics. METHODS: The 12-patient SECRET-HF phase 1 trial has thus been designed to determine the feasibility and safety of repeated intravenous injections of the extracellular vesicle (EV)-enriched secretome of cardiovascular progenitor cells differentiated from pluripotent stem cells in severely symptomatic patients with drug-refractory left ventricular (LV) dysfunction secondary to non-ischemic dilated cardiomyopathy. Here we report the case of the first treated patient (baseline NYHA class III; LV Ejection Fraction:25%) in whom a dose of 20 × 109 particles/kg was intravenously infused three times three weeks apart. FINDINGS: In addition to demonstrating the feasibility of producing a cardiac cell secretome compliant with Good Manufacturing Practice standards, this case documents the excellent tolerance of its repeated delivery, without any adverse events during or after infusions. Six months after the procedure, the patient is in NYHA Class II with improved echo parameters, a reduced daily need for diuretics (from 240 mg to 160 mg), no firing from the previously implanted automatic internal defibrillator and no alloimmunization against the drug product, thereby supporting its lack of immunogenicity. INTERPRETATION: The rationale underlying the intravenous route is that the infused EV-enriched secretome may act by rewiring endogenous immune cells, both circulating and in peripheral organs, to take on a reparative phenotype. These EV-modified immune cells could then traffic to the heart to effect tissue repair, including mitigation of inflammation which is a hallmark of cardiac failure. FUNDING: This trial is funded by the French Ministry of Health (Programme Hospitalier de Recherche CliniqueAOM19330) and the "France 2030" National Strategy Program (ANR-20-F2II-0003). It is sponsored by Assistance Publique-Hôpitaux de Paris.


Assuntos
Insuficiência Cardíaca , Secretoma , Humanos , Insuficiência Cardíaca/terapia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/etiologia , Secretoma/metabolismo , Masculino , Vesículas Extracelulares/metabolismo , Pessoa de Meia-Idade , Resultado do Tratamento
10.
J Proteome Res ; 23(6): 2160-2168, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38767394

RESUMO

Resistance is a major problem with effective cancer treatment and the stroma forms a significant portion of the tumor mass but traditional drug screens involve cancer cells alone. Cancer-associated fibroblasts (CAFs) are a major tumor stroma component and its secreted proteins may influence the function of cancer cells. The majority of secretome studies compare different cancer or CAF cell lines exclusively. Here, we present the direct characterization of the secreted protein profiles between CAFs and KRAS mutant-cancer cell lines from colorectal, lung, and pancreatic tissues using multiplexed mass spectrometry. 2573 secreted proteins were annotated, and differential analysis highlighted understudied CAF-enriched secreted proteins, including Wnt family member 5B (WNT5B), in addition to established CAF markers, such as collagens. The functional role of CAF secreted proteins was explored by assessing its effect on the response to 97 anticancer drugs since stromal cells may cause a differing cancer drug response, which may be missed on routine drug screening using cancer cells alone. CAF secreted proteins caused specific effects on each of the cancer cell lines, which highlights the complexity and challenges in cancer treatment and so the importance to consider stromal elements.


Assuntos
Fibroblastos Associados a Câncer , Secretoma , Humanos , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Secretoma/metabolismo , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Espectrometria de Massas , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Proteômica/métodos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética
11.
PLoS One ; 19(5): e0292978, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38728307

RESUMO

Endosalpingiosis (ES) and endometriosis (EM) refer to the growth of tubal and endometrial epithelium respectively, outside of their site of origin. We hypothesize that uterine secretome factors drive ectopic growth. To test this, we developed a mouse model of ES and EM using tdTomato (tdT) transgenic fluorescent mice as donors. To block implantation factors, progesterone knockout (PKO) tdT mice were created. Fluorescent lesions were present after oviduct implantation with and without WT endometrium. Implantation was increased (p<0.05) when tdt oviductal tissue was implanted with endometrium compared to oviductal tissue alone. Implantation was reduced (p<0.0005) in animals implanted with minced tdT oviductal tissue with PKO tdT endometrium compared to WT endometrium. Finally, oviductal tissues was incubated with and without a known implantation factor, leukemia inhibitory factor (LIF) prior to and during implantation. LIF promoted lesion implantation. In conclusion, endometrial derived implantation factors, such as LIF, are necessary to initiate ectopic tissue growth. We have developed an animal model of ectopic growth of gynecologic tissues in a WT mouse which will potentially allow for development of new prevention and treatment modalities.


Assuntos
Endometriose , Endométrio , Útero , Animais , Feminino , Camundongos , Endometriose/metabolismo , Endometriose/patologia , Endometriose/genética , Útero/metabolismo , Endométrio/metabolismo , Fator Inibidor de Leucemia/metabolismo , Fator Inibidor de Leucemia/genética , Secretoma/metabolismo , Camundongos Transgênicos , Modelos Animais de Doenças , Tubas Uterinas/metabolismo , Progesterona/metabolismo , Camundongos Knockout , Implantação do Embrião/fisiologia
12.
Front Immunol ; 15: 1232070, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38638443

RESUMO

Chronic liver diseases, such as non-alcoholic steatohepatitis (NASH)-induced cirrhosis, are characterized by an increasing accumulation of stressed, damaged, or dying hepatocytes. Hepatocyte damage triggers the activation of resident immune cells, such as Kupffer cells (KC), as well as the recruitment of immune cells from the circulation toward areas of inflammation. After infiltration, monocytes differentiate into monocyte-derived macrophages (MoMF) which are functionally distinct from resident KC. We herein aim to compare the in vitro signatures of polarized macrophages and activated hepatic stellate cells (HSC) with ex vivo-derived disease signatures from human NASH. Furthermore, to shed more light on HSC activation and liver fibrosis progression, we investigate the effects of the secretome from primary human monocytes, macrophages, and NK cells on HSC activation. Interleukin (IL)-4 and IL-13 treatment induced transforming growth factor beta 1 (TGF-ß1) secretion by macrophages. However, the supernatant transfer did not induce HSC activation. Interestingly, PMA-activated macrophages showed strong induction of the fibrosis response genes COL10A1 and CTGF, while the supernatant of IL-4/IL-13-treated monocytes induced the upregulation of COL3A1 in HSC. The supernatant of PMA-activated NK cells had the strongest effect on COL10A1 induction in HSC, while IL-15-stimulated NK cells reduced the expression of COL1A1 and CTGF. These data indicate that other factors, aside from the well-known cytokines and chemokines, might potentially be stronger contributors to the activation of HSCs and induction of a fibrotic response, indicating a more diverse and complex role of monocytes, macrophages, and NK cells in liver fibrosis progression.


Assuntos
Células de Kupffer , Hepatopatia Gordurosa não Alcoólica , Humanos , Células de Kupffer/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Interleucina-13/metabolismo , Secretoma , Macrófagos , Cirrose Hepática , Células Matadoras Naturais/metabolismo
13.
Mol Biol Rep ; 51(1): 570, 2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38658405

RESUMO

INTRODUCTION: Spinal cord injury (SCI) leads to significant destruction of nerve tissue, causing the degeneration of axons and the formation of cystic cavities. This study aimed to examine the characteristics of human umbilical cord-derived mesenchymal stem cells (HUCMSCs) cultured in a serum-free conditioned medium (CM) and assess their effectiveness in a well-established hemitransection SCI model. MATERIALS AND METHODS: In this study, HUCMSCs cultured medium was collected and characterized by measuring IL-10 and identifying proteomics using mass spectroscopy. This collected serum-free CM was further used in the experiments to culture and characterize the HUMSCs. Later, neuronal cells derived from CM-enriched HUCMSC were tested sequentially using an injectable caffeic acid-bioconjugated gelatin (CBG), which was further transplanted in a hemitransection SCI model. In vitro, characterization of CM-enriched HUCMSCs and differentiated neuronal cells was performed using flow cytometry, immunofluorescence, electron microscopy, and post-transplant analysis using immunohistology analysis, qPCR, in vivo bioluminescence imaging, and behavioral analysis using an infrared actimeter. RESULTS: The cells that were cultured in the conditioned media produced a pro-inflammatory cytokine called IL-10. Upon examining the secretome of the conditioned media, the Kruppel-like family of KRAB and zinc-finger proteins (C2H2 and C4) were found to be activated. Transcriptome analysis also revealed an increased expression of ELK-1, HOXD8, OTX2, YY1, STAT1, ETV7, and PATZ1 in the conditioned media. Furthermore, the expression of Human Stem-101 confirmed proliferation during the first 3 weeks after transplantation, along with the migration of CBG-UCNSC cells within the transplanted area. The gene analysis showed increased expression of Nestin, NeuN, Calb-2, Msi1, and Msi2. The group that received CBG-UCNSC therapy showed a smooth recovery by the end of week 2, with most rats regaining their walking abilities similar to those before the spinal cord injury by week 5. CONCLUSIONS: In conclusion, the CBG-UCNSC method effectively preserved the integrity of the transplanted neuronal-like cells and improved locomotor function. Thus, CM-enriched cells can potentially reduce biosafety risks associated with animal content, making them a promising option for clinical applications in treating spinal cord injuries.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Traumatismos da Medula Espinal , Transcriptoma , Cordão Umbilical , Traumatismos da Medula Espinal/terapia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/genética , Células-Tronco Mesenquimais/metabolismo , Meios de Cultivo Condicionados/farmacologia , Cordão Umbilical/citologia , Cordão Umbilical/metabolismo , Humanos , Animais , Transplante de Células-Tronco Mesenquimais/métodos , Transcriptoma/genética , Ratos , Secretoma/metabolismo , Diferenciação Celular , Neurônios/metabolismo , Modelos Animais de Doenças , Interleucina-10/genética , Interleucina-10/metabolismo , Células Cultivadas , Proteômica/métodos
14.
Stem Cell Res Ther ; 15(1): 121, 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38664697

RESUMO

BACKGROUND: Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer and, despite its adverse effects, chemotherapy is the standard systemic treatment option for TNBC. Since, it is of utmost importance to consider the combination of different agents to achieve greater efficacy and curability potential, MSC secretome is a possible innovative alternative. METHODS: In the present study, we proposed to investigate the anti-tumor effect of the combination of a chemical agent (paclitaxel) with a complex biological product, secretome derived from human Uterine Cervical Stem cells (CM-hUCESC) in TNBC. RESULTS: The combination of paclitaxel and CM-hUCESC decreased cell proliferation and invasiveness of tumor cells and induced apoptosis in vitro (MDA-MB-231 and/or primary tumor cells). The anti-tumor effect was confirmed in a mouse tumor xenograft model showing that the combination of both products has a significant effect in reducing tumor growth. Also, pre-conditioning hUCESC with a sub-lethal dose of paclitaxel enhances the effect of its secretome and in combination with paclitaxel reduced significantly tumor growth and even allows to diminish the dose of paclitaxel in vivo. This effect is in part due to the action of extracellular vesicles (EVs) derived from CM-hUCESC and soluble factors, such as TIMP-1 and - 2. CONCLUSIONS: In conclusion, our data demonstrate the synergistic effect of the combination of CM-hUCESC with paclitaxel on TNBC and opens an opportunity to reduce the dose of the chemotherapeutic agents, which may decrease chemotherapy-related toxicity.


Assuntos
Proliferação de Células , Células-Tronco Mesenquimais , Paclitaxel , Secretoma , Neoplasias de Mama Triplo Negativas , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Humanos , Feminino , Animais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Secretoma/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Apoptose/efeitos dos fármacos , Colo do Útero/metabolismo , Colo do Útero/patologia , Colo do Útero/efeitos dos fármacos
15.
Int J Mol Sci ; 25(8)2024 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-38673927

RESUMO

Domestic cat blastocysts cultured without the zona pellucida exhibit reduced implantation capacity. However, the protein expression profile has not been evaluated in these embryos. The objective of this study was to evaluate the protein expression profile of domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were generated: (1) domestic cat embryos generated by IVF and cultured in vitro (zona intact, (ZI)) and (2) domestic cat embryos cultured in vitro without the zona pellucida (zona-free (ZF group)). The cleavage, morula, and blastocyst rates were estimated at days 2, 5 and 7, respectively. Day 7 blastocysts and their culture media were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The UniProt Felis catus database was used to identify the standard proteome. No significant differences were found in the cleavage, morula, or blastocyst rates between the ZI and ZF groups (p > 0.05). Proteomic analysis revealed 22 upregulated and 20 downregulated proteins in the ZF blastocysts. Furthermore, 14 proteins involved in embryo development and implantation were present exclusively in the culture medium of the ZI blastocysts. In conclusion, embryo culture without the zona pellucida did not affect in vitro development, but altered the protein expression profile and release of domestic cat blastocysts.


Assuntos
Blastocisto , Proteômica , Zona Pelúcida , Animais , Blastocisto/metabolismo , Zona Pelúcida/metabolismo , Gatos , Proteômica/métodos , Técnicas de Cultura Embrionária , Secretoma/metabolismo , Feminino , Fertilização in vitro , Proteoma/metabolismo , Desenvolvimento Embrionário , Espectrometria de Massas em Tandem , Cromatografia Líquida
16.
Stem Cell Res Ther ; 15(1): 119, 2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38659070

RESUMO

BACKGROUND: Adipose stromal cells (ASC) are a form of mesenchymal stromal cells that elicit effects primarily via secreted factors, which may have advantages for the treatment of injury or disease. Several previous studies have demonstrated a protective role for MSC/ASC on mitigating acute kidney injury but whether ASC derived factors could hasten recovery from established injury has not been evaluated. METHODS: We generated a concentrated secretome (CS) of human ASC under well-defined conditions and evaluated its ability to improve the recovery of renal function in a preclinical model of acute kidney injury (AKI) in rats. 24 h following bilateral ischemia/reperfusion (I/R), rats were randomized following determination of plasma creatinine into groups receiving vehicle -control or ASC-CS treatment by subcutaneous injection (2 mg protein/kg) and monitored for evaluation of renal function, structure and inflammation. RESULTS: Renal function, assessed by plasma creatinine levels, recovered faster in ASC-CS treated rats vs vehicle. The most prominent difference between the ASC-CS treated vs vehicle was observed in rats with the most severe degree of initial injury (Pcr > 3.0 mg/dl 24 h post I/R), whereas rats with less severe injury (Pcr < 2.9 mg/dl) recovered quickly regardless of treatment. The quicker recovery of ASC-treated rats with severe injury was associated with less tissue damage, inflammation, and lower plasma angiopoietin 2. In vitro, ASC-CS attenuated the activation of the Th17 phenotype in lymphocytes isolated from injured kidneys. CONCLUSIONS: Taken together, these data suggest that ASC-CS represents a potent therapeutic option to improve established AKI.


Assuntos
Injúria Renal Aguda , Inflamação , Injúria Renal Aguda/terapia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Ratos , Humanos , Inflamação/patologia , Inflamação/metabolismo , Masculino , Secretoma/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Ratos Sprague-Dawley , Injeções Subcutâneas , Rim/metabolismo , Rim/patologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/terapia , Células Estromais/metabolismo
17.
Cells ; 13(7)2024 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-38607010

RESUMO

Extracellular vesicles (EVs) are secreted from many tumors, including glioblastoma multiforme (GBM), the most common and lethal brain tumor in adults, which shows high resistance to current therapies and poor patient prognosis. Given the high relevance of the information provided by cancer cell secretome, we performed a proteomic analysis of microvesicles (MVs) and exosomes (EXOs) released from GBM-derived stem cells (GSCs). The latter, obtained from the brain of GBM patients, expressed P2X7 receptors (P2X7Rs), which positively correlate with GBM growth and invasiveness. P2X7R stimulation of GSCs caused significant changes in the EV content, mostly ex novo inducing or upregulating the expression of proteins related to cytoskeleton reorganization, cell motility/spreading, energy supply, protection against oxidative stress, chromatin remodeling, and transcriptional regulation. Most of the induced/upregulated proteins have already been identified as GBM diagnostic/prognostic factors, while others have only been reported in peripheral tumors. Our findings indicate that P2X7R stimulation enhances the transport and, therefore, possible intercellular exchange of GBM aggressiveness-increasing proteins by GSC-derived EVs. Thus, P2X7Rs could be considered a new druggable target of human GBM, although these data need to be confirmed in larger experimental sets.


Assuntos
Vesículas Extracelulares , Glioblastoma , Receptores Purinérgicos P2X7 , Secretoma , Humanos , Linhagem Celular Tumoral , Vesículas Extracelulares/metabolismo , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/patologia , Proteoma/metabolismo , Proteômica , Receptores Purinérgicos P2X7/metabolismo
18.
Biochim Biophys Acta Mol Cell Res ; 1871(5): 119736, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38663552

RESUMO

The crosstalk between lung cancer cells and cancer-associated fibroblast (CAF) is pivotal in cancer progression. Heat shock protein family D member 1 (HSPD1) is a potential prognostic biomarker associated with the tumor microenvironment in lung adenocarcinoma (LUAD). However, the role of HSPD1 in CAF activation remains unclear. This study established stable HSPD1-knockdown A549 lung cancer cells using a lentivirus-mediated shRNA transduction. A targeted label-free proteomic analysis identified six significantly altered secretory proteins in the shHSPD1-A549 secretome compared to shControl-A549. Functional enrichment analysis highlighted their involvement in cell-to-cell communication and immune responses within the tumor microenvironment. Additionally, most altered proteins exhibited positive correlations and significant prognostic impacts on LUAD patient survival. Investigations on the effects of lung cancer secretomes on lung fibroblast WI-38 cells revealed that the shControl-A549 secretome stimulated fibroblast proliferation, migration, and CAF marker expression. These effects were reversed upon the knockdown of HSPD1 in A549 cells. Altogether, our findings illustrate the role of HSPD1 in mediating CAF induction through secretory proteins, potentially contributing to the progression and aggressiveness of lung cancer.


Assuntos
Fibroblastos Associados a Câncer , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Células A549 , Proliferação de Células , Secretoma/metabolismo , Microambiente Tumoral , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Proteômica/métodos , Chaperonina 60 , Proteínas Mitocondriais
19.
Biomed Pharmacother ; 174: 116536, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38569274

RESUMO

Diabetic kidney disease (DKD) is a leading cause of kidney failure. However, the involvement of renal fibroblasts and their communications with renal epithelial cells during DKD remain poorly understood. We investigated the potential role of renal proximal tubular epithelial cells (PTECs) in renal fibroblast activation that might lead to DKD. Additionally, the protective effects of curcumin, a known antioxidant, against renal fibroblast activation induced by high glucose-treated PTECs were investigated. Secretome was collected from HK-2 PTECs under normal glucose, high glucose, high glucose pretreated/cotreated with curcumin, or osmotic control condition for 24 h. Such secretome was then used to treat BHK-21 renal fibroblasts for 24 h. BHK-21 cells treated with high glucose-induced secretome had increased levels of fibroblast activation markers, including spindle index, F-actin, α-smooth muscle actin (α-SMA), fibronectin, collagen I, matrix metalloproteinase-2 (MMP-2) and MMP-9, as compared with normal glucose and osmotic control conditions. However, all these increases were successfully mitigated by curcumin. In addition, high glucose markedly increased intracellular reactive oxygen species (ROS) and transforming growth factor-ß (TGF-ß) secretion, but did not affect the secretion of platelet-derived growth factor A (PDGFA) and interleukin-1ß (IL-1ß), in HK-2 renal cells as compared with normal glucose and osmotic control conditions. Both intracellular ROS and secreted TGF-ß levels were successfully mitigated by curcumin. Therefore, curcumin prevents the high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation, at least in part, via mitigating intracellular ROS and TGF-ß secretion.


Assuntos
Curcumina , Fibroblastos , Glucose , Espécies Reativas de Oxigênio , Fator de Crescimento Transformador beta , Curcumina/farmacologia , Glucose/toxicidade , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Animais , Secretoma/efeitos dos fármacos , Secretoma/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Nefropatias Diabéticas/metabolismo , Antioxidantes/farmacologia
20.
Sci Rep ; 14(1): 8379, 2024 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-38600116

RESUMO

Macrophage responses to activation are fluid and dynamic in their ability to respond appropriately to challenges, a role integral to host defence. While bacteria can influence macrophage differentiation and polarization into pro-inflammatory and alternatively activated phenotypes through direct interactions, many questions surround indirect communication mechanisms mediated through secretomes derived from gut bacteria, such as lactobacilli. We examined effects of secretome-mediated conditioning on THP-1 human monocytes, focusing on the ability of the Lacticaseibacillus rhamnosus R0011 secretome (LrS) to drive macrophage differentiation and polarization and prime immune responses to subsequent challenge with lipopolysaccharide (LPS). Genome-wide transcriptional profiling revealed increased M2-associated gene transcription in response to LrS conditioning in THP-1 cells. Cytokine and chemokine profiling confirmed these results, indicating increased M2-associated chemokine and cytokine production (IL-1Ra, IL-10). These cells had increased cell-surface marker expression of CD11b, CD86, and CX3CR1, coupled with reduced expression of the M1 macrophage-associated marker CD64. Mitochondrial substrate utilization assays indicated diminished reliance on glycolytic substrates, coupled with increased utilization of citric acid cycle intermediates, characteristics of functional M2 activity. LPS challenge of LrS-conditioned THP-1s revealed heightened responsiveness, indicative of innate immune priming. Resting stage THP-1 macrophages co-conditioned with LrS and retinoic acid also displayed an immunoregulatory phenotype with expression of CD83, CD11c and CD103 and production of regulatory cytokines. Secretome-mediated conditioning of macrophages into an immunoregulatory phenotype is an uncharacterized and potentially important route through which lactic acid bacteria and the gut microbiota may train and shape innate immunity at the gut-mucosal interface.


Assuntos
Lacticaseibacillus rhamnosus , Monócitos , Humanos , Monócitos/metabolismo , Secretoma , Lipopolissacarídeos , Citocinas/metabolismo , Quimiocinas/metabolismo , Imunidade
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