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INTRODUCTION: Uremic retention solutes have been alleged to induce the apoptotic program of different cell types, including peripheral blood mononuclear leukocytes (PBL), which may contribute to uremic leukopenia and immune dysfunction. METHODS: The molecular effects of these solutes were investigated in uremic PBL (u-PBL) and mononuclear cell lines (THP-1 and K562) exposed to the high molecular weight fraction of uremic plasma (u-HMW) prepared by in vitro ultrafiltration with 50 kDa cut-off microconcentrators. RESULTS: u-PBL show reduced cell viability and increased apoptotic death compared to healthy control PBL (c-PBL). u-HMW induce apoptosis both in u-PBL and c-PBL, as well as in mononuclear cell lines, also stimulating cellular H2O2 formation and secretion, IRE1-α-mediated endoplasmic reticulum stress signaling, and JNK/cJun pathway activation. Also, u-HMW induce autophagy in THP-1 monocytes. u-PBL were characterized by the presence in their cellular proteome of the main proteins and carbonylation targets of u-HMW, namely albumin, transferrin, and fibrinogen, and by the increased expression of receptor for advanced glycation end-products, a scavenger receptor with promiscuous ligand binding properties involved in leukocyte activation and endocytosis. CONCLUSIONS: Large uremic solutes induce abnormal endocytosis and terminal alteration of cellular proteostasis mechanisms in PBL, including UPR/ER stress response and autophagy, ultimately activating the JNK-mediated apoptotic signaling of these cells. These findings describe the suicidal role of immune cells in facing systemic proteostasis alterations of kidney disease patients, a process that we define as the immuno-proteostasis response of uremia.
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Leucócitos Mononucleares , Proteostase , Humanos , Leucócitos Mononucleares/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas , Apoptose/fisiologiaRESUMO
Tendinopathies are common disabling conditions in equine and human athletes. The etiology is still unclear, although reactive oxygen species (ROS) and oxidative stress (OS) seem to play a crucial role. In addition, OS has been implicated in the failure of tendon lesion repair. Platelet-rich plasma (PRP) is rich in growth factors that promote tissue regeneration. This is a promising therapeutic approach in tendon injury. Moreover, growing evidence has been attributed to PRP antioxidant effects that can sustain tissue healing. In this study, the potential antioxidant effects of PRP in tenocytes exposed to oxidative stress were investigated. The results demonstrated that PRP reduces protein and lipid oxidative damage and protects tenocytes from OS-induced cell death. The results also showed that PRP was able to increase nuclear levels of redox-dependent transcription factor Nrf2 and to induce some antioxidant/phase II detoxifying enzymes (superoxide dismutase 2, catalase, heme oxygenase 1, NAD(P)H oxidoreductase quinone-1, glutamate cysteine ligase catalytic subunit and glutathione, S-transferase). Moreover, PRP also increased the enzymatic activity of catalase and glutathione S-transferase. In conclusion, this study suggests that PRP could activate various cellular signaling pathways, including the Nrf2 pathway, for the restoration of tenocyte homeostasis and to promote tendon regeneration and repair following tendon injuries.
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Antioxidantes , Fator 2 Relacionado a NF-E2 , Animais , Plaquetas , Catalase , Cavalos , TenócitosRESUMO
Beyond the consolidated role in degrading and recycling cellular waste, the autophagic- and endo-lysosomal systems play a crucial role in extracellular release pathways. Lysosomal exocytosis is a process leading to the secretion of lysosomal content upon lysosome fusion with plasma membrane and is an important mechanism of cellular clearance, necessary to maintain cell fitness. Exosomes are a class of extracellular vesicles originating from the inward budding of the membrane of late endosomes, which may not fuse with lysosomes but be released extracellularly upon exocytosis. In addition to garbage disposal tools, they are now considered a cell-to-cell communication mechanism. Autophagy is a cellular process leading to sequestration of cytosolic cargoes for their degradation within lysosomes. However, the autophagic machinery is also involved in unconventional protein secretion and autophagy-dependent secretion, which are fundamental mechanisms for toxic protein disposal, immune signalling and pathogen surveillance. These cellular processes underline the crosstalk between the autophagic and the endosomal system and indicate an intersection between degradative and secretory functions. Further, they suggest that the molecular mechanisms underlying fusion, either with lysosomes or plasma membrane, are key determinants to maintain cell homeostasis upon stressing stimuli. When they fail, the accumulation of undigested substrates leads to pathological consequences, as indicated by the involvement of autophagic and lysosomal alteration in human diseases, namely lysosomal storage disorders, age-related neurodegenerative diseases and cancer. In this paper, we reviewed the current knowledge on the functional role of extracellular release pathways involving lysosomes and the autophagic- and endo-lysosomal systems, evaluating their implication in health and disease.
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Autofagia , Exocitose , Vesículas Extracelulares/fisiologia , Lisossomos/fisiologia , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Endossomos/fisiologia , Exossomos/fisiologia , HumanosRESUMO
Oxidative stress is considered to be a key factor of the pathogenesis of Parkinson's disease, a multifactorial neurodegenerative disorder characterized by reduced dopaminergic neurons in the substantia nigra pars compacta and accumulated protein aggregates. Rotenone is a worldwide-used pesticide that induces the most common features of Parkinson's by direct inhibition of the mitochondrial complex I. Rotenone-induced Parkinson's models, as well as brain tissues from Parkinson's patients, are characterized by the presence of both lipid peroxidation and protein oxidation markers resulting from the increased level of free radical species. Oxidation introduces several modifications in protein structure, including carbonylation and nitrotyrosine formation, which severely compromise cell function. Due to the link existing between oxidative stress and Parkinson's disease, antioxidant molecules could represent possible therapeutic tools for this disease. In this study, we evaluated the effect of curcumin, a natural compound known for its antioxidant properties, in dopaminergic PC12 cells treated with rotenone, a cell model of Parkinsonism. Our results demonstrate that the treatment of PC12 cells with rotenone causes severe protein damage, with formation of both carbonylated and nitrotyrosine-derived proteins, whereas curcumin (10 µM) co-exposure exerts protective effects by reducing the levels of oxidized proteins. Curcumin also promotes proteasome activation, abolishing the inhibitory effect exerted by rotenone on this degradative system.
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Curcumina/farmacologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Praguicidas/toxicidade , Rotenona/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Modelos Biológicos , Células PC12 , Carbamilação de Proteínas/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
The monocarbonyl analogue of curcumin (1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one (C1) has been used as a specific activator of the master gene transcription factor EB (TFEB) to correlate the activation of this nuclear factor with the increased activity of lysosomal glycohydrolases and their recruitment to the cell surface. The presence of active lysosomal glycohydrolases associated with the lipid microdomains has been extensively demonstrated, and their role in glycosphingolipid (GSL) remodeling in both physiological and pathological conditions, such as neurodegenerative disorders, has been suggested. Here, we demonstrate that Jurkat cell stimulation elicits TFEB nuclear translocation and an increase of both the expression of hexosaminidase subunit beta (HEXB), hexosaminidase subunit alpha (HEXA), and galactosidase beta 1 (GLB1) genes, and the recruitment of ß-hexosaminidase (Hex, EC 3.2.1.52) and ß-galactosidase (Gal, EC 3.2.1.23) on lipid microdomains. Treatment of Jurkat cells with the curcumin analogue C1 also resulted in an increase of both lysosomal glycohydrolase activity and their targeting to the cell surface. Similar effects of C1 on lysosomal glycohydrolase expression and their recruitment to lipid microdomains was observed by treating the SH-SY5Y neuroblastoma cell line; the effects of C1 treatment were abolished by TFEB silencing. Together, these results clearly demonstrate the existence of a direct link between TFEB nuclear translocation and the transport of Hex and Gal from lysosomes to the plasma membrane.
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Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Membrana Celular/metabolismo , Curcumina/análogos & derivados , Curcumina/farmacologia , Hexosaminidases/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , beta-Galactosidase/metabolismo , Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Exocitose/efeitos dos fármacos , Humanos , Células Jurkat , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Fito-Hemaglutininas/farmacologia , Transporte Proteico/efeitos dos fármacosRESUMO
Osteochondrosis is a failure of the endochondral ossification that affects developing joints in humans and several animal species. It is a localized idiopathic joint disorder characterized by focal chondronecrosis and growing cartilage retention, which can lead to the formation of fissures, subchondral bone cysts, or intra-articular fragments. Osteochondrosis is a complex multifactorial disease associated with extracellular matrix alterations and failure in chondrocyte differentiation, mainly due to genetic, biochemical, and nutritional factors, as well as traumas. This study describes the main proteomic alterations occurring in chondrocytes isolated from osteochondrotic cartilage fragments. A comparative analysis performed on equine osteochondrotic and healthy chondrocytes showed 26 protein species as differentially represented. In particular, quantitative changes in the extracellular matrix, cytoskeletal and chaperone proteins, and in cell adhesion and signaling molecules were observed in osteochondrotic cells, compared to healthy controls. Functional group analysis annotated most of these proteins in "growth plate and cartilage development", while others were included in "glycolysis and gluconeogenesis", "positive regulation of protein import", "cell-cell adhesion mediator activity", and "mitochondrion nucleoid". These results may help to clarify some chondrocyte functional alterations that may play a significant role in determining the onset and progression of equine osteochondrosis and, being related, of human juvenile osteochondrosis.
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Condrócitos/citologia , Doenças dos Cavalos/patologia , Osteocondrose/patologia , Proteoma/análise , Proteoma/metabolismo , Animais , Células Cultivadas , Condrócitos/metabolismo , Doenças dos Cavalos/metabolismo , Cavalos , Masculino , Osteocondrose/metabolismo , ProteômicaRESUMO
OBJECTIVE: To assess the effects of xylazine and dexmedetomidine on equine chondrocytes, in vitro. STUDY DESIGN: Prospective, experimental study. STUDY MATERIAL: Equine articular chondrocytes from five male horses. METHODS: Chondrocytes were isolated from healthy equine articular cartilage of the metacarpo/metatarsophalangeal joints. Cell viability was assessed using the WST-8 assay by exposing chondrocytes to xylazine (0.5, 1, 2, 4, 8, 16.6, 25, 50 mg mL-1) or dexmedetomidine (0.001, 0.005, 0.01, 0.05, 0.175, 0.25 mg mL-1) for 15, 30 and 60 minutes. Based on the results of these tests, cells were treated with xylazine (1, 4, 25 mg mL-1) or dexmedetomidine (0.05, 0.175, 0.25 mg mL-1) for 15 minutes to further evaluate: cell viability by neutral red uptake; cell membrane integrity by lactate dehydrogenase release and by fluorescence microscopy with Hoechst 33342 and propidium iodide (PI), and apoptosis by flow cytometry using double staining with annexin V-fluorescein isothiocyanate/PI and by cell morphology. RESULTS: Both drugs reduced cell viability in a dose-dependent manner. Specifically, all xylazine concentrations, except 0.5 mg mL-1 and 1 mg mL-1, significantly reduced cell viability, whereas the effects of dexmedetomidine were evident only at 0.175 mg mL-1 and 0.25 mg mL-1. The highest concentrations of xylazine (25 mg mL-1) and dexmedetomidine (0.25 mg mL-1) caused loss of membrane integrity. Cell morphology and flow cytometry analyses demonstrated signs of late apoptosis in xylazine-treated cells, and signs of late apoptosis and necrosis in dexmedetomidine-treated cells. CONCLUSIONS AND CLINICAL RELEVANCE: This study offers new insights into the potential chondrotoxicity induced by dexmedetomidine and xylazine. Therefore, the intra-articular administration of α2-agonists should be conducted with care, especially for doses of ≥ 4 mg mL-1 of xylazine and 0.175 mg mL-1 and 0.25 mg mL-1 of dexmedetomidine.
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Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Cartilagem Articular/citologia , Condrócitos/efeitos dos fármacos , Dexmedetomidina/farmacologia , Xilazina/farmacologia , Agonistas de Receptores Adrenérgicos alfa 2/administração & dosagem , Animais , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cavalos , Técnicas In Vitro/veterinária , Masculino , Estudos Prospectivos , Sais de Tetrazólio/farmacologiaRESUMO
Since the discovery of the endocannabinoid system and due to the empirical evidence of the therapeutic effects on several illnesses both in humans and animals that follow the administration of exogenous cannabinoids (i.e., phytocannabinoids), numerous studies have been conducted. These investigations aimed to identify the expression and distribution of cannabinoid receptors in healthy and pathologic organs and tissues of different animal species and to define the interactions of phytocannabinoids with these receptors. In the last decade, pharmacokinetics, efficacy and tolerability of many Cannabis derivatives formulations, mainly containing cannabidiol, in the main species of veterinary interest, have been also investigated. This manuscript summarizes the findings reported by the scientific studies published so far on the molecular mode of action of the main phytocannabinoids, the localization of cannabinoid receptors in organs and tissues, as well as the pharmacokinetics, efficacy and tolerability of Cannabis derivatives in dogs, cats, horses and other species of veterinary interest. A deep knowledge of these issues is crucial for the use of phytocannabinoids for therapeutic purposes in animal species.
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Background: Meningioma is the most common tumor of the central nervous system of dogs. For this tumor, surgery remains the treatment of choice, either alone or in combination with radiotherapy. Unfortunately, chemotherapeutic strategies are practically absent in dogs and palliative therapies are the only option to surgery. Somatostatin receptor subtype 2 (SSTR2) is expressed in canine meningioma. Since the potent cell-proliferation inhibiting effect of somatostatin (SST), the aim of this study was to investigate in vitro the effects of octreotide, as SST analog, in the viability of canine meningioma. Methods: Four surgical canine meningiomas were used in this study to establish cell cultures. Expression of SSTR2 was verified with immunolabelling in FFPE samples and cell cultures. The effects of octreotide on cell viability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). After 24 hours they were exposed to different concentrations of octreotide (0.1 nM, 1 nM, 10 nM, 100 nM) for 24 and 48 hours. Results: All meningiomas consisted of grade I tumors. The cultured neoplastic cells expressed SSTR2 from 80% to 100%. Octreotide significantly increased cell death after 48 hours of continuous exposure, with 10 and 100 nM octreotide doses. The percentage of cell viability was 80.92â ±â 4.9 and 80.49â ±â 3.61, compared to the control, respectively, consistent with decreased cell viability of about 20% for both doses. Conclusions: Octreotide reduced the alive neoplastic cultured cells of low-grade canine meningioma in a dose-dependent pattern with continuous exposition for 48 hours. These results support an alternative systemic treatment of meningioma with octreotide in the dog.
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The D-enantiomer of lactic acid (D-lactate) is normally produced from bacterial fermentation in the gastrointestinal tract in mammals. In humans, increased D-lactate concentrations are related to gastrointestinal disease, including short bowel syndrome and malabsorptive syndrome. Similarly, increased D-lactate concentrations have been described in calves affected by diarrhea, in cats with gastrointestinal diseases, and in dogs with parvoviral enteritis. The purpose of the present study was to measure the serum D-lactate concentrations in dogs with inflammatory bowel disease (IBD). We retrospectively reviewed data from the database of the VTH of Perugia University, and dogs affected by IBD with serum samples stored at -80 °C were considered eligible for inclusion. A total of 18 dogs with IBD and 10 healthy dogs were included in the study. The dogs with IBD were divided into three subcategories based on the severity of the disease. Serum D-lactate concentrations (µM) were determined using a commercially available colorimetric assay kit (D-Lactate Colorimetric Assay Kit; Catalog #K667-100, BioVision Inc., Milpitas, CA, USA). Our results showed no significant difference (p > 0.05) in the serum concentrations of D-lactate between dogs with various degrees of IBD and healthy dogs. However, the wide variability of the D-lactate concentrations in dogs with IBD and evidence of increased serum D-lactate concentrations in dogs with confirmed dysbiosis encourage further studies on this topic to understand potential factors influencing the serum D-lactate concentrations in dogs affected by IBD.
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Oral melanomas are the most common oral malignancies in dogs and are characterized by an aggressive nature, invasiveness, and poor prognosis. With biological and genetic similarities to human oral melanomas, they serve as a valuable spontaneous comparative model. Primary cell cultures are widely used in human medicine and, more recently, in veterinary medicine to study tumorigenesis, cancer progression, and innovative therapeutic approaches. This study aims to establish two- and three-dimensional primary cell lines from oral canine melanomas using fine-needle aspiration as a minimally invasive sampling method. For this study, samples were collected from six dogs, represented by four primary oral melanomas and five lymph nodal metastases. The cells were digested to obtain single-cell suspensions, seeded in flasks, or processed with Matrigel® to form organoids. The cell cultures were characterized through flow cytometry using antibodies against Melan-A, PNL2, and Sox-10. This technique offers a minimally invasive means to obtain cell samples, particularly beneficial for patients that are ineligible for surgical procedures, and enables the establishment of in vitro models crucial for comparative studies in mucosal melanoma oncology. To the best of our knowledge, this is the first work establishing neoplastic primary cell cultures via fine-needle aspiration in dogs.
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Local anaesthetics (LAs) can have detrimental effects on rat, bovine, canine, and human tendon tissues and cells. Currently, there has been no available data on the impact of these drugs on equine tenocytes. Even if LA injection for managing painful tendon conditions in horses is limited, it is usually used via intra-articular, intrasynovial, perineural, and intrathecal as well as for lameness examinations. In this in vitro study, the cytotoxic effects of LAs, including lidocaine, mepivacaine, and bupivacaine on equine tenocytes, in the presence and absence of platelet rich plasma (PRP), were investigated. PRP accelerates tissue healing and can exert cytoprotective effects on different cell types exposed to different stressful conditions, including drugs. Results indicated that the exposure to LAs significantly reduced tenocytes viability in dose- and time-dependent manners while PRP was able to counteract their cytotoxic effects. Furthermore, microscopy and flow cytometry analyses revealed apoptosis and necrosis in equine tenocytes exposed to these drugs, that were both reduced when PRP was in the medium. These findings highlight the importance of considering the tenocyte toxicity associated with intrathecal and intraneural LA injections, as they might affect tenocytes or reduce the efficacy of associated therapies. Moreover, this study also highlights the protective effects of PRP, which could make LA injections safer.
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Anestésicos Locais , Bupivacaína , Lidocaína , Mepivacaína , Plasma Rico em Plaquetas , Tenócitos , Animais , Cavalos , Tenócitos/efeitos dos fármacos , Anestésicos Locais/farmacologia , Anestésicos Locais/toxicidade , Bupivacaína/toxicidade , Lidocaína/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Apoptose/efeitos dos fármacosRESUMO
Edible plant and fruit-derived nanovesicles (NVs) are membrane-enclosed particles with round-shape morphology and signaling functions, which resemble mammalian cell-derived extracellular vesicles. These NVs can transmit cross-kingdom signals as they contain bioactive molecules and exert biological effects on mammalian cells. Their properties and stability in the gastrointestinal tract suggest NVs as a promising nutraceutical tool. In this study, we have demonstrated for the first time the presence of NVs in olive vegetation water (OVW), a waste by-product generated during olive oil production. Biophysical characterization by scanning electron microscopy, cryo-transmission electron microscopy, and nanoparticle tracking analysis revealed the presence in OVW of NVs having size and morphology similar to that of vesicles isolated from edible plants. Integrated lipidomic, metabolomic, and proteomic analyses showed that OVW-NVs carry a set of lipids, metabolites and proteins which have recognized antioxidant and anti-inflammatory activities. The nature of biomolecules identified in OVW-NVs suggests that these vesicles could exert beneficial effects on mammalian cells and could be used in the nutraceutical and food industries. The successful isolation of OVW-NVs and the characterization of their features strengthen the idea that agricultural waste might represent a source of NVs having features similar to NVs isolated from edible plants/fruits.
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The characterization of the protein and lipid cargo of milk extracellular vesicles from different mammal species is crucial for understanding their biogenesis and biological functions, as well as for a comprehensive description of the nutritional aspects of animal milk for human diet. In fact, milk EVs have been reported to possess relevant biological effects, but the molecules/biochemical pathways underlying these effects have been poorly investigated. The biochemical characterization is an important initial step for the potential therapeutic and diagnostic use of natural or modified milk EVs. The number of studies analysing the protein and lipid composition of milk EVs is limited compared to that investigating the nucleic acid cargo. Here, we revised the literature regarding the protein and lipid content of milk EVs. Until now, most investigations have shown that the biochemical cargo of EVs is different with respect to that of other milk fractions. In addition, even if these studies derived mostly from bovine and human milk EVs, comparison between milk EVs from different animal species and milk EVs biochemical composition changes due to different factors including lactation stages and health status is also beginning to be reported.
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Tuberous sclerosis complex (TSC) is a rare, multisystem genetic disorder that leads to the development of benign tumors in multiple organs and neurological symptoms. TSC clinical manifestations show a great heterogenicity, with most patients presenting severe neuropsychiatric and neurological disorders. TSC is caused by loss-of-function mutations in either TSC1 or TSC2 genes, leading to overexpression of the mechanistic target of rapamycin (mTOR) and, consequently, abnormal cellular growth, proliferation and differentiation as well as to cell migration defects. Beside the growing interest, TSC remains a disorder poorly understood, with limited perspectives in the field of therapeutic strategies. Here we used murine postnatal subventricular zone (SVZ) neural stem progenitor cells (NSPCs) deficient of Tsc1 gene as a TSC model to unravel novel molecular aspects of the pathophysiology of this disease. 2D-DIGE-based proteomic analysis detected 55 differently represented spots in Tsc1-deficient cells, compared to wild-type counterparts, which were associated with 36 protein entries after corresponding trypsinolysis and nanoLC-ESI-Q-Orbitrap-MS/MS analysis. Proteomic results were validated using various experimental approaches. Bioinformatics associated differently represented proteins with oxidative stress and redox pathways, methylglyoxal biosynthesis, myelin sheath, protein S-nitrosylation and carbohydrate metabolism. Because most of these cellular pathways have already been linked to TSC features, these results were useful to clarify some molecular aspects of TSC etiopathogenesis and suggested novel promising therapeutic protein targets. SIGNIFICANCE: Tuberous Sclerosis Complex (TSC) is a multisystemic disorder caused by inactivating mutations of TSC1 or TSC2 genes, which induce overactivation of the mTOR component. The molecular mechanisms underlying the pathogenesis of TSC remain unclear, probably due to complexity of mTOR signaling network. To have a picture of protein abundance changes occurring in TSC disorder, murine postnatal subventricular zone (SVZ) neural stem progenitor cells (NSPCs) deficient of Tsc1 gene were used as a model of disease. Thus, Tsc1-deficient SVZ NSPCs and wild-type cells were comparatively evaluated by proteomics. This analysis evidenced changes in the abundance of proteins involved in oxidative/nitrosative stress, cytoskeleton remodelling, neurotransmission, neurogenesis and carbohydrate metabolism. These proteins might clarify novel molecular aspects of TSC etiopathogenesis and constitute putative molecular targets for novel therapeutic management of TSC-related disorders.
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Células-Tronco Neurais , Esclerose Tuberosa , Camundongos , Humanos , Animais , Esclerose Tuberosa/genética , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteômica , Espectrometria de Massas em Tandem , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria, which contain different cargo molecules and mediate several biological processes. Recent studies have shown that OMVs are involved in antibiotic-resistance (AR) mechanisms by including ß-lactamase enzymes in their lumen. Since no studies have as yet been conducted on Salmonella enterica subs. enterica serovar Infantis' OMVs, the aim of the work was to collect OMVs from five S. Infantis ß-lactam resistant strains isolated from a broiler meat production chain and to investigate whether ß-lactamase enzymes are included in OMVs during their biogenesis. OMVs were isolated by means of ultrafiltration and a Nitrocefin assay quantified the presence of ß-lactamase enzymes in the OMVs. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to identify the OMVs. The results showed that all strains release spherical OMVs, ranging from 60 to 230 nm. The Nitrocefin assay highlighted the presence of ß-lactamase enzymes within the OMVs. This suggests that ß-lactamase enzymes also get packaged into OMVs from bacterial periplasm during OMV biogenesis. An investigation into the possible role played by OMVs in AR mechanisms would open the door for an opportunity to develop new, therapeutic strategies.
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Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.
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Fumar Cigarros , Vesículas Extracelulares , Humanos , Espécies Reativas de Oxigênio/metabolismo , Fumar Cigarros/efeitos adversos , Linhagem Celular , Células Epiteliais/metabolismo , Proteínas/metabolismo , Nicotiana/efeitos adversos , Vesículas Extracelulares/metabolismo , Fosfolipídeos/metabolismo , Fosfolipídeos/farmacologia , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologiaRESUMO
Introduction: Extracellular vesicles (EVs) are nanometric-membrane-bound sub-cellular structures, which can be recovered from milk. Milk EVs have drawn increasing interest due to their potential biomedical applications, therefore it is important to investigate their impact on key immune cells, such as macrophages. Methods: In this work, the immunomodulatory effects of goat milk EVs on untreated (moMФ) and classically activated (moM1) porcine monocyte-derived macrophages were investigated using flow cytometry, ELISA, and gene expression assays. Results: These particles were efficiently internalized by macrophages and high doses (60 mg protein weight) triggered the upregulation of MHC I and MHC II DR on moMФ, but not on moM1. In moMФ, exposure to low doses (0.6 mg) of mEVs enhanced the gene expression of IL10, EBI3, and IFNB, whereas high doses up-regulated several pro-inflammatory cytokines. These nanosized structures slightly modulated cytokine gene expression on moM1. Accordingly, the cytokine (protein) contents in culture supernatants of moMФ were mildly affected by exposure to low doses of mEVs, whereas high doses promoted the increased release of TNF, IL-8, IL-1a, IL-1b, IL-1Ra, IL-6, IL-10, and IL-12. The cytokines content in moM1 supernatants was not critically affected. Discussion: Overall, our data support a clinical application of these molecules: they polarized macrophages toward an M1-like phenotype, but this activation seemed to be controlled, to prevent potentially pathological over-reaction to stressors.
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Vesículas Extracelulares , Leite , Animais , Suínos , Leite/metabolismo , Macrófagos , Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , CabrasRESUMO
[This corrects the article DOI: 10.3389/fimmu.2023.1209898.].
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BACKGROUND: The applications of proteomic strategies to ovine medicine remain limited. The definition of serum proteome may be a good tool to identify useful protein biomarkers for recognising sub-clinical conditions and overt disease in sheep. Findings from bovine species are often directly translated for use in ovine medicine. In order to characterize normal protein patterns and improve knowledge of molecular species-specific characteristics, we generated a two-dimensional reference map of sheep serum. The possible application of this approach was tested by analysing serum protein patterns in ewes with mild broncho-pulmonary disease, which is very common in sheep and in the peripartum period which is a stressful time, with a high incidence of infectious and parasitic diseases. RESULTS: This study generated the first reference 2-DE maps of sheep serum. Overall, 250 protein spots were analyzed, and 138 identified.Compared with healthy sheep, serum protein profiles of animals with rhino-tracheo-bronchitis showed a significant decrease in protein spots identified as transthyretin, apolipoprotein A1 and a significant increase in spots identified as haptoglobin, endopin 1b and alpha1B glycoprotein.In the peripartum period, haptoglobin, alpha-1-acid glycoprotein, apolipoprotein A1 levels rose, while transthyretin content dropped. CONCLUSIONS: This study describes applications of proteomics in putative biomarker discovery for early diagnosis as well as for monitoring the physiological and metabolic situations critical for ovine welfare.