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OBJECTIVES: To isolate cancer stem cells (CSC) from a metastatic oral squamous cell carcinoma (OSCC) cell line and investigate their in vitro and in vivo phenotypic characteristics. MATERIALS AND METHODS: Subpopulations with individual staining intensities for CD44 and CD326 were isolated from the OSCC cell line LN-1A by FACS: CD44Low/CD326- (CSC-M1), CD44Low/CD326High (CSC-E), and CD44High/CD326- (CSC-M2). Proliferation, clonogenic potential, adhesion, migration, epithelial-mesenchymal transition markers, and sensitivity to cisplatin and TVB-3166 were analyzed in vitro. Tumor formation and metastasis were assessed by subcutaneous and orthotopic inoculations into BALB/c mice. RESULTS: E-cadherin levels were higher in CSC-E cells while vimentin and Slug more produced by CSC-M2 cells. CSC-M1 and CSC-M2 subpopulations showed higher proliferation, produced more colonies, and have stronger adhesion to the extracellular matrix. All cell lines established tumors; however, CSC-E and CSC-M2 formed larger masses and produced more metastases. CONCLUSION: The CSC subpopulations here described show increased cancer capabilities in vitro, tumorigenic and metastatic potential in vivo, and may be exploited in the search for novel therapeutic targets for OSCC.
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The Guadiana Basin is a transnational basin, presenting historical contamination with potentially toxic metals (PTM), which origin can be both natural and anthropogenic. This study explores the use of a set of observational, chemical and ecotoxicological assays with Heterocypris incongruens, Vibrio fischeri, Pseudokirchneriella subcapitata, Thamnocephalus platyurus, identifying the most sensitive to be included in a toolbox to analyze the quality of freshwater sediments related to this type of contamination. The study included the analysis of a reservoir and streams sediments of Guadiana basin, in two consecutive years with different climate conditions 2017 (dry year) and 2018 (normal year). The results showed high chemical variability along the basin, with greater contamination with PTM in the reservoir sediments. The calculated Enrichment Factors (EF) indicated high anthropogenic contamination by Cd, followed by Pb (EF > 1.5). The geoaccumulation index (Igeo) revealed that the sediments were severely polluted with Cd, and slightly polluted with Pb and Cu, inducing a higher sublethal toxicity to Heterocypris incongruens. Among the parameters evaluated, and after the use of multivariate statistical techniques, the toolbox for assessing sediments quality, in similar climate and geological conditions, should include the analysis of: meteorology, land use/cover in the area, granulometry, organic matter content, PTM concentrations, contamination indices (e.g., Igeo and EF), and sublethal bioassays with H. incongruens (total sediment analysis) and Vibrio fisheri luminescence inhibition (pore water analysis).
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Metais Pesados , Poluentes Químicos da Água , Poluentes Químicos da Água/toxicidade , Poluentes Químicos da Água/análise , Cádmio/análise , Chumbo/análise , Monitoramento Ambiental/métodos , Água Doce , Sedimentos Geológicos/análise , Metais Pesados/toxicidade , Metais Pesados/análise , Medição de RiscoRESUMO
BACKGROUND: Declining humoral immunity in coronavirus disease 2019 (COVID-19) patients and possible reinfection have raised concern. Mucosal immunity, particularly salivary antibodies, may be short lived although long-term studies are lacking. METHODS: Using a multiplex bead-based array platform, we investigated antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins in 256 saliva samples from convalescent patients 1-9 months after symptomatic COVID-19 (nâ =â 74, cohort 1), undiagnosed individuals with self-reported questionnaires (nâ =â 147, cohort 2), and individuals sampled prepandemic (nâ =â 35, cohort 3). RESULTS: Salivary IgG antibody responses in cohort 1 (mainly mild COVID-19) were detectable up to 9 months postrecovery, with high correlations between spike and nucleocapsid specificity. At 9 months, IgG remained in blood and saliva in most patients. Salivary IgA was rarely detected at this time point. In cohort 2, salivary IgG and IgA responses were significantly associated with recent history of COVID-19-like symptoms. Salivary IgG tolerated temperature and detergent pretreatments. CONCLUSIONS: Unlike SARS-CoV-2 salivary IgA that appeared short lived, specific saliva IgG appeared stable even after mild COVID-19, as for blood serology. This noninvasive saliva-based SARS-CoV-2 antibody test with home self-collection may be a complementary alternative to conventional blood serology.
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Anticorpos Antivirais/imunologia , COVID-19/imunologia , Imunoglobulina G/imunologia , SARS-CoV-2/imunologia , Saliva/imunologia , Adulto , Idoso , Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Adulto JovemRESUMO
Water is a scarce resource in the Mediterranean region where adverse climatic conditions promoting water shortages tend to increase with climate change. Under water scarcity conditions and high atmosphere evaporative demand, the risks of decreased water quality, and land salinization are major threats to the sustainability of irrigated agriculture in this region. The assessment of the quality of irrigation water is increasingly important to ensure the maintenance of long-term salt balance at a crop, farm, and regional scale. This study is focused on the spatial and temporal variability of water quality for irrigation in the Alqueva reservoir (Southern Portugal). The assessment was performed every 2 months during a distinctive drought year (2017) and included inorganic ions (Na+, Ca2+, Mg2+, K+, NH4+, Cl-, F-, SO42-, NO3-, and NO2-), pH, and electric conductivity (ECW) of water. Sodium adsorption ratio (SAR) was calculated, and potential soil permeability problems were estimated. The assessment showed significantly higher values of the physicochemical parameters in the most upstream sites, located near tributaries inflows, and an upward trend in ion concentrations throughout the year, with significantly higher concentrations of Na+, Mg2+, Cl-, and SO42, registered through May to November, reflecting the severe drought felt in the summer, autumn, and winter. The evaluation of water quality for irrigation indicated a slight to moderate risk of reduced infiltration rates, which should be considered whenever sprinkler irrigation is used, mainly in fine-textured soils, which are prevalent in the irrigated area. The multivariate statistical approach, using principal component analysis and factor analysis, identified two principal components related to salinity and nutrient concentrations. The cluster analysis revealed three groups of similarity between samples pointing to a more time- than space-controlled pattern. Overall, the temporal dynamics of the water physicochemical parameters could indicate that an abnormal annual distribution of precipitation and temperature may distort seasonal differences. To prevent water and soil degradation, a more frequent assessment of the water quality should be considered, allowing for the selection of appropriate soil and water management measures in irrigated areas.
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Irrigação Agrícola , Secas , Monitoramento Ambiental/métodos , Qualidade da Água , Água/química , Agricultura , Mudança Climática , Região do Mediterrâneo , Portugal , Salinidade , Estações do Ano , Sódio/análise , Solo/química , Abastecimento de ÁguaRESUMO
Adipose-derived stromal/stem cells (ASCs) are promising candidates for cell-based therapies. However, the lack of markers able to unequivocally identify these cells, the differential expression of cell surface molecules among stromal progenitors from different tissues and cellular alterations caused by culture are phenomena that need to be comprehensively addressed in order to improve ASC purification and consequently refine our knowledge about their function and therapeutic efficiency. In this study, we investigated the potential of CD271, a marker used for purification of bone marrow-derived mesenchymal stem cells, on enriching ASCs from CD34+ stromal cells of human adipose tissue. Putative ASC populations were sorted based on CD271 expression (CD45- CD31- CD34+ CD271+ and CD45- CD31- CD34+ CD271- cells) and compared regarding their clonogenic efficiency, proliferation, immunophenotypic profile, and multilineage potential. To shed light on their native identity, we also interrogated the expression of key perivascular cell markers in freshly isolated cells. CD271- cells displayed twofold higher clonogenic efficiency than CD271+ cells. Upon culture, the progeny of both populations displayed similar immunophenotypic profile and in vitro adipogenic and chondrogenic potentials, while CD271+ cells produced more calcified extracellular matrix. Interestingly, uncultured freshly isolated CD271+ cells displayed higher expression of pericyte-associated markers than CD271- cells and localized in the inner region of the perivascular wall. Our results demonstrate that cells with in vitro ASC traits can be obtained from both CD271+ and CD271- stromal populations of human adipose tissue. In addition, gene expression profiling and in situ localization analyses indicate that the CD271+ population displays a pericytic phenotype.
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Tecido Adiposo/metabolismo , Antígenos CD34/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/biossíntese , Receptores de Fator de Crescimento Neural/biossíntese , Tecido Adiposo/citologia , Adulto , Feminino , Humanos , Masculino , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
BACKGROUND: In an attempt to increase the therapeutic potential for myocardial regeneration, there is a quest for new cell sources and types for cell therapy protocols. The pathophysiology of heart diseases may affect cellular characteristics and therapeutic results. METHODS: To study the proliferative and differentiation potential of mesenchymal stem cells (MSC), isolated from bone marrow (BM) of sternum, we made a comparative analysis between samples of patients with ischemic (IHD) or non-ischemic valvular (VHD) heart diseases. We included patients with IHD (n = 42) or VHD (n = 20), with average age of 60 years and no differences in cardiovascular risk factors. BM samples were collected (16.4 ± 6 mL) and submitted to centrifugation with Ficoll-Paque, yielding 4.5 ± 1.5 × 107 cells/mL. RESULTS: Morphology, immunophenotype and differentiation ability had proven that the cultivated sternal BM cells had MSC features. The colony forming unit-fibroblast (CFU-F) frequency was similar between groups (p = 0.510), but VHD samples showed positive correlation to plated cells vs. CFU-F number (r = 0.499, p = 0.049). The MSC culture was established in 29% of collected samples, achieved passage 9, without significant difference in expansion kinetics between groups (p > 0.05). Dyslipidemia and the use of statins was associated with culture establishment for IHD patients (p = 0.049 and p = 0.006, respectively). CONCLUSIONS: Together, these results show that the sternum bone can be used as a source for MSC isolation, and that ischemic or valvular diseases do not influence the cellular yield, culture establishment or in vitro growth kinetics.
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Técnicas de Cultura de Células/métodos , Doenças das Valvas Cardíacas/patologia , Células-Tronco Mesenquimais/citologia , Isquemia Miocárdica/patologia , Esterno/citologia , Idoso , Diferenciação Celular , Proliferação de Células , Separação Celular , Forma Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feminino , Humanos , Imunofenotipagem , Cinética , Masculino , Pessoa de Meia-IdadeRESUMO
Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology.
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Tecido Adiposo/citologia , Diferenciação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Pericitos/citologia , Células-Tronco/citologia , Transcriptoma/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , HumanosRESUMO
High dose immunosuppression followed by autologous hematopoietic stem cell transplantation (AHSCT) induces prolonged clinical remission in multiple sclerosis (MS) patients. However, how patient immune profiles are associated with clinical outcomes has not yet been completely elucidated. In this study, 37 MS patients were assessed for neurological outcomes, thymic function and long-term immune reconstitution after AHSCT. Patients were followed for a mean (SD) of 68.5 (13.9) months post-transplantation and were retrospectively clustered into progression- and non-progression groups, based on Expanded Disease Status Scale (EDSS) outcomes at last visit. After AHSCT, both patient groups presented increased regulatory T-cell subset counts, early expansion of central- and effector-memory CD8(+)T-cells and late thymic reactivation. However, the non-progression group presented early expansion of PD-1(+)CD8(+)T-cells and of PD-1-expressing CD19(+) B-cells. Here, we suggest that along with increased numbers of regulatory T-cell subsets, PD-1 inhibitory signaling is one possible immunoregulatory mechanism by which AHSCT restores immune tolerance in MS patients.
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Transplante de Células-Tronco Hematopoéticas/métodos , Esclerose Múltipla Recidivante-Remitente/terapia , Linfócitos T/imunologia , Timo/imunologia , Adulto , Antígenos CD19/imunologia , Antígenos CD19/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Progressão da Doença , Feminino , Humanos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/imunologia , Avaliação de Resultados em Cuidados de Saúde , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Estudos Retrospectivos , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fatores de Tempo , Transplante Autólogo , Adulto JovemRESUMO
This study aimed to assess the potential impact on soil porewater, surface and groundwater from the beneficial application of organic wastes to soil, using their eluates and acute bioassays with aquatic organisms and plants: luminescence inhibition of Vibrio fischeri (15 and 30 min), Daphnia magna immobilization (48 h), Thamnocephalus platyurus survival (24 h), and seed germination of Lolium perenne (7 d) and Lactuca sativa (5 d). Some organic wastes' eluates promoted high toxic responses, but that toxicity could not be predicted by their chemical characterization, which is compulsory by regulatory documents. In fact, when organisms were exposed to the water-extractable chemical compounds of the organic wastes, the toxic responses were more connected to the degree of stabilization of the organic wastes, or to the treatment used to achieve that stabilization, than to their contaminant load. That is why the environmental risk assessment of the use of organic wastes as soil amendments should integrate bioassays with eluates, in order to correctly evaluate the effects of the most bioavailable fraction of all the chemical compounds, which can be difficult to predict from the characterization required in regulatory documents. According to our results, some rapid and standardized acute bioassays can be suggested to integrate a Tier 1 ecotoxicological evaluation of organic wastes with potential to be land applied, namely luminescence inhibition of V. fischeri, D. magna immobilization, and the germination of L. perenne and L. sativa.
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Ecotoxicologia/métodos , Compostos Orgânicos/toxicidade , Resíduos/efeitos adversos , Poluentes Químicos da Água/toxicidade , Poluição Química da Água/análise , Aliivibrio fischeri/efeitos dos fármacos , Animais , Bioensaio/métodos , Daphnia/efeitos dos fármacos , Germinação/efeitos dos fármacos , Lactuca/efeitos dos fármacos , Lolium/efeitos dos fármacos , Portugal , Solo , Poluentes do Solo/toxicidade , Águas Residuárias/toxicidade , Poluição Química da Água/efeitos adversosRESUMO
BACKGROUND: The therapeutic use of human embryonic stem cells (hESCs) is dependent on an efficient cryopreservation protocol for long-term storage. The aim of this study was to determine whether the combination of three cryoprotecting reagents using two freezing systems might improve hESC recovery rates with maintenance of hESC pluripotency properties for potential cell therapy application. METHODS: Recovery rates of hESC colonies which were frozen in three cryoprotective solutions: Me2SO/HES/SR medium, Defined-medium® and Me2SO/SFB in medium solution were evaluated in ultra-slow programmable freezing system (USPF) and a slow-rate freezing system (SRF). The hESC pluripotency properties after freezing-thawing were evaluated. RESULTS: We estimated the distribution frequency of survival colonies and observed that independent of the freezing system used (USPF or SRF) the best results were obtained with Me2SO/HES/SR as cryopreservation medium. We showed a significant hESC recovery colonies rate after thawing in Me2SO/HES/SR medium were 3.88 and 2.9 in USPF and SRF, respectively. The recovery colonies rate with Defined-medium® were 1.05 and 1.07 however in classical Me2SO medium were 0.5 and 0.86 in USPF and SRF, respectively. We showed significant difference between Me2SO/HES/SR medium×Defined-medium® and between Me2SO/HES/SR medium×Me2SO medium, for two cryopreservation systems (P<0.05). CONCLUSION: We developed an in house protocol using the combination of Me2SO/HES/SR medium and ultra-slow programmable freezing system which resulted in hESC colonies that remain undifferentiated, maintain their in vitro and in vivo pluripotency properties and genetic stability. This approach may be suitable for cell therapy studies.
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Criopreservação/métodos , Dimetil Sulfóxido/farmacologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/fisiologia , Derivados de Hidroxietil Amido/farmacologia , Substitutos do Plasma/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Crioprotetores/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Congelamento , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes/citologiaRESUMO
Recent studies have demonstrated the role of adenosine (ADO) in sickle-cell anemia (SCA). ADO is produced by CD39 and CD73 and converted to inosine by adenosine deaminase (ADA). We evaluated the effects of hydroxycarbamide (HU) treatment on the modulation of adenosine levels in SCA patients. The expressions of CD39, CD73, and CD26 were evaluated by flow cytometry on blood cells in 15 HU-treated and 17 untreated patients and 10 healthy individuals. RNA was extracted from monocytes, and ADA gene expression was quantified by real-time PCR. ADA activity was also evaluated. We found that ADA transcripts were two times higher in monocytes of HU-treated patients, compared with untreated (P = 0.039). Monocytes of HU-treated patients expressed CD26, while monocytes of controls and untreated patients did not (P = 0.023). In treated patients, a lower percentage of T lymphocytes expressed CD39 compared with untreated (P = 0.003), and the percentage of T regulatory (Treg) cells was reduced in the treated group compared with untreated (P = 0.017) and controls (P = 0.0009). Besides, HU-treated patients displayed increased ADA activity, compared with untreated. Our results indicate a novel mechanism of action of HU mediated by the reduction of adenosine levels and its effects on pathophysiological processes in SCA.
Assuntos
Adenosina/metabolismo , Anemia Falciforme/metabolismo , Antidrepanocíticos/farmacologia , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/metabolismo , Hidroxiureia/farmacologia , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Adolescente , Adulto , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Antidrepanocíticos/uso terapêutico , Apirase/genética , Apirase/metabolismo , Células Sanguíneas/patologia , Estudos de Casos e Controles , Criança , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxiureia/uso terapêutico , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , Adulto JovemRESUMO
The presence of pesticides in aquatic ecosystems poses significant risks to non-target organisms, necessitating monitoring and environmental risk assessment. This study aimed to evaluate the dynamics and environmental risk of pesticides in a hydro-agricultural area with intensive agricultural practices, in the Mediterranean region (South of Portugal). Seasonality and location influenced pesticide numbers and concentrations, with the highest levels observed during the dry season. Triazines, phenylureas, and organophosphates were the predominant pesticide classes, with terbuthylazine, bentazone, terbutryn, diazinon, and metolachlor exhibiting the highest detection frequencies (68 % to 72 %). Notably, 44 % of the quantified pesticides are no longer authorized in Portugal, with 33 % posing a high environmental risk. Some insecticides, including imidacloprid, methiocarb, and malathion, were occasionally detected at concentrations that posed high risks to the aquatic ecosystem (RQ ≥ 1). Irgarol, an algicide used in irrigation canals, presented a high risk in 91 % of the analysed samples. The study's distribution profile of pesticides revealed a significant transportation of these compounds from reservoirs to irrigation hydrants, establishing them as a secondary source of crop and environmental contamination. Additionally, the assessment of spatial distribution and environmental risk allowed for the identification of specific pollutants in different locations, prioritizing them based on their ecotoxicological risk to aquatic ecosystems. These findings reinforce the importance of implementing management measures at the level of hydro-agricultural areas, helping to stop the cycle of pesticide contamination. Only this type of strategy will make it possible to protect water quality, biodiversity and the health of citizens, contributing to the European Union's objectives of improving the condition of freshwater bodies and promoting the sustainable use of pesticides.
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Praguicidas , Poluentes Químicos da Água , Praguicidas/análise , Ecossistema , Monitoramento Ambiental , Poluentes Químicos da Água/análise , Agricultura , Abastecimento de ÁguaRESUMO
BACKGROUND AND OBJECTIVES: Bone marrow mesenchymal stromal cells (BM-MSCs) are key elements of the hematopoietic niche and participate in the regulatory mechanisms of hematopoietic stem cells (HSCs). Hematological diseases can affect MSCs and their functions. However, the dysregulations caused by sickle cell disease (SCD) are not fully elucidated. This work explored changes in BM-MSCs and their relationship with age using sickle cell mice (Townes-SS). MATERIALS AND METHODS: BM-MSCs were isolated from Townes-SS, and control groups 30- and 60-day-old Townes-AA and C57BL/6 J. RESULTS: The BM-MSCs showed no morphological differences in culture and demonstrated a murine MSC-like immunophenotypic profile (Sca-1+, CD29+, CD44+, CD90.2+, CD31-, CD45-, and CD117-). Subsequently, all BM-MSCs were able to differentiate into adipocytes and osteocytes in vitro. Finally, 30-day-old BM-MSCs of Townes-SS showed higher expression of genes related to the maintenance of HSCs (Cxcl12, Vegfa, and Angpt1) and lower expression of pro-inflammatory genes (Tnfa and Il-6). However, 60-day-old BM-MSCs of Townes-SS started to show expression of genes related to reduced HSC maintenance and increased expression of pro-inflammatory genes. CONCLUSION: These results indicates age as a modifying factor of gene expression of BM-MSCs in the context of SCD.
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Anemia Falciforme , Células-Tronco Mesenquimais , Humanos , Animais , Camundongos , Medula Óssea , Camundongos Endogâmicos C57BL , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação CelularRESUMO
The outbreak and pandemic of COVID-19 forced people into extreme isolation and social distancing, with significant limitations on various activity sectors, including sports. This study aimed to assess the psychological health status of athletes during sports lockdown. Additionally, we intend to verify the mediating role of sleep disorders in stress perception and subjective happiness. Our sample was composed of 1492 Portuguese athletes from eight different team sports. During sports lockdown, athletes were found to have high stress levels and low subjective happiness levels and experience sleep disorders. Finally, these results conclude that sports lockdowns harm athletes' psychological health and well-being. Pay cuts to athletes are an extra stress factor that exacerbate these adverse effects on psychological health. Finally, sleep is a mediator variable between stress perception and subjective happiness levels. This study's significant contributions, limitations, and future directions are discussed in the conclusion.
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Analytical methods based on QuEChERS and liquid chromatography-tandem mass spectrometry were developed for the determination of 42 polar and semi-polar pesticides in three representative vegetable matrices, viz. corn, grapes and alfalfa. A 26-2 fractional factorial experimental design was used to cost-effectively optimize the extraction procedure. The optimal analytical approaches were validated in terms of linearity, sensitivity, trueness, and precision. Most of the target pesticides showed a relative recovery of 70-120 %. Moreover, limits of detection (between 0.01 ng/g and 20 ng/g) were below the maximum residue levels set for the target analytes in nearly all cases. Analysis of real samples showed the presence of five pesticides in grapes and alfalfa at concentrations between the method LOQs and 40 ng/g. To the authors' knowledge, these methods are the first ever described for the determination of 6, 13 and 24 of the 42 target pesticides measured in corn, grapes and alfalfa, respectively.
Assuntos
Praguicidas , Vitis , Medicago sativa , Zea mays , Cromatografia LíquidaRESUMO
Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro. The marker CD69 is a target of canonical nuclear factor kappa-B (NF-κB) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3(+) T cells were activated and cultured in the presence or absence of MSCs. CD4(+) cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 (BAY) and a siRNA against v-rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canonical and non-canonical NF-κB signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69(+) cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69(+) cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signalling.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Lectinas Tipo C/metabolismo , Ativação Linfocitária/imunologia , Células-Tronco Mesenquimais/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Lectinas Tipo C/genética , Análise em Microsséries , NF-kappa B/genética , Nitrilas , Transplante de Células-Tronco de Sangue Periférico/métodos , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Sulfonas , Linfócitos T Reguladores/metabolismo , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismoRESUMO
BACKGROUND AIMS: Mesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues. METHODS: MSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays. RESULTS: Post-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure. CONCLUSIONS: A method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.
Assuntos
Criopreservação/métodos , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Adipócitos/citologia , Animais , Diferenciação Celular , Proliferação de Células , Forma Celular , Sobrevivência Celular , Análise Citogenética , Humanos , Imunofenotipagem , Recém-Nascido , Osteócitos/citologia , Xenobióticos/análiseRESUMO
The coronavirus pandemic has exposed a global tendency throughout history to blame immigrants for propagating epidemics. Chinese individuals were thus targeted during past public health crises in Peru, but during the current coronavirus pandemic racist notions painting people of Chinese descent as "agents of contagion" diminished significantly. Here we examine three major epidemics (yellow fever, the bubonic plague, and covid-19) to demonstrate the current and somewhat surprising shift in negative attitudes toward the Chinese community. Peruvians' refusal to embrace derogatory terms (the "Chinese virus") or target individuals of Asian descent constitutes an intriguing case at a moment when xenophobic discourse is rampant in the Western hemisphere.