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1.
Front Immunol ; 12: 677025, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34504487

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global crisis; however, our current understanding of the host immune response to SARS-CoV-2 infection remains limited. Herein, we performed RNA sequencing using peripheral blood from acute and convalescent patients and interrogated the dynamic changes of adaptive immune response to SARS-CoV-2 infection over time. Our results revealed numerous alterations in these cohorts in terms of gene expression profiles and the features of immune repertoire. Moreover, a machine learning method was developed and resulted in the identification of five independent biomarkers and a collection of biomarkers that could accurately differentiate and predict the development of COVID-19. Interestingly, the increased expression of one of these biomarkers, UCHL1, a molecule related to nervous system damage, was associated with the clustering of severe symptoms. Importantly, analyses on immune repertoire metrics revealed the distinct kinetics of T-cell and B-cell responses to SARS-CoV-2 infection, with B-cell response plateaued in the acute phase and declined thereafter, whereas T-cell response can be maintained for up to 6 months post-infection onset and T-cell clonality was positively correlated with the serum level of anti-SARS-CoV-2 IgG. Together, the significantly altered genes or biomarkers, as well as the abnormally high levels of B-cell response in acute infection, may contribute to the pathogenesis of COVID-19 through mediating inflammation and immune responses, whereas prolonged T-cell response in the convalescents might help these patients in preventing reinfection. Thus, our findings could provide insight into the underlying molecular mechanism of host immune response to COVID-19 and facilitate the development of novel therapeutic strategies and effective vaccines.


Assuntos
COVID-19/genética , COVID-19/imunologia , Leucócitos Mononucleares/química , Transcriptoma , Adulto , Idoso , Anticorpos Antivirais/sangue , Linfócitos B/imunologia , Biomarcadores/sangue , COVID-19/sangue , COVID-19/virologia , China , Estudos de Coortes , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/fisiologia , Análise de Sequência de RNA , Linfócitos T/imunologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/imunologia
2.
J Infect Dis ; 224(5): 777-782, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34467988

RESUMO

We analyzed plasma levels of interferons (IFNs) and cytokines, and expression of IFN-stimulated genes in peripheral blood mononuclear cells in patients with coronavirus disease 2019 of varying disease severity. Patients hospitalized with mild disease exhibited transient type I IFN responses, while intensive care unit patients had prolonged type I IFN responses. Type II IFN responses were compromised in intensive care unit patients. Type III IFN responses were induced in the early phase of infection, even in convalescent patients. These results highlight the importance of early type I and III IFN responses in controlling coronavirus disease 2019 progression.


Assuntos
COVID-19/imunologia , Interferon Tipo I/imunologia , Interferon gama/imunologia , Interferons/imunologia , COVID-19/sangue , Quimiocinas/sangue , Citocinas/sangue , Humanos , Interferon Tipo I/sangue , Interferon Tipo I/genética , Interferon gama/sangue , Interferon gama/genética , Interferons/sangue , Leucócitos Mononucleares/imunologia , SARS-CoV-2/isolamento & purificação
3.
Egypt J Immunol ; 28(3): 145-156, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34453786

RESUMO

Human tumors including colorectal cancers (CRC) are often infiltrated by immune cells predominantly T lymphocytes especially regulatory T (Treg) cells expressing the forkhead box protein 3 (Foxp3). It has been suggested that CD25+CD4+Foxp3+ regulatory T cells (Tregs) might hamper effective immunosurveillance of emerging cancer cell. The aim of this study was to measure the frequency of total CD4+CD25+ Tregs & CD4+CD25+Foxp3+ subset of Treg cells in peripheral blood of Egyptian CRC patients and their correlation with the tumor stage, histopathology of the tumor and lymph node affection. A total of 31 CRC patients were enrolled in the study. The tumor was categorized using a TNM staging system. Peripheral blood samples were collected within the first 24 h of surgery. The frequency of total CD4+CD25+ Tregs & CD4+CD25+ Foxp3+ subset of Treg cells in peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry and absolute count was determined. High frequency of Tregs was detected in cancer patients with distal margin involvement (44-48 cells/µL) compared with those with free distal margin (5-32 cells/µL). Similarly, higher frequency of Tregs were detected (16-44 cells/µL) in cancers with lymph node involvement compared with cancers without lymph node involvement (5-32 cells/µL). Higher frequency of CD4+CD25+Foxp3+ Tregs were found in mucinous adenocarcinomas than in other histopathological types, although both observations were statistically insignificant. The median value for total absolute lymphocyte count/ µL was 639, out of which CD4+CD25+ subset constituted 35 cells, and about half of this subset were Foxp3+Tregs. In conclusion, CD4+CD25+Foxp3+ Tregs may be a useful marker for predicting invasion, metastasis, and prognosis of colorectal cancer in Egyptian patients.


Assuntos
Neoplasias Colorretais , Leucócitos Mononucleares , Egito , Citometria de Fluxo , Fatores de Transcrição Forkhead , Humanos , Subunidade alfa de Receptor de Interleucina-2 , Subpopulações de Linfócitos T , Linfócitos T Reguladores
4.
J Transl Med ; 19(1): 362, 2021 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-34419106

RESUMO

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease with a complicated pathogenesis, and its aetiology has not been clearly unveiled. The lack of effective diagnosis and treatment methods makes it necessary to explore the molecular mechanism of SLE. We aimed to identify some critical signalling pathways and key competing endogenous RNAs (ceRNAs) underlying the molecular mechanism of SLE and to map out the systematic signalling networks by integrating the data on different kinds of RNAs. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from both SLE patients and healthy subjects, RNA was extracted from the PBMCs, and RNA libraries including ribosomal RNA-depleted strand-specific libraries and small RNA libraries were built for deep RNA sequencing (RNA-seq). RNA-seq yielded differential expression profiles of lncRNAs/circRNAs/miRNAs/mRNAs related to SLE. The DAVID database (v. 6.8) was employed for Gene Ontology (GO) and KEGG pathway analysis. ceRNA networks (circRNA/lncRNA-miRNA-mRNA) were constructed and visualized using Cytoscape software (v. 3.5.0). The TargetScan and miRanda databases were used to predict target relationships in ceRNA networks. qRT-PCR was used to verify our data. RESULTS: Differential expression of ceRNAs related to SLE was detected in SLE patients' PBMCs: 644 mRNAs (384 upregulated, 260 downregulated), 326 miRNAs (223 upregulated, 103 downregulated), 221 lncRNAs (79 upregulated, 142 downregulated), and 31 circRNAs (21 upregulated, 10 downregulated). We drew ceRNA signalling networks made up of the differentially expressed mRNAs/miRNAs/lncRNAs/circRNAs mentioned above, and the hub genes included IRF5, IFNAR2, TLR7, IRAK4, STAT1, STAT2, C2, and Tyk2. These hub genes were involved in ceRNA signalling pathways, such as the IL-17 signalling pathway and type I interferon signalling pathway. CONCLUSIONS: We explored the differential expression profiles of various kinds of ceRNAs and integrated signalling networks constructed by ceRNAs. Our findings offer new insights into the pathogenesis of SLE and hint at therapeutic strategies.


Assuntos
Lúpus Eritematoso Sistêmico , MicroRNAs , RNA Longo não Codificante , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Fatores Reguladores de Interferon , Leucócitos Mononucleares , Lúpus Eritematoso Sistêmico/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética
5.
Elife ; 102021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34424199

RESUMO

The exact immunopathophysiology of community-acquired pneumonia (CAP) caused by SARS-CoV-2 (COVID-19) remains clouded by a general lack of relevant disease controls. The scarcity of single-cell investigations in the broader population of patients with CAP renders it difficult to distinguish immune features unique to COVID-19 from the common characteristics of a dysregulated host response to pneumonia. We performed integrated single-cell transcriptomic and proteomic analyses in peripheral blood mononuclear cells from a matched cohort of eight patients with COVID-19, eight patients with CAP caused by Influenza A or other pathogens, and four non-infectious control subjects. Using this balanced, multi-omics approach, we describe shared and diverging transcriptional and phenotypic patterns-including increased levels of type I interferon-stimulated natural killer cells in COVID-19, cytotoxic CD8 T EMRA cells in both COVID-19 and influenza, and distinctive monocyte compositions between all groups-and thereby expand our understanding of the peripheral immune response in different etiologies of pneumonia.


Assuntos
COVID-19/imunologia , Infecções Comunitárias Adquiridas/imunologia , Influenza Humana/imunologia , Análise de Célula Única , Adulto , Feminino , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade
6.
Food Res Int ; 147: 110526, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34399504

RESUMO

Sorbus aucuparia L. is a source of edible fruits appreciated for their nutritional and medicinal properties. In this work some bioactivity mechanisms were evaluated, which might be connected with the traditional application of rowanberries in cardiovascular complications of diabetes. With the use of a panel of chemical and biological in vitro models the rowanberry extracts were proved to significantly inhibit the formation of advanced glycation end products, neutralise multiple oxidants generated in vivo, increase the non-enzymatic antioxidant capacity of human plasma and protect plasma components (proteins and lipids) against oxidative/nitrative damage at in vivo-relevant levels (1-5 µg/mL). Moreover, the extracts were found safe in cytotoxicity tests on the peripheral blood mononuclear cells. The comprehensive phytochemical profiling of the extracts (RP/HILIC-UHPLC-PDA-ESI-MS3, HPLC-PDA, and UV-spectrophotometric methods) led to the identification of 51 phenolics, including caffeic and ferulic acids pseudodepsides (34 compounds, prevailing isomers of chlorogenic acid and cynarin, total content up to 269.4 mg/g), flavonols (mostly quercetin glycosides, up to 5.8 mg/g), flavan-3-ol derivatives (proanthocyanidin oligomers and polymers, up to 17.0 mg/g), and simple phenolic acids. The experiments on model constituents of the extracts and correlation studies were used to evaluate contribution of polyphenols to the observed effects. Taking into account the possible additive and synergistic effects, the co-occurrence of various compounds was indicated as partly responsible for biological activity of the fruits. Considering both the composition and activity parameters, the methanol-water (1:1, v/v) extract and its concentrated phenolic fractions appeared to be the most advantageous for biological application.


Assuntos
Sorbus , Frutas , Humanos , Leucócitos Mononucleares , Estresse Oxidativo , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia
7.
Molecules ; 26(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34443554

RESUMO

Rheumatoid arthritis (RA) is an autoimmune inflammatory joint disease with complex pathogenesis associated with cytokine dysregulation. Macrophage migration inhibitory factor (MIF) plays a role in systemic inflammation and joint destruction in RA and could be associated with the secretion of other immune-modulatory cytokines such as IL-25, IL-31, and IL-33. For the above, our main aim was to evaluate the IL-25, IL-31, and IL-33 secretion from recombinant human MIF (rhMIF)-stimulated peripheral blood mononuclear cells (PBMC) of RA patients. The rhMIF and lipopolysaccharide (LPS) plus rhMIF stimuli promote the secretion of IL-25, IL-31, and IL-33 (p < 0.05) from PBMC of RA patients. The study groups, the different stimuli, and the interaction between both showed a statistically significant effect on the secretion of IL-25 (p < 0.05) and IL-31 (p < 0.01). The study of the effect of the RA patient treatments and their interaction with the effect of stimuli did not show an interaction between them. In conclusion, our study generates new evidence for the role of MIF in the secretion of IL-25, IL-31, and IL-33 and its immunomodulatory effect on RA.


Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Interleucina-17/metabolismo , Interleucina-33/metabolismo , Interleucinas/metabolismo , Oxirredutases Intramoleculares/metabolismo , Leucócitos Mononucleares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Adulto , Feminino , Humanos , Imunomodulação/efeitos dos fármacos , Oxirredutases Intramoleculares/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Fatores Inibidores da Migração de Macrófagos/farmacologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/farmacologia
8.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 33(7): 779-785, 2021 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-34412744

RESUMO

OBJECTIVE: To verify the specific differentiated subsets of monocytes in sepsis, and to screen and construct the differential gene set of monocytes used for early diagnosis of sepsis. METHODS: Patients with sepsis admitted to Guangdong Provincial People's Hospital from June 2020 to March 2021 were enrolled, and peripheral blood mononuclear cells (PBMC) were extracted. Single-cell sequencing technology and pseudo-time analysis were used to verify the differential subsets of monocytes. Bioinformatics methods were used to analyze the expression of genes in differential subsets of monocytes and screen out differential genes for the preliminary construction of a candidate differential gene set. The digital polymerase chain reaction (PCR) technology was used to verify the candidate differential genes in PBMC of sepsis patients and sepsis human myeloid leukemia mononuclear cells (THP-1) models, and the Venn diagram was used to construct the final differential gene set of monocytes. Gene Expression Omnibus (GEO) database was used to validate the differential gene set of monocytes. RESULTS: (1) The results of cell annotation and pseudo-time analysis showed that the differentiation of NEAT1+CD163+ monocyte occurred in the early stage of sepsis was significantly different from other subsets, which validated that NEAT1+CD163+ monocyte was the characteristic subset in the pathological process of sepsis. (2) Twenty-two differential genes related to sepsis were screened out from the gene expression of NEAT1+CD163+ monocyte. After further verification by digital PCR, basic leucine zipper ATF-like transcription factor (BATF), JUNB proto-oncogene, carcinoembryonic antigen-related cell adhesion molecule 4 (CEACAM4), chromosome 9 open reading frame 95 (C9orf95), G protein subunit alpha 15 (GNA15), complement C3a receptor 1 (C3AR1), transforming growth factor beta 1 (TGFB1) and mitochondrial carrier homolog 1 (MTCH1) were screened out to construct the final differential gene set of monocytes. (3) The external validation results showed that C9orf95 gene had no data in GSE154918 and GSE133822 from GEO, it was excluded during validation. In GSE154918, the expressions of BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1 in the sepsis group were significantly higher than those in the healthy control group (log2expression level: BATF was 12.78±0.08 vs. 11.39±0.35, JUNB was 16.88±0.07 vs. 16.04±0.03, CEACAM4 was 14.73±0.08 vs. 13.77±0.05, GNA15 was 13.16±0.06 vs. 12.30±0.04, C3AR1 was 14.62±0.13 vs. 12.87±0.05, TGFB1 was 16.95±0.05 vs. 16.57±0.36, MTCH1 was 14.80±0.02 vs. 14.61±0.15, all P < 0.05). In GSE133822, the expressions of BATF, CEACAM4, GNA15, and C3AR1 in the sepsis group were significantly higher than those in the health control group (log2expression level: BATF was 8.66±0.16 vs. 7.92±0.14, CEACAM4 was 9.20±0.16 vs. 8.36±0.20, GNA15 was 10.66±0.18 vs. 10.13±0.16, C3AR1 was 11.49±0.27 vs. 10.48±0.16, all P < 0.05), while the expressions of JUNB, TGFB1, and MTCH1 were not statistically different between two groups. The results of gene set variation analysis (GSVA) showed that the enrichment scores of monocytes differential gene set of sepsis group were significantly higher than those of the healthy control group in both GSE154918 (0.38±0.04 vs. -0.44±0.02) and GSE133822 (0.56±0.02 vs. 0.20±0.05, both P < 0.01). Receiver operator characteristic curve (ROC curve) analysis showed that the differential gene set of monocytes had a reliable diagnostic value for early sepsis with the area under ROC curve (AUC) of 0.993 [95% confidence interval (95%CI) was 0.980-1.000] in GSE154918 and 0.944 (95%CI was 0.873-1.000) in GSE133822. CONCLUSIONS: A differential gene set of monocytes (BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1) screened out by single-cell sequencing and digital PCR technology has a reliable diagnostic value for the early sepsis, and may provide a new idea for the early diagnosis of sepsis.


Assuntos
Monócitos , Sepse , Diagnóstico Precoce , Humanos , Leucócitos Mononucleares , Reação em Cadeia da Polimerase , Sepse/diagnóstico , Sepse/genética , Tecnologia
9.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360706

RESUMO

For the treatment of severe COVID-19, supplementation with human plasma-purified α-1 antitrypsin (AAT) to patients is currently considered. AAT inhibits host proteases that facilitate viral entry and possesses broad anti-inflammatory and immunomodulatory activities. Researchers have demonstrated that an interaction between SARS-CoV-2 spike protein (S) and lipopolysaccharides (LPS) enhances pro-inflammatory responses in vitro and in vivo. Hence, we wanted to understand the potential anti-inflammatory activities of plasma-derived and recombinant AAT (recAAT) in a model of human total peripheral blood mononuclear cells (PBMCs) exposed to a combination of CHO expressed trimeric spike protein and LPS, ex vivo. We confirmed that cytokine production was enhanced in PBMCs within six hours when low levels of LPS were combined with purified spike proteins ("spike"). In the presence of 0.5 mg/mL recAAT, however, LPS/spike-induced TNF-α and IL-1ß mRNA expression and protein release were significantly inhibited (by about 46-50%) relative to LPS/spike alone. Although without statistical significance, recAAT also reduced production of IL-6 and IL-8. Notably, under the same experimental conditions, the plasma-derived AAT preparation Respreeza (used in native and oxidized forms) did not show significant effects. Our findings imply that an early pro-inflammatory activation of human PBMCs is better controlled by the recombinant version of AAT than the human plasma-derived AAT used here. Considering the increasing clinical interest in AAT therapy as useful to ameliorate the hyper-inflammation seen during COVID-19 infection, different AAT preparations require careful evaluation.


Assuntos
Anti-Inflamatórios/farmacologia , Leucócitos Mononucleares/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , alfa 1-Antitripsina/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/imunologia , Células CHO , COVID-19/terapia , Células Cultivadas , Cricetulus , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/toxicidade , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , alfa 1-Antitripsina/química , alfa 1-Antitripsina/imunologia
10.
Front Immunol ; 12: 716075, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34394120

RESUMO

The existence of asymptomatic and re-detectable positive coronavirus disease 2019 (COVID-19) patients presents the disease control challenges of COVID-19. Most studies on immune responses in COVID-19 have focused on moderately or severely symptomatic patients; however, little is known about the immune response in asymptomatic and re-detectable positive (RP) patients. Here we performed a comprehensive analysis of the transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) from 48 COVID-19 patients which included 8 asymptomatic, 13 symptomatic, 15 recovered and 12 RP patients. The weighted gene co-expression network analysis (WGCNA) identified six co-expression modules, of which the turquoise module was positively correlated with the asymptomatic, symptomatic, and recovered COVID-19 patients. The red module positively correlated with symptomatic patients only and the blue and brown modules positively correlated with the RP patients. The analysis by single sample gene set enrichment analysis (ssGSEA) revealed a lower level of IFN response and complement activation in the asymptomatic patients compared with the symptomatic, indicating a weaker immune response of the PBMCs in the asymptomatic patients. In addition, gene set enrichment analysis (GSEA) analysis showed the enrichment of TNFα/NF-κB and influenza infection in the RP patients compared with the recovered patients, indicating a hyper-inflammatory immune response in the PBMC of RP patients. Thus our findings could extend our understanding of host immune response during the progression of COVID-19 disease and assist clinical management and the immunotherapy development for COVID-19.


Assuntos
Doenças Assintomáticas , COVID-19/imunologia , Portador Sadio/imunologia , Leucócitos Mononucleares/imunologia , SARS-CoV-2/imunologia , Transcriptoma/genética , Adulto , Portador Sadio/virologia , Ativação do Complemento/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/imunologia , Influenza Humana/complicações , Interferons/sangue , Interferons/imunologia , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Transcriptoma/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto Jovem
11.
J Transl Med ; 19(1): 331, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344401

RESUMO

BACKGROUND: Biomarkers for distinguishing psoriatic arthritis (PsA) from psoriasis without arthritis (PsO) are still lacking. METHODS: We applied isobaric tags for relative and absolute quantification (iTRAQ) and LC-MS/MS to analyze the proteome profile of peripheral blood mononuclear cells (PBMCs) collected from patients with PsO, patients with PsA, and healthy controls. Bioinformatics analysis and western blotting were performed to identify and validate differentially expressed proteins. RESULTS: We identified 389, 199, 291, and 60 significantly differentially expressed proteins (adj.p < 0.05) in the comparison of all psoriatic patients versus healthy controls, PsO group versus healthy controls, PsA group versus healthy controls, and PsA group versus PsO group, respectively. Among these proteins, 14 proteins may represent promising biomarkers for PsA: SIRT2, NAA50, ARF6, ADPRHL2, SF3B6, SH3KBP1, UBA3, SCP2, RPS5, NUDT5, NCBP1, SYNE1, NDUFB7, HTATSF1. Furthermore, western blotting confirmed that SIRT2 expression was significantly higher in PBMCs from PsA patients than PsO and healthy controls, and was negatively correlated with the phosphorylation of p38 mitogen-activated protein kinase (p-p38MAPK; p = 0.006, r = - 0.582). CONCLUSIONS: This pilot study provided a broad characterization of the proteome of PBMCs in PsA as compared to PsO and healthy controls, which may help to provide prospective strategies for PsA diagnosis.


Assuntos
Artrite Psoriásica , Psoríase , Cromatografia Líquida , Glicosídeo Hidrolases , Humanos , Leucócitos Mononucleares , Projetos Piloto , Estudos Prospectivos , Proteômica , Espectrometria de Massas em Tandem
12.
Curr Protoc ; 1(8): e215, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34370403

RESUMO

Current methods for the determination of cell-mediated cytotoxic activity in blood samples usually isolate peripheral blood mononuclear cells by density gradient centrifugation or alternatively use erythrocyte lysis. Both centrifugation and red cell lysis can cause cellular depletion and cell dysfunction, resulting in erroneous measurements. To address limitations of current assays, we developed an improved strategy to determine cellular cytotoxicity using flow cytometry. Viable nucleic acid stains are used to identify live nucleated cells and discriminate them from non-nucleated erythrocytes, platelets, and debris while avoiding lysing and washing steps to maintain cell functionality. To detect target cells, we have used two different labeling approaches. In the first approach, EGFP-labeled K562 human chronic myelogenous leukemia cells provide a "ready-to-use" target without the need of additional for labeling or staining. For the second approach, we perform parallel cytotoxicity assays in the presence of wild-type K562 cells previously loaded with a fluorescent dye that has spectral properties similar to those of EGFP. Given the importance of cytotoxic assays and the deleterious effects of current sample preparation methods, the aim of this study was to adapt this "untouched cells" flow cytometry method to study cytotoxic activity using unlysed whole blood samples and fluorescent target cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for cell-mediated cytotoxic activity determination in unlysed whole blood Basic Protocol 2: Protocol preparation, sample acquisition, and gating strategy for flow cytometric identification of cell-mediated cytotoxic activity using unlysed whole blood samples Support Protocol 1: Optimization of the performance of target cell labeling approaches Support Protocol 2: Assessment of the linearity and reproducibility of cytotoxicity assays.


Assuntos
Células Matadoras Naturais , Leucócitos Mononucleares , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Reprodutibilidade dos Testes
13.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445558

RESUMO

Cell-based therapy is a highly promising treatment paradigm in ischemic disease due to its ability to repair tissue when implanted into a damaged site. These therapeutic effects involve a strong paracrine component resulting from the high levels of bioactive molecules secreted in response to the local microenvironment. Therefore, the secreted therapeutic can be modulated by preconditioning the cells during in vitro culturing. Herein, we investigated the potential use of magnetic resonance imaging (MRI) probes, the "iron-quercetin complex" or IronQ, for preconditioning peripheral blood mononuclear cells (PBMCs) to expand proangiogenic cells and enhance their secreted therapeutic factors. PBMCs obtained from healthy donor blood were cultured in the presence of the iron-quercetin complex. Differentiated preconditioning PBMCs were characterized by immunostaining. An enzyme-linked immunosorbent assay was carried out to describe the secreted cytokines. In vitro migration and tubular formation using human umbilical vein endothelial cells (HUVECs) were completed to investigate the proangiogenic efficacy. IronQ significantly increased mononuclear progenitor cell proliferation and differentiation into spindle-shape-like cells, expressing both hematopoietic and stromal cell markers. The expansion increased the number of colony-forming units (CFU-Hill). The conditioned medium obtained from IronQ-treated PBMCs contained high levels of interleukin 8 (IL-8), IL-10, urokinase-type-plasminogen-activator (uPA), matrix metalloproteinases-9 (MMP-9), and tumor necrosis factor-alpha (TNF-α), as well as augmented migration and capillary network formation of HUVECs and fibroblast cells, in vitro. Our study demonstrated that the IronQ-preconditioning PBMC protocol could enhance the angiogenic and reparative potential of non-mobilized PBMCs. This protocol might be used as an adjunctive strategy to improve the efficacy of cell therapy when using PBMCs for ischemic diseases and chronic wounds. However, in vivo assessment is required for further validation.


Assuntos
Movimento Celular , Fibroblastos/fisiologia , Ferro/farmacologia , Leucócitos Mononucleares/fisiologia , Neovascularização Fisiológica , Quercetina/farmacologia , Cicatrização , Adulto , Antioxidantes/farmacologia , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/citologia , Humanos , Leucócitos Mononucleares/citologia , Oligoelementos/farmacologia , Adulto Jovem
14.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361776

RESUMO

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus, and 432 proteins by Lentinula edodes. Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.


Assuntos
Antineoplásicos/química , Antioxidantes/química , Antivirais/química , Proteínas Fúngicas/química , Pleurotus/química , Proteoma/química , Cogumelos Shiitake/química , Aminoácidos/química , Aminoácidos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Antivirais/isolamento & purificação , Antivirais/farmacologia , Benzotiazóis/antagonistas & inibidores , Compostos de Bifenilo/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Misturas Complexas/química , Flavonoides/química , Flavonoides/isolamento & purificação , Proteínas Fúngicas/classificação , Proteínas Fúngicas/isolamento & purificação , Humanos , Lectinas/química , Lectinas/isolamento & purificação , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Especificidade de Órgãos , Fenóis/química , Fenóis/isolamento & purificação , Picratos/antagonistas & inibidores , Pleurotus/metabolismo , Cultura Primária de Células , Proteoma/classificação , Proteoma/isolamento & purificação , Serina Proteases/química , Serina Proteases/isolamento & purificação , Cogumelos Shiitake/metabolismo , Ácidos Sulfônicos/antagonistas & inibidores , Superóxido Dismutase/química , Superóxido Dismutase/isolamento & purificação , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/isolamento & purificação , Vitaminas/química , Vitaminas/isolamento & purificação , Água/química
15.
Viruses ; 13(8)2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34452355

RESUMO

BACKGROUND: New coronavirus SARS-CoV-2, a causative agent of the COVID-19 pandemic, has been circulating among humans since November 2019. Multiple studies have assessed the qualitative and quantitative characteristics of virus-specific immunity in COVID-19 convalescents, however, some aspects of the development of memory T-cell responses after natural SARS-CoV-2 infection remain uncovered. METHODS: In most of published studies T-cell immunity to the new coronavirus is assessed using peptides corresponding to SARS-CoV-1 or SARS-CoV-2 T-cell epitopes, or with peptide pools covering various parts of the viral proteins. Here, we determined the level of CD4+ and CD8+ memory T-cell responses in COVID-19 convalescents by stimulating PBMCs collected 1 to 6 months after recovery with sucrose gradient-purified live SARS-CoV-2. IFNγ production by the central and effector memory helper and cytotoxic T cells was assessed by intracellular cytokine staining assay and flow cytometry. RESULTS: Stimulation of PBMCs with live SARS-CoV-2 revealed IFNγ-producing T-helper effector memory cells with CD4+CD45RA-CCR7- phenotype, which persisted in circulation for up to 6 month after COVID-19. In contrast, SARS-CoV-2-specific IFNγ-secreting cytotoxic effector memory T cells were found at significant levels only shortly after the disease, but rapidly decreased over time. CONCLUSION: The stimulation of immune cells with live SARS-CoV-2 revealed a rapid decline in the pool of effector memory CD8+, but not CD4+, T cells after recovery from COVID-19. These data provide additional information on the development and persistence of cellular immune responses after natural infection, and can inform further development of T cell-based SARS-CoV-2 vaccines.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Memória Imunológica , Interferon gama/imunologia , Leucócitos Mononucleares/imunologia , SARS-CoV-2/imunologia , COVID-19/virologia , Epitopos de Linfócito T/imunologia , Humanos , Leucócitos Mononucleares/virologia , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Linfócitos T Citotóxicos/imunologia
16.
Nutrients ; 13(7)2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34371884

RESUMO

The dietary supplement, trans-resveratrol and hesperetin combination (tRES-HESP), induces expression of glyoxalase 1, countering the accumulation of reactive dicarbonyl glycating agent, methylglyoxal (MG), in overweight and obese subjects. tRES-HESP produced reversal of insulin resistance, improving dysglycemia and low-grade inflammation in a randomized, double-blind, placebo-controlled crossover study. Herein, we report further analysis of study variables. MG metabolism-related variables correlated with BMI, dysglycemia, vascular inflammation, blood pressure, and dyslipidemia. With tRES-HESP treatment, plasma MG correlated negatively with endothelial independent arterial dilatation (r = -0.48, p < 0.05) and negatively with peripheral blood mononuclear cell (PBMC) quinone reductase activity (r = -0.68, p < 0.05)-a marker of the activation status of transcription factor Nrf2. For change from baseline of PBMC gene expression with tRES-HESP treatment, Glo1 expression correlated negatively with change in the oral glucose tolerance test area-under-the-curve plasma glucose (ΔAUGg) (r = -0.56, p < 0.05) and thioredoxin interacting protein (TXNIP) correlated positively with ΔAUGg (r = 0.59, p < 0.05). Tumor necrosis factor-α (TNFα) correlated positively with change in fasting plasma glucose (r = 0.70, p < 0.001) and negatively with change in insulin sensitivity (r = -0.68, p < 0.01). These correlations were not present with placebo. tRES-HESP decreased low-grade inflammation, characterized by decreased expression of CCL2, COX-2, IL-8, and RAGE. Changes in CCL2, IL-8, and RAGE were intercorrelated and all correlated positively with changes in MLXIP, MAFF, MAFG, NCF1, and FTH1, and negatively with changes in HMOX1 and TKT; changes in IL-8 also correlated positively with change in COX-2. Total urinary excretion of tRES and HESP metabolites were strongly correlated. These findings suggest tRES-HESP counters MG accumulation and protein glycation, decreasing activation of the unfolded protein response and expression of TXNIP and TNFα, producing reversal of insulin resistance. tRES-HESP is suitable for further evaluation for treatment of insulin resistance and related disorders.


Assuntos
Hesperidina/administração & dosagem , Resistência à Insulina , Obesidade/terapia , Sobrepeso/terapia , Resveratrol/administração & dosagem , Adulto , Pressão Sanguínea/efeitos dos fármacos , Índice de Massa Corporal , Proteínas de Transporte/sangue , Correlação de Dados , Estudos Cross-Over , Suplementos Nutricionais , Método Duplo-Cego , Quimioterapia Combinada , Dislipidemias/sangue , Dislipidemias/terapia , Feminino , Transtornos do Metabolismo de Glucose/sangue , Transtornos do Metabolismo de Glucose/terapia , Glicosilação/efeitos dos fármacos , Humanos , Inflamação , Mediadores da Inflamação/sangue , Leucócitos Mononucleares/metabolismo , Masculino , Obesidade/sangue , Sobrepeso/sangue , Aldeído Pirúvico/sangue , Fator de Necrose Tumoral alfa/sangue
17.
Microb Pathog ; 158: 105114, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34333072

RESUMO

Understanding the pathogenesis of SARS-CoV-2 is essential for developing effective treatment strategies. Viruses hijack the host metabolism to redirect the resources for their replication and survival. The influence of SARS-CoV-2 on host metabolism is yet to be fully understood. In this study, we analyzed the transcriptomic data obtained from different human respiratory cell lines and patient samples (nasopharyngeal swab, peripheral blood mononuclear cells, lung biopsy, bronchoalveolar lavage fluid) to understand metabolic alterations in response to SARS-CoV-2 infection. We explored the expression pattern of metabolic genes in the comprehensive genome-scale network model of human metabolism, Recon3D, to extract key metabolic genes, pathways, and reporter metabolites under each SARS-CoV-2-infected condition. A SARS-CoV-2 core metabolic interactome was constructed for network-based drug repurposing. Our analysis revealed the host-dependent dysregulation of glycolysis, mitochondrial metabolism, amino acid metabolism, nucleotide metabolism, glutathione metabolism, polyamine synthesis, and lipid metabolism. We observed different pro- and antiviral metabolic changes and generated hypotheses on how the host metabolism can be targeted for reducing viral titers and immunomodulation. These findings warrant further exploration with more samples and in vitro studies to test predictions.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Leucócitos Mononucleares , Biologia de Sistemas , Transcriptoma
18.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360753

RESUMO

Activation of innate immunity and low-grade inflammation contributes to hyperglycemia and an onset of Type 2 Diabetes Mellitus (T2DM). Interleukin-2 (IL-2), leptin, High Mobility Group Box-1 (HMGB-1), and increased glucose concentrations are mediators of these processes also by modulating peripheral blood mononuclear cells (PBMCs) response. The aim of this study was to investigate if HMGB-1 and IL-2 turn on PBMCs and their leptin secretion. In isolated human PBMCs and their subpopulations from healthy individuals and naïve T2DM patients, leptin release, pro-inflammatory response and Toll-like Receptors (TLRs) activation was measured. After treatment with IL-2 and HMGB1, NK (Natural Killer) have the highest amount of leptin secretion, whilst NK-T have the maximal release in basal conditions. TLR4 (TAK242) and/or TLR2 (TLR2-IgA) inhibitors decreased leptin secretion after IL-2 and HMGB1 treatment. A further non-significant increase in leptin secretion was reported in PBMCs of naive T2DM patients in response to IL-2 and HMGB-1 stimulation. Finally, hyperglycemia or hyperinsulinemia might stimulate leptin secretion from PBMCs. The amount of leptin released from PBMCs after the different treatments was enough to stimulate the secretion of IL-1ß from monocytes. Targeting leptin sera levels and secretion from PBMCs could represent a new therapeutic strategy to counteract metabolic diseases such as T2DM.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Proteína HMGB1/farmacologia , Hiperglicemia/metabolismo , Hiperinsulinismo/metabolismo , Interleucina-2/farmacologia , Leptina/metabolismo , Leucócitos Mononucleares/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Hiperglicemia/patologia , Hiperinsulinismo/patologia , Leucócitos Mononucleares/patologia
19.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361047

RESUMO

Chemoresistance of germ cell tumors (GCTs) represents an intensively studied property of GCTs that is the result of a complicated multifactorial process. One of the driving factors in this process is the tumor microenvironment (TME). Intensive crosstalk between the DNA damage/DNA repair pathways and the TME has already been reported. This study aimed at evaluating the interplay between the immune TME and endogenous DNA damage levels in GCT patients. A cocultivation system consisting of peripheral blood mononuclear cells (PBMCs) from healthy donors and GCT cell lines was used in an in vitro study. The patient cohort included 74 chemotherapy-naïve GCT patients. Endogenous DNA damage levels were measured by comet assay. Immunophenotyping of leukocyte subpopulations was performed using flow cytometry. Statistical analysis included data assessing immunophenotypes, DNA damage levels and clinicopathological characteristics of enrolled patients. The DNA damage level in PBMCs cocultivated with cisplatin (CDDP)-resistant GCT cell lines was significantly higher than in PBMCs cocultivated with their sensitive counterparts. In GCT patients, endogenous DNA damage levels above the cutoff value were independently associated with increased percentages of natural killer cells, CD16-positive dendritic cells and regulatory T cells. The crosstalk between the endogenous DNA damage level and specific changes in the immune TME reflected in the blood of GCT patients was revealed. The obtained data contribute to a deeper understanding of ongoing interactions in the TME of GCTs.


Assuntos
Dano ao DNA , Leucócitos Mononucleares/imunologia , Neoplasias Testiculares/imunologia , Microambiente Tumoral/imunologia , Adulto , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/imunologia , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Humanos , Leucócitos Mononucleares/classificação , Masculino , Pessoa de Meia-Idade , Neoplasias Testiculares/tratamento farmacológico , Neoplasias Testiculares/genética , Neoplasias Testiculares/patologia
20.
Zh Nevrol Psikhiatr Im S S Korsakova ; 121(7. Vyp. 2): 82-89, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34387452

RESUMO

OBJECTIVE: To investigate the direct effect of D1-like dopaminergic receptors antagonist on Th17-cells function in multiple sclerosis (MS) in vitro. MATERIAL AND METHODS: Forty-one relapsing-remitting MS patients and twenty-five healthy subjects were examined. The functional activity of Th17-cells was assessed by the ability to produce IL-17 and IFN-γ by peripheral blood mononuclear cells (PBMCs) and CD4+ T cells, stimulated with microbeads coated with anti-CD3/anti-CD28-antibodies. To study the involvement of D1-like dopaminergic receptors in modulation of Th17-cell function, the samples of PBMCs or CD4+ T-cells were cultured in the presence of dopamine and/or specific D1-like dopaminergic receptors antagonist (SCH23390). Cytokine levels in cell culture supernatants were measured by ELISA. RESULTS: The production of IL-17 and IFN-γ by stimulated PBMCs were higher in MS patients during relapse than in MS patients during clinical remission or in healthy subjects. The production of cytokines by stimulated PBMCs or CD4+ T-cells in MS patients during clinical remission and healthy subjects was comparable. Dopamine reduced the production of cytokines by PBMCs and CD4+ T-cells in all groups. Blockade of D1-like dopaminergic receptors did not affect the dopamine-mediated cytokine suppression in MS patients and healthy subjects. Blockade of D1-like dopaminergic receptors directly suppressed cytokine production by PBMCs and CD4+ T-cells in MS patients and healthy subjects. CONCLUSIONS: Dopamine and blockade of D1-like dopaminergic receptors have an inhibitory effect on Th17-cell function in MS. The activation of D2-like dopaminergic receptors could mediate the inhibitory effect of dopamine on Th17-cells.


Assuntos
Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Humanos , Leucócitos Mononucleares , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Receptores Dopaminérgicos , Células Th17
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