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1.
Chinese Medical Journal ; (24): 1310-1316, 2021.
Article in English | WPRIM | ID: wpr-878104

ABSTRACT

BACKGROUND@#Epigenetics, especially DNA methylation, plays an important role in the pathogenesis of primary Sjogren syndrome (pSS). Our study aimed to reveal the role of DNA methylation in peripheral monocytes of pSS patients.@*METHODS@#A total of 11 pSS patients and five age-matched healthy controls (HCs) were included in this study. Monocytes were isolated from peripheral blood mononuclear cells using magnetic microbeads. DNA methylation profiles were generated using Human Methylation 850K BeadChips.@*RESULTS@#In monocytes from pSS patients, we identified 2819 differentially methylated positions (DMPs), comprising 1977 hypomethylated- and 842 hypermethylated-DMPs, corresponding to 1313 unique genes when compared with HCs. IFI44L, MX1, PAARP9, and IFITM1, which influence the interferon (IFN) signaling pathway, were among the genes hypomethylated in pSS. Functional analysis of genes with a minimum of two DMPs showed involvement in antigen binding, transcriptional regulation, cell adhesion, IFN-γ pathway, type I IFN pathway, antigen presentation, Epstein-Barr virus infection, human T-lymphotropic virus type 1 virus infection, and metabolic disease-related pathways. In addition, patients with higher serum IgG levels exhibited enrichment in Notch signaling and metabolic-related pathways. Upon comparing monocytes with salivary gland epithelial cells, an important overlap was observed in the cell cycle, cell senescence, and interleukin-17 signaling pathways. The differentially methylated genes were more enriched in the ribosome- and AMP-activated protein kinase signaling pathway in anti-Ro/SSA and anti-La/SSB autoantibodies double-positive patients.@*CONCLUSION@#Genome-wide DNA methylation profiling revealed significant differences in DNA methylation in monocytes isolated from patients with pSS.


Subject(s)
DNA Methylation/genetics , Epstein-Barr Virus Infections , Herpesvirus 4, Human , Humans , Leukocytes, Mononuclear , Monocytes , Sjogren's Syndrome/genetics
2.
Clinics ; 76: e2432, 2021. tab, graf
Article in English | LILACS | ID: biblio-1153954

ABSTRACT

OBJECTIVES: Telomeres are a terminal "DNA cap" that prevent chromosomal fusion and degradation. However, aging is inherent to life, and so is the loss of terminal sequences. Telomerase is a specialized reverse transcriptase encoded by self-splicing introns that counteract chromosome erosion. Telomerase activity is observed during early embryonic development, but after the blastocyst stage, the expression of telomerase reduces. The consequences of either insufficient or unrestrained telomerase activity underscore the importance of ongoing studies aimed at elucidating the regulation of telomerase activity in humans. In the present study, we aimed to standardize a simplified telomerase repeat-amplification protocol (TRAP) assay to detect telomerase activity in unstimulated and PHA-stimulated mononuclear cells. METHODS and RESULTS: Our optimized qPCR-based can efficiently evaluate telomerase activity. Quantification of protein and DNA between unstimulated and PHA-stimulated peripheral blood mononuclear cells revealed cellular activation and cell-cycle entry. The assay also showed that relative telomerase activity is significantly different between these two conditions, supporting the applicability of the assay. Furthermore, our findings corroborated that telomerase activity decreases with age. CONCLUSIONS: Telomeres and telomerase are implicated in aging and development of chronic diseases and cancer; however, difficulty in accessing commercial kits to investigate these aspects is a critical constraint in health surveillance studies. Our optimized assay was successfully used to differentiate telomerase activity between unstimulated and stimulated cells, clearly showing the reactivation of telomerase upon cell activation. This assay is affordable, reproducible, and can be executed in resource-limited settings.


Subject(s)
Humans , Female , Pregnancy , Telomerase/genetics , Telomerase/metabolism , Neoplasms , Aging , Leukocytes, Mononuclear/metabolism , Chronic Disease , Cost-Benefit Analysis
3.
Braz. j. med. biol. res ; 54(8): e10850, 2021. tab, graf
Article in English | LILACS | ID: biblio-1249328

ABSTRACT

The conversion of adenosine to inosine is catalyzed by adenosine deaminase (ADA) (EC 3.5.4.4), which has two isoforms in humans (ADA1 and ADA2) and belongs to the zinc-dependent hydrolase family. ADA modulates lymphocyte function and differentiation, and regulates inflammatory and immune responses. This study investigated ADA activity in lymphocyte-rich peripheral blood mononuclear cells (PBMCs) in the absence of disease. The viability of lymphocyte-rich PBMCs isolated from humans and kept in 0.9% saline solution at 4-8°C was analyzed over 20 h. The incubation time and biochemical properties of the enzyme, such as its Michaelis-Menten constant (Km) and maximum velocity (Vmax), were characterized through the liberation of ammonia from the adenosine substrate. Additionally, the presence of ADA protein on the lymphocyte surface was determined by flow cytometry using an anti-CD26 monoclonal human antibody, and the PBMCs showed long-term viability after 20 h. The ADA enzymatic activity was linear from 15 to 120 min of incubation, from 2.5 to 12.5 µg of protein, and pH 6.0 to 7.4. The Km and Vmax values were 0.103±0.051 mM and 0.025±0.001 nmol NH3·mg-1·s-1, respectively. Zinc and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) inhibited enzymatic activity, and substrate preference was given to adenosine over 2′-deoxyadenosine and guanosine. The present study provides the biochemical characterization of ADA in human lymphocyte-rich PBMCs, and indicates the appropriate conditions for enzyme activity quantification.


Subject(s)
Humans , Adenosine Deaminase , Dipeptidyl Peptidase 4 , Leukocytes, Mononuclear , Adenine , Lymphocytes
4.
Article in Chinese | WPRIM | ID: wpr-880131

ABSTRACT

OBJECTIVE@#To analyze the changes in the gene expression profile of T cells in CML patients after TCRζ up-regulation expression, and to explore the molecular mechanism of T cell reactivation after transgenic up-regulation of TCRζ.@*METHODS@#The peripheral blood mononuclear cells(PBMCs) from 3 newly untreated chronic-stage CML patients were collected, and the CD3@*RESULTS@#A total of 2248 differentially-expressed genes were obtained, including 553 up-regulated genes and 1695 down-regulated genes in experimental group as compared with those in control group (P<0.05) . The GO and KEGG enrichment analyses showed that differentially expressed genes involved in the biological processes related to T cell immune function, such as TCR signaling pathway, T cell proliferation and activation. Some of core genes involved in promoting the TCR signaling pathway, T cell proliferation, activation and apoptosis pathways were significantly up-regulated, while some core genes involved in inhibiting T cell activation were significantly down-regulated.@*CONCLUSION@#The molecular mechanism of the significantly improved T cell activation and proliferation ability in CML patients after TCRζ up-regulation may be related to the differential transcripts mediated signaling pathways of T cell activation, proliferation and apoptosis.


Subject(s)
Humans , Leukocytes, Mononuclear , Lymphocyte Activation , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes , Up-Regulation
5.
Rev. Assoc. Med. Bras. (1992) ; 66(12): 1712-1717, Dec. 2020. graf
Article in English | LILACS, SES-SP | ID: biblio-1143676

ABSTRACT

SUMMARY OBJECTIVE: This study aimed to investigate the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1) expression and its role in cytokine production from peripheral blood mononuclear cells (PBMCs) in patients with coronary artery disease (CAD) and non-CAD participants (NCAD). METHODS: Blood samples were taken from 15 patients with CAD and 15 NCAD individuals. The plasma was used for biochemical analyses. MALAT1 and CD36 expressions were evaluated in the isolated peripheral blood mononuclear cells (PBMCs) by real-time PCR. Furthermore, the levels of inflammatory cytokines e.g. interleukin (IL)-6, IL-10, and IL-22 were measured in the supernatants of the cultured PBMCs by flow cytometry. RESULTS: The levels of MALAT1 and CD36 were not significantly different between the CAD and NCAD groups. However, a lower level of MALAT1 and CD36 was observed in PBMCs of vitamin D deficient (<15 ng/ml) CAD and NCAD participants. Furthermore, the vitamin D deficient (<15 ng/ml) group showed a significantly higher plasma level of IL-6, IL-10, and IL-22 compared to the non-deficient (≥15 ng/ml) group. In addition, significant positive correlations were found between CD36, IL-22, and fasting blood sugar (FBS) with MALAT1. CONCLUSION: Given that in vitamin D deficient individuals a decreased level of MALAT1 was associated with CD36 expression and increased IL-22 production, vitamin D supplementation may play a role in reducing MALAT1/CD36/IL-22 mediated complications such as T2DM and CAD, especially in vitamin D deficiency.


RESUMO OBJETIVO: O objetivo deste estudo foi investigar a expressão do RNA longo não codificante lncRNA MALAT1 e o seu papel na produção de citocinas a partir de células mononucleares do sangue periférico (PBMCs) em pacientes com doença arterial coronariana (DAC) e participantes sem DAC (NDAC). MÉTODOS: Amostras de sangue foram coletadas de 15 pacientes com DAC e 15 indivíduos NCAD. O plasma foi usado para análises bioquímicas. As expressões de MALAT1 e CD36 foram avaliadas nas células mononucleares do sangue periférico (PBMCs) isoladas por PCR em tempo real. Além disso, os níveis de citocinas inflamatórias, como a interleucina (IL)-6, IL-10 e IL-22 foram medidas no sobrenadante da cultura de PBMCs por citometria de fluxo. RESULTADOS: Os níveis de MALAT1 e CD36 não foram significativamente diferentes entre os grupos DAC e NDAC. No entanto, um nível inferior de MALAT1 e CD36 foi observado nas PBMCs de participantes com deficiência de vitamina D (< 15 ng/ml) tanto no grupo DAC quanto no NDAC. Além disso, o grupo com deficiência de vitamina D (< 15 ng/ml) apresentou um nível plasmático significativamente maior de IL-6, IL-10 e IL-22 em comparação com o grupo sem a deficiência (≥15 ng/ml). Além disso, foram encontradas correlações positivas significativas entre CD36, IL-22, e glicemia de jejum (GJ) e o MALAT1. CONCLUSÃO: Dado que em indivíduos com deficiência de vitamina D a diminuição do nível de MALAT1 foi associada com a expressão de CD36 e produção aumentada de IL-22, a suplementação de vitamina D pode ter um papel importante na redução de complicações mediadas por MALAT1/CD36/IL-22, tais como DMT2 e DAC, especialmente em casos de deficiência de vitamina D.


Subject(s)
Humans , Coronary Artery Disease , RNA, Long Noncoding , Vitamin D , Leukocytes, Mononuclear , Cytokines
6.
Arq. gastroenterol ; 57(4): 366-374, Oct.-Dec. 2020. tab, graf
Article in English | LILACS | ID: biblio-1142336

ABSTRACT

ABSTRACT BACKGROUND: During the Helicobacter pylori (HP) infection, the infiltration of the leukocytes into stomach mucosa is directed by locally produced chemokines that play a decisive role in infection outcome. The CagA is the most potent virulence factor of HP, so that the infection with CagA + strains is associated with more severe complications than infection with CagA - HP. OBJECTIVE: The aim was to determine the expression of chemokines CXCL10, CCL17, CCL20 and CCL22, and their receptors by CagA + HP- and CagA - HP-derived crude extract (HP-CE)-stimulated peripheral blood mononuclear cells (PBMCs) from peptic ulcer (PU) patients. METHODS: The serum and the PBMCs were collected from 20 HP-infected PU patients, 20 HP-infected asymptomatic subjects (HIA) and 20 non-infected healthy subjects (NHS). The PBMCs were cultured in absence of stimulator or with 10 µg CagA + HP crude extract (CagA + CE), 10 µg CagA - HP crude extract (CagA - CE). Chemokines and receptors were measured by ELISA and real time-PCR respectively. RESULTS: In PU patients, the production of chemokines CXCL10, CCL17, CCL20 and CCL22, and the expression of chemokine receptors CXCR3, CCR4 and CCR6 by CagA + CE-induced PBMCs were significantly higher than non-stimulated and CagA - CE stimulated cultures. The CXCL10 production by CagA + CE stimulated PBMCs from HIA subjects was significantly higher than the equal cultures from PU and NHS groups. The CCL17 and the CCL20 production by non-stimulated, CagA + CE stimulated, and CagA - CE stimulated PBMCs from PU subjects were significantly higher than the equal cultures from NHS and HIA groups. The CCL22 production by non-stimulated, CagA + CE stimulated and CagA - CE stimulated PBMCs from NHS group were significantly higher than the equal cultures from HIA and PU groups. The CagA + CE stimulated PBMCs from HIA subjects expressed lower amounts of CCR6 in comparison with CagA + CE stimulated PBMCs from NHS and PU groups. The serum levels CXCL10 and CCL20 in PU and HIA groups were significantly higher than NHS subjects. NHS and HIA groups displayed higher serum levels of CCL22 in comparison with PU patients. CONCLUSION: Results indicated that the CagA status of bacterium influence the expression of chemokines and receptors by HP-CE stimulated PBMCs from PU patients.


RESUMO CONTEXTO: Durante a infecção por Helicobacter pylori (HP), a infiltração dos leucócitos na mucosa estomacal é dirigida por quimiocinas produzidas localmente que desempenham um papel decisivo no resultado da infecção. O CagA é o fator de virulência mais potente do HP, de modo que a infecção com cepas CagA + está associada a complicações mais graves do que a infecção com CagA - HP. OBJETIVO: O objetivo foi determinar a expressão das quimiocinas CXCL10, CCL17, CCL20 e CCL22, e seus receptores por CagA + HP- e CagA - extrato bruto (EB) derivado de HP (HP-EB) de células mononucleares do sangue periférico (CMSP) de pacientes com úlcera péptica (UP). MÉTODOS: O soro e as CMSP foram coletados de 20 pacientes com UP infectados pelo HP, 20 indivíduos assintomáticos infectados pelo HP (AI-HP) e 20 indivíduos saudáveis não infectados pelo HP (NI-HP). As CMSP foram cultivadas na ausência de estimulador ou com extrato bruto CagA + HP de 10 μg (CagA + EB), 10 μg CagA - extrato bruto HP (CagA - EB). Quimiocinas e receptores foram medidos por ELISA e PCR em tempo real, respectivamente. RESULTADOS: Em pacientes com UP a produção de quimiocinas CXCL10, CCL17, CCL20 e CCL22, e a expressão dos receptores de quimiocina CXCR3, CCR4 e CCR6 por CagA + CMSP induzidos pelo EB foram significativamente maiores do que as culturas não estimuladas e CagA - EB estimulados. A produção de CXCL10 por CagA + EB estimulou as CMSP de sujeitos AI-HP em proporção significativamente maior do que as culturas iguais dos grupos UP e NI-HP. A produção de CCL17 e CCL20 por grupos não estimulados, CagA + EB estimulado, e CagA - EB estimulou CMSP de sujeitos com UP e foram significativamente superiores às culturas iguais dos grupos NI-HP e AI-HP. A produção de CCL22 por grupos não estimulados, CagA + EB estimulado e CagA - EB estimulado pelo grupo NI-HP foram significativamente maiores do que as culturas iguais dos grupos AI-HP e PU. O CagA + EB estimulou as CMSP dos sujeitos do AI-HP, expressando menores quantidades de CCR6 em comparação com as CMSP estimuladas pelo CagA + EB de grupos NI-HP e UP. Os níveis sanguíneos de CXCL10 e CCL20 nos grupos UP e AI-HP foram significativamente superiores aos dos sujeitos do NI-HP. Os grupos NI-HP e AI-HP apresentaram níveis sanguíneos mais elevados de CCL22 em comparação com pacientes com UP. CONCLUSÃO: Os resultados indicaram que o estado CagA da bactéria influencia a expressão de quimiocinas e receptores por HP-EB estimulados CMSP de pacientes com UP.


Subject(s)
Humans , Peptic Ulcer , Helicobacter pylori , Bacterial Proteins , Leukocytes, Mononuclear , Helicobacter Infections , Virulence Factors , Chemokine CCL17 , Chemokine CCL20 , Chemokine CCL22 , Leukocytes , Antigens, Bacterial
7.
Electron. j. biotechnol ; 47: 17-28, sept. 2020. ilus, graf
Article in English | LILACS | ID: biblio-1253006

ABSTRACT

BACKGROUND: Cichoric acid (CA) is extracted from Echinacea purpurea. It is well known and widely used for its immunological function. However, the effect of CA on peripheral blood mononuclear cells (PBMCs) from yaks is still unclear. This study investigated the potential influences of CA on the proliferation, cytokine induction, and apoptosis of PBMCs from Datong yak in vivo, and aimed to provide a basis for exploring the pharmacological activities of CA on yaks. RESULTS: In this study, CA promoted PBMCs proliferation by combining concanavalin A (Con A) and exhibited a dose-dependent effect as demonstrated by a Cell Counting Kit-8. The concentration of 60 µg/ml CA was the best and promoted the transformation from the G0/G1 phase to the S and G2/M phases with Con A. Furthermore, 60 µg/ml CA significantly increased IL-2, IL-6, and IFN-γ levels and PCNA, CDK4 and Bcl-2 expression levels, but it significantly inhibited the TP53, Bax, and Caspase-3 expression levels. Transcriptome analysis revealed a total of 6807 differentially expressed genes (DEGs) between the CA treatment and control groups. Of these genes, 3788 were significantly upregulated and 3019 were downregulated. Gene Ontology and pathway analysis revealed that DEGs were enriched in cell proliferation and immune function signaling pathways. The expression level of some transcription factors (BTB, Ras, RRM_1, and zf-C2H2) and genes (CCNF, CCND1, and CDK4) related to PBMCs proliferation in yaks were significantly promoted after CA treatment. By contrast, anti-proliferation-associated genes (TP53 and CDKN1A) were inhibited. CONCLUSIONS: In summary, CA could regulate the immune function of yaks by promoting proliferation and inhibiting inflammation and apoptosis of PBMCs.


Subject(s)
Animals , Cattle , Succinates/pharmacology , Caffeic Acids/pharmacology , Leukocytes, Mononuclear/drug effects , Echinacea/chemistry , Cell Proliferation/drug effects , Transcription Factors , Enzyme-Linked Immunosorbent Assay , Leukocytes, Mononuclear/cytology , Blotting, Western , Cytokines , Apoptosis/drug effects , Concanavalin A/pharmacology , Real-Time Polymerase Chain Reaction , RNA-Seq
8.
Article in Chinese | WPRIM | ID: wpr-827994

ABSTRACT

To study the effect of Huangqin Qingre Chubi Capsules containing serum on the protein expressions of AMPK and FoxO3 a in peripheral blood mononuclear cells of patients with rheumatoid arthritis(RA), in order to explore the mechanism of anti-oxidation. Peripheral anticoagulant was collected from patients and normal people. Monocytes(PBMC) were isolated through density gradient centrifugation, and the logarithmic phase cells were cultured. Drug containing serum was prepared through intragastric admini-stration to SD rats. The rats were divided into five groups, namely normal group, model group, AMPK blocker group(compound C 10 μmol·L~(-1)), medium-dose HQC+AMPK blocker group, and middle-dose HQC group. The cell inhibition rate was calculated by MTT method. The levels of IL-1β, IL-4, LPO, MDA, SOD and TAOC were detected by ELISA. The expressions of AMPK, p-AMPK, p-FoxO3 a and FoxO3 a were detected by Western blot. The HQC containing serum had an inhibitory effect on human monocytes in peripheral blood. The best concentration was observed in middle-dose HQC, and the best time was 24 hours. Middle-dose HQC group was better than model group, AMPK blocker group and middle-dose HQC + AMPK blocker group in terms of increase of SOD, p-AMPK, p-FoxO3 a and decrease of LPO. It was better than model group and AMPK blocker group in terms of increase of IL-4, TAOC, AMPK, FoxO3 a and decrease of IL-1β, MDA. The differences were statistically significant(P<0.05 or P<0.01). The HQC containing serum may increase the levels of TAOC and SOD, decrease the level of MDA and LPO, activate AMPK, directly phosphorylate FOXO3 a, enhance its transcriptional activity, and improve the state of oxidative stress in RA patients.


Subject(s)
AMP-Activated Protein Kinases , Animals , Arthritis, Rheumatoid , Capsules , Forkhead Box Protein O3 , Humans , Leukocytes, Mononuclear , Oxidative Stress , Rats , Rats, Sprague-Dawley , Scutellaria baicalensis
9.
Article in Chinese | WPRIM | ID: wpr-827191

ABSTRACT

OBJECTIVE@#To study the correlation of the expression alteration of Tim-3 with the T cell and B cell dysfunction in peripheral blood of multiple myeloma (MM) patients.@*METHODS@#30 patients diagnosed as MM from October 2016 to October 2018 were selected and enrolled in MM group, and 30 healthy persons whose sex and age was matched with the MM patients were selected and enrolled in healthy control group (HC). The blood samples from MM patients and HC were collected, and the peripheral blood mononuclear cells (PBMNC) were separated by density gradient centrifugation, then the serum was kept for further study. The ratios of CD3CD4Tim-3T cells, CD3CD8Tim-3T cells and the CD19+CD20-CD38+B cells were analysed by flow cytometry (FCM),and the concentration of T cell-related cytokines IFN-γ, TNF-αand B cell-related antibodies IgA, IgM and IgG were measured by ELISA. At the same time, the differences of the ratios of CCD3CD4Tim-3T, CD3CD8Tim-3T cells and plasmablast and the concentration of IFN-γ, TNF-α, IgA, IgM and IgG between the MM patient and HC were estimated, and the correlation of the ratio of CD3CD4Tim-3T, CD3CD8Tim-3T cells with the ratio of plasmablast and the concentration of IFN-γ, TNF-α, IgA, IgM and IgG in MM patients were analyzed.@*RESULTS@#The ratio of CD3CD4Tim-3T, CD3CD8Tim-3T cells increased in MM patients, while the ratio of CD19+CD20- CD38+B cells and the concentration of IFN-γ, TNF-α, IgA, IgM and IgG decreased in MM patients. And there was a negative correlation of the ratio of CD3CD4Tim-3T cells with CD19+CD20-CD38+B cells and the concentration of IFN-γ, IgA, IgM and IgG in MM patients, while the ratio of CD3CD8Tim-3T cells just negatively correlated with the concentration of TNF-α.@*CONCLUSION@#Expression of Tim-3 on CD4 and CD8 cells elevates in the peripheral blood of MM patients, which also correlates with the function suppression of T and B cells.


Subject(s)
B-Lymphocytes , CD8-Positive T-Lymphocytes , Hepatitis A Virus Cellular Receptor 2 , Metabolism , Humans , Leukocytes, Mononuclear , Multiple Myeloma
10.
Article in Chinese | WPRIM | ID: wpr-827186

ABSTRACT

OBJECTIVE@#To explore the effect of miR-335-5p/ADCY3 interaction on the lymphocyte function in the patients with aplastic anemia (AA).@*METHODS@#Blood samples were collected from 22 healthy volunteers (HC) and 50 AA patients including 38 severe AA (SAA) and 12 non-severe AA (NSAA). Peripheral blood mononuclear cells (PBMNC) were isolated. The expression of miR-335-5p and ADCY3 mRNA was detected by using RT-PCR. Negative control miR-335-5p (NC group) and miR-335-5p mimic (mimic group) were transfected to AA-PBMNC by using RNAimax reagent, respectively. The proliferative ability, activation and cytokines of CD4 T and CD8 T cells were measured by flow cytometry. Dual-luciferase reporter assay was used to verify the targeted relationship between miR-335-5p and target gene.@*RESULTS@#The expression of miR-335-5p was significantly downregulated in SAA-PBMNC and NSAA-PBMNC compared with HC-PBMNC (0.08±0.01 vs 0.74±0.10, P<0.01; 0.17±0.02 vs 0.74±0.10, P<0.01). Meanwhile, the expression of miR-335-5p in SAA-PBMNC was very statistically significantly lower than that in NSAA-PBMNC (P<0.01). Compared with NC group, upregulation of miR-335-5p in vitro could significantly inhibited the proliferation of CD4 T and CD8 T cells in AA-PBMNC (P<0.05 and P<0.05, respectively). And, upregulating miR-335-5p in AA-PBMNC could significantly inhibited the activation of CD4 and CD8 T cells (P<0.01 and P<0.01, respectively). The ratio of CD4TNFα T, CD8IFNγ+T and CD8TNFα T cell by up-regulating the expression of miR-335-5p from AA-PBMNC in vitro was also significantly lower (P<0.01, P<0.05 and P<0.05, respectively). In addition, the expression of ADCY3 was higher in AA-PBMNC than that in HC-PBMNC (1.70±0.15 vs 0.76±0.12, P<0.01). Furthermore, by means of dual-luciferase reporter assay, the luciferase activity of ADCY3'UTR wildtype could be inhibited by miR-335-5p.@*CONCLUSIONS@#The expression of miR-335-5p was significantly downregulated in AA, and that correlates with disease severity. Up-regulating miR-335-5p can correct the hyperimmune status in AA patients by targeting ADCY3. These changes may relates with the strengthen of inhibition for targeted gene ADCY3.


Subject(s)
Anemia, Aplastic , Genetics , CD8-Positive T-Lymphocytes , Humans , Leukocytes, Mononuclear , Lymphocyte Count , MicroRNAs , Genetics
11.
Journal of Experimental Hematology ; (6): 1338-1343, 2020.
Article in Chinese | WPRIM | ID: wpr-827115

ABSTRACT

OBJECTIVE@#To explore the value of has-microRNA-155 (miR-155) in peripheral blood mononuclear cells (PBMNC) in prognostic evaluation of elderly patients with primary immune thrombocytopenia (PITP).@*METHODS@#One hundred and thirty elderly PITP patients and 60 healthy volunteers in our hospital were selected. The relative expression level of miR-155 in PBMNC was detected by RT-PCR. Unconditional logistic multivariate regression analysis was used to analyze the relationship between miR-155 expression and prognosis of PITP patients, and Kaplan-Meier was further used to analyze the relationship between miR-155 and PITP recurrence.@*RESULTS@#The relative expression level of miR-155 in PBMNC of elderly PITP patients was significantly higher than that in healthy volunteers, and increased significantly with the severity of the disease (P<0.05). The overall effective rate of elderly PITP patients with miR-155 low-expression was significantly higher than that in the patients with miR-155 high-expression (96.92% vs 72.31%) by after treatment with glucocorticoid. Multivariate analysis showed that miR-155 was an independent risk factor for PITP patients. Elderly patients with high expression of miR-155 showed a higher risk of recurrence.@*CONCLUSION@#miR-155 in PBMNC has a high accuracy for PITP diagnosis, and the elderly patients with high level of miR-155 show a poor prognosis and a higher risk of recurrence.


Subject(s)
Aged , Biomarkers, Tumor , Humans , Kaplan-Meier Estimate , Leukocytes, Mononuclear , MicroRNAs , Neoplasm Recurrence, Local , Prognosis , Purpura, Thrombocytopenic, Idiopathic
12.
J. appl. oral sci ; 28: e20200124, 2020. tab, graf
Article in English | LILACS, BBO | ID: biblio-1134800

ABSTRACT

Abstract Objectives To evaluate apoptotic levels of peripheral blood mononuclear cells (PBMCs) and apoptotic regulatory proteins (Bax and Bcl-2) in lymphocyte subsets of oral cancer (OC) patients and healthy controls (HC). Methodology The percentage of apoptotic cells and lymphocyte counts were measured in the first cohort using PBMCs obtained from 23 OC patients and 6 HC. In the second cohort, (OC, 33; HC, 13), the mean fluorescence intensity (MFI) of Bax and Bcl-2 in CD19+ B, CD4+ T, CD8+ T, and CD16+56+ natural killer (NK) cells was determined via flow cytometry. Results The percentage of apoptotic cells was higher in the PBMCs of OC patients than in HC patients, particularly in patients with stage IV cancer (p<0.05). However, lymphocyte counts were significantly lower in stage IV patients (p<0.05). NK CD19+ B and CD16+56+ cell counts were significantly lower in OC patients compared with HC patients (p<0.001 and p<0.01, respectively), but CD4+ T cells were interestingly significantly higher in OC patients (p<0.001). While Bax MFI was slightly higher, Bcl-2 MFI was significantly lower for all four lymphocyte subsets in OC samples, particularly in stage IV patients, when compared with HC. Consequently, Bax/Bcl-2 ratios showed an upward trend from HC to OC patients, particularly those in stage IV. We found similar trends in Bax and Bcl-2 MFI for tumor stage, tumor size, and lymph node involvement. Conclusions The increased lymphocyte apoptosis in stage IV OC patients may be related to higher Bax levels and lower Bcl-2 levels. The Bax/Bcl-2 ratio in lymphocytes may be useful to determine the prognosis of OC patients, and could be considered a mean for supportive treatment in the future.


Subject(s)
Humans , Mouth Neoplasms , Leukocytes, Mononuclear , Killer Cells, Natural , Lymphocyte Subsets , Apoptosis , Flow Cytometry
13.
Journal of Experimental Hematology ; (6): 2051-2055, 2020.
Article in Chinese | WPRIM | ID: wpr-880014

ABSTRACT

OBJECTIVE@#To investigate the effect of IL-27 on Th17 cells in patients with henoch-schönlein purpura(HSP) in order to further elucidate the pathogenesis.@*METHODS@#Fifty patients with HSP treated in our hospital from April 2019 to July 2019 were selected as HSP group, and 30 volunteers underwent physical examination at the same time were selected as control group. The proportion of Th17 cells in peripheral blood of HSP group and healthy control group was determined by flow cytometry (FCM). A total of 27 HSP patients were selected, and candidate peripheral blood mononuclear lymphocytes (PBMC) were co-cultured with exogenous rhIL-27, and the ratio of Th17 cells was detected by flow cytometry.@*RESULTS@#The proportion of Th17 cells in the peripheral blood of HSP patients with acute phase was (1.57±0.54)%, which was significantly higher than that of the control group (0.86±0.40)% (t=-6.298, P<0.001), and the proportion of Th17 cells was decreased significantly after rhIL-27 co-culture (1.39%±0.52% vs 0.98%±0.44%)(P<0.05).@*CONCLUSION@#IL-27 can reduce the level of Th17 cells in patients with HSP, which may be involved in the pathogenic process of HSP and play a protective role in the development of the disease.


Subject(s)
Humans , Interleukin-27 , Leukocytes, Mononuclear , Patients , Purpura, Schoenlein-Henoch , Th17 Cells
14.
Journal of Experimental Hematology ; (6): 1873-1879, 2020.
Article in Chinese | WPRIM | ID: wpr-879986

ABSTRACT

OBJECTIVE@#To investigate the down-regulation effect of let-7b-5p on the expression of FTO in acute myeloid leukemia cell line THP-1 and inhibitory effect on THP-1 proliferation via m@*METHODS@#The acute myeloid leukemia cell line THP-1 and the normal human peripheral blood mononuclear cells (PBMNC) were selected as subjects. The expression of let-7b-5p and FTO mRNA in those cells was detected by qPCR, further the expression of FTO protein in those cells was detected by Western blot. And, the luciferase reporter gene assay was used to verify the targeting effect of let-7b-5p on FTO. Finally, THP-1 cells were transfected respectively with let-7b-5p mimic, and PBMNC with let-7b-5p inhibitor, there after the C-MYC mRNA m@*RESULTS@#Compared with PBMNC, the expression of let-7b-5p in THP-1 significantly decreased, while the expression of FTO was significantly increased (P<0.05). After transfection with let-7b-5p mimic combined with FTO 3'-UTR, the luciferase activity of transfected THP-1 cells significantly decreased, but the luciferase activity significantly increased after transfection with mutant 3'-UTR, which was significantly different from the negative control group(blank vector) (P<0.05). Let-7b-5p inhibitor down-regulated c-MYC mRNA m@*CONCLUSION@#Human acute myeloid leukemia cell line THP-1 low expresses the let-7b-5p, which regulates c-MYC expression through let-7b-5p-/FTO-/m


Subject(s)
Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Cell Line, Tumor , Cell Proliferation , Humans , Leukemia , Leukocytes, Mononuclear , MicroRNAs/genetics , Signal Transduction , THP-1 Cells
15.
Journal of Experimental Hematology ; (6): 1842-1847, 2020.
Article in Chinese | WPRIM | ID: wpr-879981

ABSTRACT

OBJECTIVE@#To investigate the effect of GÖ6976 on the proliferation of chronic myeloid leukemia cells and its toxic effect on normal cells and mice, so as to provide experimental basis for the effectiveness and safety of its clinical application.@*METHODS@#Different concentrations of GÖ6976 were applied to the K562 cells, human peripheral blood mononuclear cells (PBMNC) and normal BaF3 cells, MTT assay was used to detect the effect on cell proliferation. BALB/C mice were used to investigate the toxicity in vivo. The general situation, body weight and the number of white blood cells in peripheral blood were monitored during administration, the blood collected from eyeballs before and after administration was used for biochemical examination, at the same time, the liver, kidney and femurs were examined pathologically.@*RESULTS@#GÖ6976 could significantly inhibit the proliferation of K562 cells, inhibition effect increased with increasing dose (r=0.9623). However, there was no significant change in the inhibitory effect on PBMNC and BaF3 cells. The pathological examination of organs in each group showed no abnormal manifestations such as inflammatory infiltration, while the change rate of leukocyte count in peripheral blood of high dose group fluctuated greatly (P<0.05), which might be related to the inhibition of intracellular protein kinase C, and no abnormality was observed in blood biochemical indexes. In the low dose group, there was no significant difference in peripheral blood leukocyte count, blood biochemical index and histopathology during administration drug as compared with the control group.@*CONCLUSION@#GÖ6976 possesses a significant inhibitory effect on the proliferation of K562 cells, and the inhibitory effect increases with increasing dose. Long-term application of 5.0 μmol/L and below concentrations of GÖ6976 shows no obvious inhibitory effect on PBMNC, BaF3 cells. Long-term application of 10 mg/kg and below concentrations of GÖ6976 shows no obvious toxic effect on BALB/c mice.


Subject(s)
Animals , Apoptosis , Carbazoles , Cell Proliferation , Humans , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Leukocytes, Mononuclear , Mice , Mice, Inbred BALB C
16.
Article in Chinese | WPRIM | ID: wpr-879935

ABSTRACT

OBJECTIVE@#To investigate the functional pathways enriched and differentially expressed genes (DEGs) in peripheral blood mononuclear cells (PBMCs) of patients with gram-positive and gram-negative sepsis.@*METHODS@#Dataset GSE9960 obtained from NCBI GEO database containing PBMC samples from 16 non-infectious systematic inflammatory response syndrome (SIRS) patients, 17 gram-positive septic patients and 18 gram-negative septic patients were included in the study. Functional pathway annotations were conducted by gene set enrichment analysis and weighted gene co-expression network analysis. DEGs were filtered and master DEGs were then validated in PBMCs of gram-positive septic, gram-negative septic and non-infectious SIRS patients.@*RESULTS@#The enriched gene sets in gram-positive sepsis and gram-negative sepsis were significantly different. The results indicated the opposite co-expression networks in SIRS and gram-negative sepsis, and the entirely different co-expression networks in gram-positive and gram-negative sepsis. Furthermore, we validated that @*CONCLUSIONS@#The results indicate that there are differences in the mechanism and pathogenesis of gram-positive and gram-negative sepsis, which may provide potential markers for sepsis diagnosis and empirical antimicrobial therapy.


Subject(s)
Biomarkers/analysis , Gene Expression Profiling , Gram-Negative Bacterial Infections/physiopathology , Gram-Positive Bacterial Infections/physiopathology , Humans , Leukocytes, Mononuclear/pathology , Sepsis/physiopathology
17.
Braz. j. infect. dis ; 23(1): 27-33, Jan.-Feb. 2019. tab, graf
Article in English | LILACS | ID: biblio-1001503

ABSTRACT

ABSTRACT Introduction: Human T-cell lymphotropic virus type 1 (HTLV-1) is sexually transmitted and causes persistent infection. This virus induces activation of the immune system and production of inflammatory cytokines. This study aimed to assess the cytokine profile and cytopathological findings in the cervicovaginal fluid of asymptomatic HTLV-1-infected women. Methods: HTLV-1-infected and uninfected women were selected at the Centro de Atendimento ao Portador de HTLV in Salvador-Brazil. None of the included HTLV-1-infected women reported any HTLV-1-associated diseases. All volunteers underwent gynecological examination to collect cervicovaginal fluid. Cytokine quantification was performed using the Cytometric Bead Array (CBA) Human Th1/Th2/Th17 kit. Light microscopy was used to evaluate cervicovaginal cytopathology. In addition, proviral load in cervicovaginal fluid and peripheral blood was measured by real-time quantitative polymerase chain reaction. Results: 112 women (63 HTLV-1-infected and 49 uninfected) were evaluated. No differences were found with respect to cytopathological cervicovaginal findings between the groups. IL-2, TNF, IL-4, IL-10, and IL-17 levels were significantly higher in cervicovaginal fluid of the HTLV-1-infected women than in uninfected women (p < 0.05). Conversely, IFN-γ was found to be lower in the HTLV-1-infected women (p < 0.001) compared to uninfected individuals. Cervicovaginal proviral load was detectable in 53% of the HTLV-1-infected women and was found to be consistently lower than the proviral load in peripheral blood. Conclusions: HTLV-1 infection induces immune activation in cervicovaginal environment, characterized by elevated concentrations of Th1, Th2, and IL17 in the cervicovaginal fluid.


Subject(s)
Humans , Female , Adult , Vagina/pathology , Body Fluids/chemistry , HTLV-I Infections/pathology , Cervix Uteri/pathology , Cytokines/analysis , Social Class , Vagina/immunology , Vagina/virology , Body Fluids/immunology , Enzyme-Linked Immunosorbent Assay , Leukocytes, Mononuclear/virology , Human T-lymphotropic virus 1/isolation & purification , HTLV-I Infections/immunology , HTLV-I Infections/virology , Cervix Uteri/immunology , Cervix Uteri/virology , Cross-Sectional Studies , Th2 Cells/immunology , Th1 Cells/immunology , Statistics, Nonparametric , Viral Load , Interleukin-17/immunology
18.
Braz. j. infect. dis ; 23(1): 22-26, Jan.-Feb. 2019. tab
Article in English | LILACS | ID: biblio-1001495

ABSTRACT

ABSTRACT Objectives: To investigate the prevalence of human polyomavirus (BK and JC viruses) infection in peripheral blood mononuclear cells of healthy blood donors. Methods: The study included 250 healthy blood donors. Five-milliliter blood was drawn into sterile EDTA tubes and PBMCs were isolated from whole blood. The isolated PBMCs were counted and stored at −70 °C for future investigation. DNA was extracted and subjected to simple, sensitive and specific semi-nested PCR as well as QPCR using both general and specific primers for different assays. Results: Of 250 blood samples, 66 (26.4%) were positive for BKV DNA (146-34,514 copies/106 cells). JC DNA was found in 45 (18%) blood samples (65-21,250 copies/106 cells). Co-infection with these viruses were found in 11 (4.4%) out of 250 blood samples. Discussion: Our study provides important data on polyomavirus infection in peripheral blood mononuclear leukocytes in immunocompetent individuals. These data indicate significant differences between the prevalence of BKV and JCV infection in healthy blood donors. The prevalence of BK and JC virus infection is higher in the age range 30-39 years compared to other age ranges.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Young Adult , Tumor Virus Infections/virology , Blood Donors , Leukocytes, Mononuclear/virology , BK Virus/isolation & purification , JC Virus/isolation & purification , Polyomavirus Infections/virology , Tumor Virus Infections/blood , Tumor Virus Infections/epidemiology , DNA, Viral/isolation & purification , Prevalence , Age Distribution , BK Virus/genetics , JC Virus/genetics , Viral Load , Polyomavirus Infections/blood , Polyomavirus Infections/epidemiology , Real-Time Polymerase Chain Reaction , Iran/epidemiology
19.
Article in English | WPRIM | ID: wpr-776887

ABSTRACT

Astragalus membranaceus may be a potential therapy for childhood asthma but its driving mechanism remains elusive. The main components of A. membranaceus were identified by HPLC. The children with asthma remission were divided into two combination group (control group, the combination of budesonide and terbutaline) and A. membranaceus group (treatment group, the combination of budesonide, terbutaline and A. membranaceus). The therapeutic results were compared between two groups after 3-month therapy. Porcine peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by using density gradient centrifugation on percoll. The levels of FoxP3, EGF-β, IL-17 and IL-23 from PBMCs and serum IgE were measured. The relative percentage of Treg/Th17 cells was determined using flow cytometry. The main components of A. membranaceus were calycosin-7-O-glucoside, isoquercitrin, ononin, calycosin, quercetin, genistein, kaempferol, isorhamnetin and formononetin, all of which may contribute to asthma therapy. Lung function was significantly improved in the treatment group when compared with a control group (P < 0.05). The efficacy in preventing the occurrence of childhood asthma was higher in the treatment group than the control group (P < 0.05). The levels of IgE, IL-17 and IL-23 were reduced significantly in the treatment group when compared with the control group, while the levels of FoxP3 and TGF-β were increased in the treatment group when compared with the control group (P < 0.05). A. membranaceus increased the percentage of Treg cells and reduced the percentage of Th17 cells. A. membranaceus is potential natural product for improving the therapeutic efficacy of combination therapy of budesonide and terbutaline for the children with asthma remission by modulating the balance of Treg/Th17 cells.


Subject(s)
Animals , Asthma , Drug Therapy , Allergy and Immunology , Astragalus propinquus , Chemistry , Budesonide , Cells, Cultured , Child , Child, Preschool , Cytokines , Metabolism , Drugs, Chinese Herbal , Pharmacology , Female , Humans , Immunologic Factors , Pharmacology , Leukocytes, Mononuclear , Metabolism , Lung , Physiology , Male , Swine , T-Lymphocytes, Regulatory , Cell Biology , Terbutaline , Th17 Cells , Cell Biology , Treatment Outcome
20.
Article in Chinese | WPRIM | ID: wpr-776807

ABSTRACT

OBJECTIVE@#To determine the expression profile of microRNA (miRNA) in peripheral blood mononuclear cells (PBMC) and immune factors in pregnant women with hepatitis B virus (HBV) infection.@*METHODS@#A total of 182 pregnant women infected with HBV were randomly selected, with 40 healthy pregnant women and 35 non-pregnant women as controls. High-throughput sequencing was used to detect RNA in the PBMC of all subjects. Indirect ELISA method was used to determine the changes of cytokines in peripheral blood samples.@*RESULTS@#Compared with the control group, 18 differentially expressed miRNA were identified in those with HBV infection (P< 0.01). Among these, miR-3607-3p, miR-20a, miR-1296, miR-153-1 and miR-X4 may directly regulate the transcriptional level of target genes including IL-10, IL-18, IL-16, MCP-1, NUP50 and CCR1. Meanwhile, peripheral blood cytokines IL-10, IL-18, IL-16 and MCP-1 were significantly increased in those with HBV infection (P<0.01), with the expression level of IL-16 and MCP-1 being strongly correlated with the viral load.@*CONCLUSION@#The expression profiles of miRNA in PBMC and cytokines in peripheral blood can change significantly during pregnancy, both may be involved in the immune response to HBV infection.


Subject(s)
Cytokines , Blood , DNA, Viral , Female , Hepatitis B , Blood , Hepatitis B virus , Humans , Leukocytes, Mononuclear , Metabolism , MicroRNAs , Blood , Pregnancy
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