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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(5): 1750-1756, 2020 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-33067985

RESUMO

OBJECTIVE: To investigate the safety and efficacy of tumor-associated antigen-specific cytotoxic T lympho- cytes (TAA-CTL) in the treatment of multiple myeloma (MM) and non-Hodgkin lymphoma (NHL). METHODS: Peripheral blood mononuclear cells (PBMNC)of patients were collected. Dendritic cells (DC) were loaded with multiple tumor-associated antigens (TAA) (NY-ESO-1, MAGE-A3, MAGE-A4, WT1, Survivin, PRAME, LMP1 and LMP2A), then co-cultured with PBMNC to induce cytotoxic T lymphocytes (CTL). The phenotypes of cell products were detected, and the disease statuse was evaluated in 7 patients during or after infusion. The changes of TAA-CTL amount in the PBMNC of patients were measured by using IFN-γ ELISpot assay. RESULTS: TAA-CTL products were generated comprising CD3+ T cells (mean 82.98%) with a mixture of CD4+ (mean 42.09%) and CD8+ (mean 25.32%) T cells. Among them, 70% expressed effectors memory markers (CD45RO+CD62L-CCR7-). Each patient received TAA-CTL infusions for 1-4 times, and none of them showed obvious adverse reactions. The clinical symptoms and laboratory or imaging examination of 5 patients achieved positive effects. After cell therapy, the spot-forming cells (SFC) levels of most patients gradually increased and the peak often appeared about 2-3 weeks after the infusion. CONCLUSION: TAA-CTLs preliminarily show its safety and efficacy in MM and NHL patients, however, a larger population sample is needed to explore its clinical application value.


Assuntos
Linfoma não Hodgkin , Mieloma Múltiplo , Células Dendríticas , Humanos , Leucócitos Mononucleares , Linfoma não Hodgkin/terapia , Mieloma Múltiplo/terapia , Linfócitos T Citotóxicos
2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(5): 1762-1768, 2020 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-33067987

RESUMO

OBJECTIVE: To investigate the effect of dasatinib on the expansion of NK cells in vitro, as well as the subsets, receptor expression and cytotoxic function of NK cells. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy adult volunteers and cultured with SCGM added IL-2 and IL-15 for expansion of NK cells. In this culture system, dasatinib of different concentrations were added. Cell counting and phenotyping by flow cytometry were used to evaluate the amplification efficiency of NK cells. FCM was used to detect the expression of receptors on the surface of NK cells and the distribution of subsets. Subsequently, degranulation assay and CFSE/7AAD based cytotoxicity assay were used to detect the effects of dasatinib on NK cytotoxicity against leukemia cell line K562 cells. RESULTS: The expansion efficiency of NK cells in vitro could be increased by dasatinib at the concentration range of 5-50 nmol/L, and the expansion efficiency of NK cells reached the peak at 20 nmol/L of dasatinib. The NK cytotoxicity against K562 cells in dasatinib cultured group at the concentration of 20 nmol/L was significantly higher than that in control group. For the cells cultured by disatinib in vitro, the MFI of CD226, NKP46 and NKG2D was up-regulated; the ratio of NKG2A+CD57- subset was down-regulated, while the ratio of NKG2A-CD57+ subset was up-regulated.The degranulation response of NKG2A-CD57+ NK cells to K562 cells was stronger than that of NKG2A+CD57- NK cells. CONCLUSION: The results shows that appropriate dose of dasatinib(20 nmol/L) can increases the amplification efficiency of NK cells, simultaneously up-regulates the expression of NK activating receptors and increases the NKG2A-CD57+ subset, which lead to the enhancement of NK cytotoxicty against leukemia cell lines.


Assuntos
Antineoplásicos , Leucócitos Mononucleares , Adulto , Antineoplásicos/farmacologia , Dasatinibe/farmacologia , Humanos , Células K562 , Células Matadoras Naturais
3.
J Appl Oral Sci ; 28: e20200124, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32901694

RESUMO

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.


Assuntos
Leucócitos Mononucleares , Neoplasias Bucais , Apoptose , Citometria de Fluxo , Humanos , Células Matadoras Naturais , Subpopulações de Linfócitos
4.
Proc Natl Acad Sci U S A ; 117(39): 24384-24391, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32913053

RESUMO

An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8+ T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2-specific CD8+ and CD4+ T cells in vitro, with CD4+ T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8+ T cell epitopes, A2/S269-277 and A2/Orf1ab3183-3191 Using peptide-HLA tetramer enrichment, direct ex vivo assessment of A2/S269 +CD8+ and A2/Orf1ab3183 +CD8+ populations indicated that A2/S269 +CD8+ T cells were detected at comparable frequencies (∼1.3 × 10-5) in acute and convalescent HLA-A*02:01+ patients. These frequencies were higher than those found in uninfected HLA-A*02:01+ donors (∼2.5 × 10-6), but low when compared to frequencies for influenza-specific (A2/M158) and Epstein-Barr virus (EBV)-specific (A2/BMLF1280) (∼1.38 × 10-4) populations. Phenotyping A2/S269 +CD8+ T cells from COVID-19 convalescents ex vivo showed that A2/S269 +CD8+ T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8+ T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/S269 +CD8+ T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8+ T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8+ T cell immunity in COVID-19.


Assuntos
Betacoronavirus/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/imunologia , Antígeno HLA-A2/imunologia , Pneumonia Viral/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T , Feminino , Humanos , Memória Imunológica , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Pandemias , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Proteínas Virais/química , Proteínas Virais/imunologia
5.
Anticancer Res ; 40(9): 4875-4883, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878775

RESUMO

BACKGROUND/AIM: Some reports showed encouraging efficacy of immune checkpoint inhibitors among patients who experienced immune-related adverse events (irAEs). Thus, characterization of T-cell repertoire and immune signatures in peripheral blood mononuclear cells (PBMCs) and tumors before and after immune checkpoint inhibitors treatment should contribute to better understanding of irAE-provoked anticancer immune responses. MATERIALS AND METHODS: We applied expression analysis of immune-related genes and T-cell receptor sequencing in tumor and PBMCs from five patients with renal cell carcinoma before combined immunotherapy and at the onset of severe irAEs. RESULTS: We found that the cluster of differentiation 8 (CD8)/forkhead box P3(FOXP3), granzyme B(GZMB)/CD3, perforin 1(PRF1)/CD3 and programmed cell death 1(PD1)/CD8 expression ratios were significantly elevated in PBMCs at the onset of irAEs. In addition, we found expansion of certain T-cell clones in metastatic tissue after irAEs, which were already increased in peripheral blood at the onset of irAEs. CONCLUSION: irAE-provoked T-cells may also circulate and attack distant tumors, leading to durable response in patients with irAEs.


Assuntos
Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/terapia , Imunoterapia/efeitos adversos , Neoplasias Renais/imunologia , Neoplasias Renais/terapia , Idoso , Antineoplásicos Imunológicos/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/imunologia , Feminino , Perfilação da Expressão Gênica , Genes Codificadores dos Receptores de Linfócitos T/genética , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia
6.
Signal Transduct Target Ther ; 5(1): 186, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32883951

RESUMO

Sterol regulatory element binding protein-2 (SREBP-2) is activated by cytokines or pathogen, such as virus or bacteria, but its association with diminished cholesterol levels in COVID-19 patients is unknown. Here, we evaluated SREBP-2 activation in peripheral blood mononuclear cells of COVID-19 patients and verified the function of SREBP-2 in COVID-19. Intriguingly, we report the first observation of SREBP-2 C-terminal fragment in COVID-19 patients' blood and propose SREBP-2 C-terminal fragment as an indicator for determining severity. We confirmed that SREBP-2-induced cholesterol biosynthesis was suppressed by Sestrin-1 and PCSK9 expression, while the SREBP-2-induced inflammatory responses was upregulated in COVID-19 ICU patients. Using an infectious disease mouse model, inhibitors of SREBP-2 and NF-κB suppressed cytokine storms caused by viral infection and prevented pulmonary damages. These results collectively suggest that SREBP-2 can serve as an indicator for severity diagnosis and therapeutic target for preventing cytokine storm and lung damage in severe COVID-19 patients.


Assuntos
Betacoronavirus/patogenicidade , Colesterol/biossíntese , Infecções por Coronavirus/genética , Síndrome da Liberação de Citocina/genética , Interações Hospedeiro-Patógeno/genética , Leucócitos Mononucleares/imunologia , Pneumonia Viral/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Betacoronavirus/imunologia , Estudos de Casos e Controles , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/virologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/mortalidade , Síndrome da Liberação de Citocina/virologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Unidades de Terapia Intensiva , Interleucina-1beta/genética , Interleucina-1beta/imunologia , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/virologia , NF-kappa B/genética , NF-kappa B/imunologia , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/mortalidade , Pneumonia Viral/virologia , Cultura Primária de Células , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/imunologia , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/imunologia , Análise de Sobrevida , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
7.
Sci Adv ; 6(31)2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32937590

RESUMO

The outbreak of the highly contagious and deadly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as coronavirus disease 2019 (COVID-19), has posed a serious threat to public health across the globe, calling for the development of effective diagnostic markers and therapeutics. Here, we report a highly reliable severity diagnostic biomarker, acetylated 676th lysine transforming growth factor-beta-induced protein (TGFBIp K676Ac). TGFBIp K676Ac was consistently elevated in the blood of patients with SARS-CoV-2 pneumonia (n = 113), especially in patients in the intensive care unit (ICU) compared to non-ICU patients. Patients' blood samples showed increased cytokines and lymphopenia, which are exemplary indicators of SARS-CoV-2 pneumonia. Treatment with TGFBIp neutralizing antibodies suppressed the cytokine storm. The increased level of TGFBIp K676Ac in ICU patients suggests the promise of this protein as a reliable severity diagnostic biomarker for severe SARS-CoV-2 disease.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/diagnóstico , Síndrome da Liberação de Citocina/diagnóstico , Proteínas da Matriz Extracelular/imunologia , Leucócitos Mononucleares/imunologia , Pneumonia Viral/diagnóstico , Processamento de Proteína Pós-Traducional , Insuficiência Respiratória/diagnóstico , Fator de Crescimento Transformador beta/imunologia , Acetilação , Anticorpos Neutralizantes/farmacologia , Betacoronavirus/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Infecções por Coronavirus/sangue , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Síndrome da Liberação de Citocina/sangue , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/patologia , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/genética , Expressão Gênica , Humanos , Unidades de Terapia Intensiva , Contagem de Leucócitos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/virologia , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/virologia , Lisina/metabolismo , NF-kappa B/genética , NF-kappa B/imunologia , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Cultura Primária de Células , Prognóstico , Insuficiência Respiratória/sangue , Insuficiência Respiratória/imunologia , Insuficiência Respiratória/patologia , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética
8.
Proc Natl Acad Sci U S A ; 117(37): 22984-22991, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868431

RESUMO

Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2' and 3' positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion.


Assuntos
Aciltransferases/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Lipídeo A/imunologia , Yersinia pestis/imunologia , Animais , Evolução Biológica , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único/imunologia , Células THP-1/imunologia , Células U937 , Yersinia pseudotuberculosis/imunologia
9.
PLoS One ; 15(9): e0237496, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881912

RESUMO

BACKGROUND: Obesity is often associated with inflammation in adipose tissue (AT) with release of mediators of atherogenesis. We postulated that it would be feasible to collect sufficient abdominal AT to quantify changes in a broad array of adaptive and innate mononuclear white cells in obese non-diabetic adults in response to a dipeptidyl protease inhibitor (DPP4i), known to inhibit activation of immune white cells. METHODS: Adults 18-55 years-of-age were screened for abdominal obesity and insulin resistance or impaired glucose tolerance but without known inflammatory conditions. Twenty-one eligible participants consented for study and were randomized 3:1 to receive sitagliptin (DPP4i) at 100mg or matching placebo daily for 28 days. Abdominal AT collected by percutaneous biopsy and peripheral blood mononuclear cell fractions were evaluated before and after treatment; plasma was stored for batch testing. RESULTS: Highly sensitive C-reactive protein, a global marker of inflammation, was not elevated in the study population. Innate lymphoid cells (ILC) type 3 (ILC-3) in abdominal AT decreased with active treatment compared with placebo (p = 0.04). Other immune white cells in AT and peripheral blood mononuclear cell (PBMC) fractions did not change with treatment compared to placebo (p>0.05); although ILC-2 declined in PBMCs (p = 0.007) in the sitagliptin treatment group. Two circulating biomarkers of atherogenesis, interferon-inducible protein-10 (IP-10) and sCD40L declined in plasma (p = 0.02 and p = 0.07, respectively) in the active treatment group, providing indirect validation of a net reduction in inflammation. CONCLUSIONS: In this pilot study, two cell types of the innate lymphoid system, ILC-3 in AT and ILC-2 PBMCs declined during treatment and as did circulating biomarkers of atherogenesis. Changes in other immune cells were not demonstrable. The study showed that sufficient abdominal AT could be obtained to quantify white cells of both innate and adaptive immunity and to demonstrate changes during therapy with an immune inhibitor. TRIAL REGISTRATION: ClinicalTrials.gov identifier (NCT number): NCT02576.


Assuntos
Gordura Abdominal/patologia , Imunidade Inata , Leucócitos Mononucleares/patologia , Obesidade/imunologia , Adulto , Biomarcadores/sangue , Estudos de Viabilidade , Feminino , Citometria de Fluxo , Humanos , Linfócitos/patologia , Masculino , Pessoa de Meia-Idade , Obesidade/sangue , Fosfato de Sitagliptina/farmacologia , Resultado do Tratamento
10.
Anticancer Res ; 40(10): 5329-5341, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988851

RESUMO

Investigation of the efficacy and mechanisms of human immuno-oncology agents has been hampered due to species-specific differences when utilizing preclinical mouse models. Peripheral blood mononuclear cell (PBMC) humanized mice provide a platform for investigating the modulation of the human immune-mediated antitumor response while circumventing the limitations of syngeneic model systems. Use of humanized mice has been stymied by model-specific limitations, some of which include the development of graft versus host disease, technical difficulty and cost associated with each humanized animal, and insufficient engraftment of some human immune subsets. Recent advances have addressed many of these limitations from which have emerged humanized models that are more clinically relevant. This review characterizes the expanded usage, advantages and limitations of humanized mice and provides insights into the development of the next generation of murine humanized models to further inform clinical applications of cancer immunotherapeutic agents.


Assuntos
Imunidade Celular/efeitos dos fármacos , Imunoterapia , Leucócitos Mononucleares/imunologia , Neoplasias/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Modelos Animais de Doenças , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos , Neoplasias/imunologia , Neoplasias/patologia
11.
Anticancer Res ; 40(10): 5355-5359, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988854

RESUMO

BACKGROUND/AIM: Recent studies indicate that chimeric antigen receptor (CAR)-T-cells seem to be superior to CAR modified NK-92 cells. One, at least partial, explanation to this discrepancy has been addressed herein, by having NK-92 cells as target cells in cytotoxicity reactions using peripheral blood mononuclear cells. MATERIALS AND METHODS: A time-resolved fluorometric assay (TDA-labeled NK-92 or K562 as target cells) was used for measuring the cytotoxic activity of blood mononuclear cells (PBMC). RESULTS: The cytotoxic capacity of the NK-92 cells was initially demonstrated by their ability to efficiently kill K562 cells. Interestingly, having PBMC as effector cells rendered the very same NK-92 cells sensitive to NK-cell mediated cytolysis. A 1:100 target:effector ratio gave 34.1% lysis compared to 72.2% lysis for K562 cells. Incubating PBMC for longer times (24 up to 48 h) potentiated their NK-activity against NK-92 cells even more, reaching a level close to that obtained with K562 cells. CONCLUSION: This study pinpoints a severe problem that has to be considered in future immune-based cancer therapies with NK-92 as well as CAR-transduced NK-92 cells.


Assuntos
Imunoterapia Adotiva , Células Matadoras Naturais/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Neoplasias/terapia , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Células K562 , Neoplasias/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia
12.
Zhonghua Bing Li Xue Za Zhi ; 49(10): 1041-1045, 2020 Oct 08.
Artigo em Chinês | MEDLINE | ID: mdl-32992420

RESUMO

Objective: To observe the expression level of histone demethyltransferase Jmjd3 in patients with pre-eclampsia (PE), and to investigate the possible mechanism of its epigenetic modification in regulating Th1/Th2 imbalance in PE patients. Methods: The mRNA levels of histone demethyltransferase Jmjd3 from peripheral blood mononuclear cells (PBMC) of PE patients and normal pregnant women were detected by RT-PCR. Peripheral serum IFN-γ and IL-4  were detected by ELISA. RT-PCR was used to detect the mRNA levels of Jmjd3, Tbx21 and Cxcr3 in the spleen of PE and control mice. Immunomagnetic beads were used to sort out the initial CD4+ T cells in the spleen of control and PE mice. Western blot was used to detect H3K27me1 and H3K27me3 levels. ChIP analysis was used for H3K27me3 demethylation modification in spleens of PE mice. Results: Compared with normal pregnant women, the mRNA level of Jmjd3 in PBMC of PE patients was significantly increased, the level of IFN-γ in serum was significantly increased, and the level of IL-4 was significantly reduced (P<0.01). Compared with normal control mice, the mRNA level of Jmjd3 in the spleen of PE mice was significantly increased, and the expression of Tbx21 and Cxcr3 was significantly increased in PE mice (P<0.01); the H3K27me3 level of CD4+ T cells in PE mice was significantly reduced (P<0.05), but H3K27me1 was not changed. ChIP analysis showed that CD4+ T cells H3K27me3 in PE group mice were in the Ifng promoter region, compared with control mice. Recruitment was significantly reduced, while recruitment in the promoter region of Il4 was significantly increased (P<0.01). Conclusions: In both PE patients and mice with PE model, the relative expression level of histone demethyltransferase Jmjd3 is significantly up-regulated, which further induces the demethylation of H3K27me3 in the Ifng promoter region and promotes the initial CD4+ T cells to Th1 cell differentiation and development, leading to an imbalance of Th1/Th2, which may be one of the important reasons for the development of preeclampsia.


Assuntos
Histonas , Histona Desmetilases com o Domínio Jumonji/fisiologia , Pré-Eclâmpsia/genética , Animais , Epigênese Genética , Feminino , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Leucócitos Mononucleares , Camundongos , Gravidez
13.
PLoS One ; 15(8): e0238132, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32853221

RESUMO

Bears do not suffer from osteoporosis during hibernation, which is associated with long-term inactivity, lack of food intake, and cold exposure. However, the mechanisms involved in bone loss prevention have scarcely been elucidated in bears. We investigated the effect of serum from hibernating Japanese black bears (Ursus thibetanus japonicus) on differentiation of peripheral blood mononuclear cells (PBMCs) to osteoclasts (OCs). PBMCs collected from 3 bears were separately cultured with 10% serum of 4 active and 4 hibernating bears (each individual serum type was assessed separately by a bear PBMCs), and differentiation were induced by treatment with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). PBMCs that were cultured with the active bear serum containing medium (ABSM) differentiated to multi-nucleated OCs, and were positive for TRAP stain. However, cells supplemented with hibernating bear serum containing medium (HBSM) failed to form OCs, and showed significantly lower TRAP stain (p < 0.001). On the other hand, HBSM induced proliferation of adipose derived mesenchymal stem cells (ADSCs) similarly to ABSM (p > 0.05), indicating no difference on cell growth. It was revealed that osteoclastogenesis of PBMCs is hindered by HBSM, implying an underlying mechanism for the suppressed bone resorption during hibernation in bears. In addition, this study for the first time showed the formation of bears' OCs in-vitro.


Assuntos
Hibernação/fisiologia , Osteoclastos/fisiologia , Osteogênese/fisiologia , Ursidae/fisiologia , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/fisiopatologia , Diferenciação Celular/fisiologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/fisiologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Osteoclastos/metabolismo , Osteoporose/metabolismo , Osteoporose/fisiopatologia , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Ursidae/metabolismo
14.
PLoS One ; 15(8): e0236338, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785215

RESUMO

Dysregulation of BCL2 is a pathophysiology observed in haematological malignancies. For implementation of available treatment-options it is preferred to know the relative quantification of BCL2 mRNA with appropriate reference genes. For the choice of reference genes-(i) Reference Genes were selected by assessing variation of >60,000 genes from 4 RNA-seq datasets of haematological malignancies followed by filtering based on their GO biological process annotations and proximity of their chromosomal locations to known disease translocations. Selected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using geNorm, NormFinder, BestKeeper and RefFinder. (ii) 43 commonly used Reference Genes were obtained from literature through extensive systematic review. Levels of BCL2 mRNA was assessed by qPCR normalized either by novel reference genes from this study or GAPDH, the most cited reference gene in literature and compared. The analysis showed PTCD2, PPP1R3B and FBXW9 to be the most unregulated genes across lymph-nodes, bone marrow and PBMC samples unlike the Reference Genes used in literature. BCL2 mRNA level shows a consistent higher expression in haematological malignancy patients when normalized by these novel Reference Genes as opposed to GAPDH, the most cited Reference Gene. These reference genes should also be applicable in qPCR platforms using Taqman probes and other model systems including cell lines and rodent models. Absence of sample from healthy-normal individual in diagnostic cases call for careful selection of Reference Genes for relative quantification of a biomarker by qPCR.BCL2 can be used as molecular diagnostics only if normalized with a set of reference genes with stable yet low levels of expression across different types of haematological malignancies.


Assuntos
Biomarcadores Tumorais/isolamento & purificação , Neoplasias Hematológicas/diagnóstico , Proteínas Proto-Oncogênicas c-bcl-2/isolamento & purificação , RNA Mensageiro/isolamento & purificação , RNA-Seq/normas , Animais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Medula Óssea/patologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Estudos de Viabilidade , Regulação Neoplásica da Expressão Gênica , Genes Essenciais , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Leucócitos Mononucleares , Proteínas Proto-Oncogênicas c-bcl-2/sangue , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/sangue , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência
15.
Science ; 369(6508): 1210-1220, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32788292

RESUMO

Coronavirus disease 2019 (COVID-19) represents a global crisis, yet major knowledge gaps remain about human immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We analyzed immune responses in 76 COVID-19 patients and 69 healthy individuals from Hong Kong and Atlanta, Georgia, United States. In the peripheral blood mononuclear cells (PBMCs) of COVID-19 patients, we observed reduced expression of human leukocyte antigen class DR (HLA-DR) and proinflammatory cytokines by myeloid cells as well as impaired mammalian target of rapamycin (mTOR) signaling and interferon-α (IFN-α) production by plasmacytoid dendritic cells. By contrast, we detected enhanced plasma levels of inflammatory mediators-including EN-RAGE, TNFSF14, and oncostatin M-which correlated with disease severity and increased bacterial products in plasma. Single-cell transcriptomics revealed a lack of type I IFNs, reduced HLA-DR in the myeloid cells of patients with severe COVID-19, and transient expression of IFN-stimulated genes. This was consistent with bulk PBMC transcriptomics and transient, low IFN-α levels in plasma during infection. These results reveal mechanisms and potential therapeutic targets for COVID-19.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Citocinas/sangue , DNA Bacteriano/sangue , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Citometria de Fluxo , Antígenos HLA-DR/análise , Humanos , Imunidade , Imunidade Inata , Imunoglobulinas/sangue , Imunoglobulinas/imunologia , Mediadores da Inflamação/sangue , Interferon Tipo I/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/sangue , Masculino , Células Mieloides/imunologia , Células Mieloides/metabolismo , Pandemias , Transdução de Sinais , Análise de Célula Única , Biologia de Sistemas , Serina-Treonina Quinases TOR/metabolismo , Transcrição Genética , Transcriptoma
16.
Immunity ; 53(3): 685-696.e3, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32783921

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic poses a current world-wide public health threat. However, little is known about its hallmarks compared to other infectious diseases. Here, we report the single-cell transcriptional landscape of longitudinally collected peripheral blood mononuclear cells (PBMCs) in both COVID-19- and influenza A virus (IAV)-infected patients. We observed increase of plasma cells in both COVID-19 and IAV patients and XIAP associated factor 1 (XAF1)-, tumor necrosis factor (TNF)-, and FAS-induced T cell apoptosis in COVID-19 patients. Further analyses revealed distinct signaling pathways activated in COVID-19 (STAT1 and IRF3) versus IAV (STAT3 and NFκB) patients and substantial differences in the expression of key factors. These factors include relatively increase of interleukin (IL)6R and IL6ST expression in COVID-19 patients but similarly increased IL-6 concentrations compared to IAV patients, supporting the clinical observations of increased proinflammatory cytokines in COVID-19 patients. Thus, we provide the landscape of PBMCs and unveil distinct immune response pathways in COVID-19 and IAV patients.


Assuntos
Infecções por Coronavirus/imunologia , Citocinas/imunologia , Influenza Humana/imunologia , Leucócitos Mononucleares/imunologia , Pneumonia Viral/imunologia , Transdução de Sinais/imunologia , Betacoronavirus/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Pandemias
17.
Cells ; 9(9)2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32847031

RESUMO

Following influenza infection, rs2248374-G ERAP2 expressing cells may transcribe an alternative spliced isoform: ERAP2/Iso3. This variant, unlike ERAP2-wt, is unable to trim peptides to be loaded on MHC class I molecules, but it can still dimerize with both ERAP2-wt and ERAP1-wt, thus contributing to profiling an alternative cellular immune-peptidome. In order to verify if the expression of ERAP2/Iso3 may be induced by other pathogens, PBMCs and MDMs isolated from 20 healthy subjects were stimulated with flu, LPS, CMV, HIV-AT-2, SARS-CoV-2 antigens to analyze its mRNA and protein expression. In parallel, Calu3 cell lines and PBMCs were in vitro infected with growing doses of SARS-CoV-2 (0.5, 5, 1000 MOI) and HIV-1BAL (0.1, 1, and 10 ng p24 HIV-1Bal/1 × 106 PBMCs) viruses, respectively. Results showed that: (1) ERAP2/Iso3 mRNA expression can be prompted by many pathogens and it is coupled with the modulation of several determinants (cytokines, interferon-stimulated genes, activation/inhibition markers, antigen-presentation elements) orchestrating the anti-microbial immune response (Quantigene); (2) ERAP2/Iso3 mRNA is translated into a protein (western blot); (3) ERAP2/Iso3 mRNA expression is sensitive to SARS-CoV-2 and HIV-1 concentration. Considering the key role played by ERAPs in antigen processing and presentation, it is conceivable that these enzymes may be potential targets and modulators of the pathogenicity of infectious diseases and further analyses are needed to define the role played by the different isoforms.


Assuntos
Aminopeptidases/genética , Betacoronavirus/imunologia , Infecções por Coronavirus/genética , Imunização/métodos , Leucócitos Mononucleares/virologia , Macrófagos/virologia , Pneumonia Viral/genética , Isoformas de Proteínas/genética , Apresentação do Antígeno/genética , Doadores de Sangue , Linhagem Celular Tumoral , Infecções por Coronavirus/virologia , Expressão Gênica/imunologia , Genótipo , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Pandemias , Pneumonia Viral/virologia , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/imunologia
18.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(4): 1338-1343, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32798423

RESUMO

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.


Assuntos
MicroRNAs , Púrpura Trombocitopênica Idiopática , Idoso , Biomarcadores Tumorais , Humanos , Estimativa de Kaplan-Meier , Leucócitos Mononucleares , Recidiva Local de Neoplasia , Prognóstico
19.
BMC Infect Dis ; 20(1): 590, 2020 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778058

RESUMO

BACKGROUND: Antiviral therapy is recommended for patients with immune-active chronic hepatitis B (CHB) to decrease the risk of liver-related complications. However, the outcomes of the pegylated IFN-α (PEG-IFN-α) therapy vary among CHB patients. We aimed to identify factors that can influence the outcomes in CHB patients who received antiviral PEG-IFN-α monotherapy. METHODS: Thirty-two CHB patients who received PEG-IFN-α monotherapy were enrolled in this study. All of the patients underwent two liver biopsies at baseline and 6 months after the initiation of the therapy. CD8+ T cells, CD4+ T cells, CD68+ mononuclear cells, and PD-1 levels in the 64 liver biopsy specimens were examined via immunofluorescence. RESULTS: The overall median frequency of CD8+ T cells in the liver tissues of 32 CHB patients significantly decreased at 6 months after the therapy initiation (p < 0.01). In the FIER (fibrosis and inflammation response with HBeAg seroconversion) group, CD8+PD-1+ T cells significantly decreased at 6 months (p < 0.05), while CD8+PD-1- T cells had no significant difference. On the contrary, in the FIENR (no fibrosis and inflammation response and HBeAg seroconversion) group, CD8+PD-1- T cells significantly decreased after 6 months of PEG-IFN-α treatment (p < 0.05), while CD8+PD-1+ T cells had no significant difference. In addition, the levels of CD68+ mononuclear cells in the FIER group showed an overall increasing trend after treatment (p < 0.05). CONCLUSIONS: The changes in the levels of CD8+PD-1+ T cells and CD68+ mononuclear cells may be related to the response to PEG-IFN-α therapy.


Assuntos
Antivirais/uso terapêutico , Linfócitos T CD8-Positivos/metabolismo , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Fígado/patologia , Polietilenoglicóis/uso terapêutico , Adulto , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Antígenos E da Hepatite B/sangue , Antígenos E da Hepatite B/imunologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Fígado/metabolismo , Masculino , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Recombinantes/uso terapêutico , Adulto Jovem
20.
Artigo em Chinês | MEDLINE | ID: mdl-32842211

RESUMO

Objective:To study the carotid intima-media thickness (IMT) level of OSA patients of different severity, the proportion of Th17 cells in peripheral blood and the mRNA level of nuclear transfer factor RORγt, and to explore the relationship between Th17 cells and atherosclerosis in OSA patients. Method:Sixty-two patients who underwent respiratory and sleep monitoring were selected and divided into three groups according to the AHI index: 15 patients in the normal control group(AHI<5), there were 22 cases in the mild group(AHI 5-15) and 25 cases in the moderate and severe group(AHI≥15). Carotid intima-media thickness(IMT) was measured in all subjects, the ratio of Th17 cells in peripheral blood monouelear cells(PBMC) were analysed by flow cytometry. The expression of RORγt mRNA were detected by real-time polymerase chain reaction. Result:The carotid IMT of patients in the normal control group, the mild OSA group and the moderate to severe OSA group were (0.74±0.21) mm, (1.09±0.23) mm and (1.60±0.30) mm, respectively. The moderate to severe group was higher than the mild group and the normal control group, the difference was statistically significant (P<0.01). The proportion of Th17 in peripheral blood of the three groups was(2.54±0.20)%, (4.34±0.30)%, and (8.27±0.31)%, respectively. The moderate to severe group was significantly higher than the mild group and the control group (P<0.01), the difference was statistically significant. The relative mRNA expression levels of RORγt in the three groups were 0.92±0.24, 2.60±0.59, and 4.93±0.72, respectively. The moderate to severe group was significantly higher than the mild group and the control group (P<0.01), the difference was statistically significant. The proportion of Th17 in peripheral blood and the relative expression of RORvt mRNA were positively correlated with the carotid IMT (r value was 0.80, 0.78, respectively, all P<0.01). Conclusion:Th17 cells differentiation is increased in OSA patients, and Th17 cells are correlated with indicators reflecting the progression of atherosclerosis. Th17 cells may be involved in the development of atherosclerosis in OSA patients.


Assuntos
Aterosclerose , Apneia Obstrutiva do Sono , Espessura Intima-Media Carotídea , Humanos , Leucócitos Mononucleares , Células Th17
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