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1.
Zhonghua Nei Ke Za Zhi ; 59(3): 200-206, 2020 Mar 01.
Artículo en Chino | MEDLINE | ID: mdl-32146746

RESUMEN

Objective: To investigate the characteristics and prognostic value of peripheral blood T lymphocyte subsets in patients with severe influenza. Methods: This was a single-center cross-sectional study in influenza patients admitted to Peking Union Medical College Hospital from August 2017 to April 2018. Peripheral blood lymphocyte subsets were detected by flow cytometry in both patients and 108 healthy controls. Influenza patients were divided into mild group and severe group. Severe patients were further classified into alive and fatal subgroups. Results: A total of 42 influenza patients were recruited in this study, including 24 severe cases (6 deaths). The remaining 18 cases were mild. The peripheral blood lymphocyte counts and lymphocyte subset counts (B, NK, CD4(+)T, CD8(+)T) in either mild patients[795 (571,1 007), 43 (23,144), 70 (47,135), 330 (256,457), 226 (148,366) cells/µl respectively] or severe patients[661 (474,1 151),92 (52,139), 54 (34,134), 373 (235,555), 180 (105,310) cells/µl respectively] were both significantly lower than those of healthy controls [1 963 (1 603,2 394),179 (119,239), 356 (231,496), 663 (531,824), 481 (341,693) cells/µl respectively]. Meanwhile, the T cells and CD8(+)T counts in fatal patients [370 (260,537) cells/µl and 87 (74,105) cells/µl] were significantly lower than those in severe and alive patients [722 (390,990) cells/µl and 222 (154,404) cells/µl]. CD8(+)HLA-DR/CD8(+)and CD8(+)CD38(+)/CD8(+)T cell activating subgroups in mild cases[(53.7±19.2)% and 74.8% (64.1%,83.7%) respectively] were significantly higher than those in severe cases[(38.5±21.7)% and 53.3% (45.3%,67.2%) respectively].Moreover,CD8(+)HLA-DR/CD8(+)count in severe and alive group was higher than that in fatal group [(46.1±19.1)% vs. (18.2±14.6)%, P<0.01]. Logistic regression analysis showed that CD8(+)T cell count (OR=0.952, 95%CI 0.910-0.997, P=0.035) and CD8(+)HLA-DR/CD8(+)T (OR=0.916, 95%CI 0.850-0.987, P=0.022) were both negatively correlated with mortality.Peripheral blood lymphocyte counts in mild cases rapidly decreased within 1 day after diagnosis, and returned to the basic level one week later. Conclusions: All peripheral blood lymphocyte subsets (T,B,NK) in patients with influenza are significantly reduced. These findings are consistent with the immunological characteristics of respiratory viral infections, in which peripheral lymphocytes (especially T cells) migrate to respiratory tract in the early stage and circulate to the peripheral blood after recovery. The activated CD8(+)T cell counts in peripheral blood are negatively correlated with the severity of disease, which could be considered as a prognostic indicator of severe influenza.


Asunto(s)
Gripe Humana/diagnóstico , Gripe Humana/inmunología , Subgrupos de Linfocitos T/citología , Linfocitos T CD8-positivos/citología , Estudios de Casos y Controles , Estudios Transversales , Citometría de Flujo , Humanos , Recuento de Linfocitos , Pronóstico , Índice de Severidad de la Enfermedad
2.
Life Sci ; 245: 117390, 2020 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-32007574

RESUMEN

AIMS: This study aimed to profile circulating T follicular helper cells (cTfh) and their effect on B cells in rheumatic heart disease (RHD). MATERIALS AND METHODS: Participants were divided into healthy control (HC, n = 30) and RHD (n = 30) groups. Percentages of cTfh subpopulations, based on CD4, CXCR5, CXCR3, CCR6, Foxp3, Ki67, and PD-1 cell markers, and of CD19+ B cell subgroups were measured by flow cytometry and compared between the groups. Also, IL-21 concentration in plasma and mitral valve were quantitated by cytometric bead array, immunofluorescence, and western blotting. KEY FINDINGS: The PD-1+ cTfh, B cells (naive B cells, plasmablasts, and plasma B cells) proportion and cTfh17/cTfh ratios in RHD group were significantly increased, compared to HC (p < 0.01 in all cases), while different types of memory B cells were diminished (p < 0.001). In RHD patients, percentages of PD-1+ cTfh and switched memory B cells were negatively correlated (r = -0.565, p = 0.009); meanwhile, percentages of plasmablasts and PD-1+ cTfh cells were positively correlated (r = 0.594, p = 0.005). Additionally, IL-21 levels in plasma and mitral valve of RHD group were higher than those in HC. Also, IL-21 levels correlated with PD-1+ cTfh(r = 0.557, p = 0.010), cTfh17 (r = 0.567, p = 0.009), and plasmablast (r = -0.5957, p = 0.005) cell proportions, and (cTh2 + cTh17)/cTfh1 ratio (r = -0.547, p = 0.013). SIGNIFICANCE: The activation of PD-1+ cTfh and cTfh17 subtype was highly correlated with plasmablast maturation and IL-21 production in rheumatic heart disease. Thus indicating the prominent role of cTfh and humoral reactivity in the immune pathogenesis of RHD.


Asunto(s)
Inmunidad Humoral , Cardiopatía Reumática/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos B/fisiología , Western Blotting , Estudios de Casos y Controles , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Persona de Mediana Edad , Cardiopatía Reumática/etiología , Linfocitos T Colaboradores-Inductores/fisiología
3.
Life Sci ; 245: 117387, 2020 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-32007575

RESUMEN

The aim of this study was to investigate the inhibition of daidzein or/and regular exercise on breast cancer and to reveal the potential biological mechanisms. BALB/c mice pretreated with regular exercise training for 20 days (15 m/min, 60 min/d) were orthotopically transplanted with mouse breast cancer cells (4T1), and then treated with daidzein (145 mg/kg) by gavage for another 22 days. Results showed that exercise or daidzein inhibited tumor growth in mice to a different degree. Particularly, co-treatment with exercise and daidzein showed an obviously synergistic inhibition on the tumor growth (P < 0.01), compared with the tumor control. Further researches indicated that the combination of exercise and daidzein synergistically mobilized and redistributed natural killer cells through upregulating the level of epinephrine and interleukin-6. Moreover, exercise combined with daidzein induces apoptosis in cancer cells via Fas/FasL-initiated mitochondrial apoptosis signaling pathway. These results suggested that regular exercise combined with daidzein may explore a candidate way to prevent and treat the breast cancer.


Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/terapia , Terapia por Ejercicio/métodos , Isoflavonas/uso terapéutico , Células Asesinas Naturales/efectos de los fármacos , Animales , Western Blotting , Línea Celular Tumoral , Terapia Combinada , Femenino , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias
4.
Life Sci ; 246: 117396, 2020 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-32035130

RESUMEN

AIMS: Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide. Decrease in NKG2D ligand levels and exhaustion of NK cells in HCC patients are major causes of immune escape, high recurrence, poor prognosis, and low overall survival. Enhancing the susceptibility of HCC to NK cells by upregulating NKG2DLs on tumor cells is an effective treatment strategy. This study aimed to identify the effect of the Anterior gradient 2 (AGR2)-derived peptide P1, which was reported to bind to HLA-A*0201 as an epitope, on both the expression of major histocompatibility complex class I-related chains A/B (MICA/B) on HCC cells and the cytotoxicity of NK cells. MAIN METHODS: The effect of P1 on MICA/B expression on HCC cells was determined by qRT-PCR, western blotting, and flow cytometry analysis. HCC cells were pre-treated with various pathway inhibitors to identify the molecular pathways associated with P1 treatment. The cytotoxicity of NK cells toward HCC was investigated by LDH cytotoxicity assay. The tumor-suppression effect of P1 was determined in vivo using a NOD/SCID mice HCC model. KEY FINDINGS: P1 significantly increased MICA/B expression on HCC cells, thereby enhancing their susceptibility to the cytotoxicity of NK cells in vitro and in vivo. Further, p38 MAPK cell signaling pathway inhibitor SB203580 significantly attenuated the effects of P1 in vivo and in vitro. SIGNIFICANCE: P1 upregulates MICA and MICB expression on HCC cells, thereby promoting their recognition and elimination by NK cells, which makes P1 an attractive novel immunotherapy agent.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Neoplasias Hepáticas/metabolismo , Mucoproteínas/fisiología , Proteínas Oncogénicas/fisiología , Animales , Western Blotting , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Interferón gamma/metabolismo , Ratones Endogámicos NOD , Ratones SCID , Mucoproteínas/metabolismo , Trasplante de Neoplasias , Proteínas Oncogénicas/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia Arriba
5.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(1): 218-224, 2020 Feb.
Artículo en Chino | MEDLINE | ID: mdl-32027280

RESUMEN

OBJECTIVE: To study the level and clinical value of Th17 cell subset in the peripheral blood of the patients with aplastic anemia (AA). METHODS: Eighty-five patients with aplastic anemia in our hospital from May 2017 to February 2018 were selected. Among them, 51 patients with poor curative effect were enrolled in group A. and the 34 patients with good curative effect were enrolled in group B, the 35 healthy persons were selected as controls. The levels of serum IL-17, IL-6, IL-4 and IFN-ß were analyzed by ELISA; the levels of peripheral blood Th17 cell subset were analyzed by flow cytometry; the expression levels of RORγt and STAT3 mRNA in lymphocytes were analyzed by RT-PCR; the expression levels of RORγt and STAT3 protein in lymphocytes were analyzed by Western blot. RESULTS: There was no significant difference in sex, age, WBC count and complications between group A and group B (P>0.05). Non-severe aplastic anemia (NSAA) in group B accounted for 23 cases, and the NSAA ratio (23/34) was significantly higher than that in group A (20/51) (P<0.05). The Hb level (86.25±7.9 g/L) and Plt count (54.7 ± 6.3×109/L) in group B were significantly higher than those in group A (P<0.05). ELISA showed that the levels of IL-17 and IL-6 in group A were significantly higher than those in control group (P<0.05); the levels of IL-17 and IL-6 in group B were significantly lower than those in group A (P<0.05); the levels of IL-4 and IFN-ß in group A were significantly lower than those in control group (P<0.05); the levels of IL-4 and IFN-ßin group B were significantly higher than those in group A (P<0.05). Flow cytometry showed that the proportion of Th17 cells in peripheral blood of group A was higher than that of group B (P<0.05). RT-PCR showed that the levels of RORγt and STAT3 mRNA in group A were significantly higher than those in control group (P<0.05), and the mRNA levels of RORγt and STAT3 in group B were significantly lower than those in group A (P<0.05). Western blot showed that the protein levels of RORγt and STAT3 in group A were significantly higher than those in control group (P<0.05), and the protein levels of RORγt and STAT3 in group B were significantly lower than those in group A (P<0.05). CONCLUSION: The Th17 cells in peripheral blood of AA patients increase, and the inflammatory reaction in the patients are enhanced. It may be related with the rise of RORγt and STAT3 gene expression, which provides a research direction for clinical treatment of AA.


Asunto(s)
Anemia Aplásica , Células Th17 , Citometría de Flujo , Humanos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , ARN Mensajero
6.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(1): 235-241, 2020 Feb.
Artículo en Chino | MEDLINE | ID: mdl-32027283

RESUMEN

OBJECTIVE: To establish a novel flow cytometric immunobead array (FCIA) for detecting plasma von Willebrand factor antigen (vWF:Ag), and to analyze the clinical value of FCIA in predicting the prognosis of patients with ischemic stroke (IS). METHODS: Anti-human vWF monoclonal antibody SZ29 IgG was coated on microspheres overnight, the diluted plasma was added after blocking, then incubated with FITC-conjugated sheep-anti-human vWF IgG polyclonal antibody, and finally detected by flow cytometry. The plasma vWF in 21 case of von Willebrand disease (vWD) and 105 controls (CTL) were detected by FCIA and ELISA, so as to carry out methodological assessment. Plasma vWF:Ag of 61 IS patients was detected by FCIA and the data of prognosis followed-up for 2-year were collected. RESULTS: The linear fitting of FCIA was good (R2=0.99) without significant difference between FCIA and ELISA. The Bland-Altman bias was 1.12% with 95% limits of agreement that spanned from -45.06% to 47.30%, and the slope of the linear regression was 0.97 (r=0.86, P<0.01). Importantly, the FCIA method was faster than ELISA, and superior to the ELISA in the detection of low levels of vWF:Ag. The levels of vWF:Ag, vWF:GPIbR and vWF:CB in IS patients were significantly higher than those in healthy controls (Z=8.36, 8.71, 6.22, respectively, P<0.01). CONCLUSION: The FCIA for detecting plasma vWF:Ag is not only an effective supplement to ELISA, but also the efficiency is faster and more sensitive, thus improves the diagnosis of type 3 vWD. Elevated levels of vWF: Ag in IS patients indicate the poor recovery of daily activities and prognosis.


Asunto(s)
Enfermedades de von Willebrand , Animales , Anticuerpos , Citometría de Flujo , Humanos , Ovinos , Accidente Cerebrovascular , Factor de von Willebrand
7.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(1): 262-266, 2020 Feb.
Artículo en Chino | MEDLINE | ID: mdl-32027287

RESUMEN

OBJECTIVE: To investigate the clinical significance of minimal residual disease (MRD) monitoring by multiparameter flow cytometry (MFC) before allogeneic hematopoietic stem cell transplantation (allo-HSCT) in acute leukemia. METHODS: 81 cases of patients with AL treated with allo-HSCT in Department of Hematology, the First Affiliated Hospital of Chongqing Medical University form July 2015 to July 2018 was selected and retorspectively analyed. of which 79 patients were in CR and two patients were in non-CR. The CR group was further divided into two groups of MRD+ and MRD- based on the MRD level prior to HSCT. RESULTS: Among 81 patients, there were statistically significant differences in the three-year overall survival(OS) (CR 82.2%: NCR 0%), cumulative relapse incidence(RI) (CR 17.7%; NCR 100%) and leukemia-free survival rate(LFS) (CR 42.3%: NCR 0%) between CR and NCR group(P<0.05). Among 79 CR patients, MRD was negative in 30 patients while positive in 49 patients, there was significant differences in the three-year overall survival between MRD- and MRD+ group. The results of univariate analysis showed that the MRD+ group showed lower LFS compared with that of MRD- group (10.5% vs 36.2%)(P<0.001,95%CI). CONCLUSION: MRD- patients shows longer LFS as compared with that of MRD+ patients, therefore, MRD monitoring by MFC before allo-HSCT is very important for the prognosis of the AL patients.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Citometría de Flujo , Humanos , Neoplasia Residual , Pronóstico , Estudios Retrospectivos , Trasplante Homólogo
8.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(1): 329-332, 2020 Feb.
Artículo en Chino | MEDLINE | ID: mdl-32027298

RESUMEN

Abstract  Chimetic antigen receptor T cells (CAR-T) are a kind of novel killer cells derived from autogenous or allogeneic T cells targeting the tumor specific antigens by gene engineering transpormation. The CAR-T cells possess the advantages of high specificity, high efficiency and non-MHC restriction, thus achieving good therapeutic effects in relapsed/refractory hematological tumors and some solid tumors. The process of CAR-T cell preparation includes cell isolation and purification, activation and differentiation, gene modification, in vitro amplification, phenotypic quality control, preservation and reinfusion. In recent years, with the rapid development of flow cytometry, this technology has been widely used in all aspects of CAR-T cell therapy, including pre-treatment screening, in vitro preparation and post-transfusion monitoring, which plays an important role in its innovative optimization.


Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Inmunoterapia Adoptiva , Citometría de Flujo , Receptores de Antígenos de Linfocitos T , Linfocitos T
9.
Exp Parasitol ; 210: 107830, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31917970

RESUMEN

Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi, which is transmitted by insects of the family Reduviidae. Since conventional treatments with nitroheterocyclic drugs show serious adverse reactions and have questionable efficiency, different research groups have investigated polypeptide-based approaches to interfere with the parasite cell cycle in other Trypanosomatids. These strategies are supported by the fact that surface players are candidates to develop surface ligands that impair function since they may act as virulence factors. In this study, we used a phage display approach to identify peptides from one library-LX8CX8 (17 aa) (where X corresponds to any amino acid). After testing different biopanning conditions using live or fixed epimastigotes, 10 clones were sequenced that encoded the same peptide, named here as EPI18. The bacteriophage expressing EPI18 binds to epimastigotes from distinct strains of T. cruzi. To confirm these results, this peptide was synthetized, biotinylated, and assayed using flow cytometry and confocal microscopy analyses. These assays confirmed the specificity of the binding capacity of EPI18 toward epimastigote surfaces. Our findings suggest that EPI18 may have potential biotechnological applications that include peptide-based strategies to control parasite transmission.


Asunto(s)
Enfermedad de Chagas/tratamiento farmacológico , Péptidos/farmacología , Trypanosoma cruzi/efectos de los fármacos , Secuencia de Aminoácidos , Bacteriófagos/aislamiento & purificación , Bioprospección , Biotinilación , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/prevención & control , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Microscopía Confocal , Microscopía Fluorescente , Biblioteca de Péptidos , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Temperatura Ambiental , Trypanosoma cruzi/genética
10.
BMC Infect Dis ; 20(1): 58, 2020 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-31952516

RESUMEN

BACKGROUND: Cytomegalovirus (CMV) infection is one of the most common opportunistic infections following organ transplantation, despite administration of CMV prophylaxis. CMV-specific T-cell immunity (TCI) has been associated with reduced rates of CMV infection. We describe for the first time clinical experience using the CMV T-Cell Immunity Panel (CMV-TCIP), a commercially available assay which measures CMV-specific CD4+ and CD8+ T-cell responses, to predict clinically significant CMV events. METHODS: Adult (> 18-year-old) patients with CMV-TCIP results and ≥ 1 subsequent assessment for CMV DNAemia were included at Brown University and the University of Maryland Medical Center-affiliated hospitals between 4/2017 and 5/2019. A clinically significant CMV event was defined as CMV DNAemia prompting initiation of treatment. We excluded indeterminate results, mostly due to background positivity, allogeneic hematopoetic cell transplant (HCT) recipients, or patients who were continued on antiviral therapy against CMV irrespective of the CMV-TCIP result, because ongoing antiviral therapy could prevent a CMV event. RESULTS: We analyzed 44 samples from 37 patients: 31 were solid organ transplant recipients, 4 had hematologic malignancies, 2 had autoimmune disorders. The CMV-protection receiver operating characteristic (ROC) area under the curve (AUC) was significant for %CMV-specific CD4+ (AUC: 0.78, P < 0.001) and borderline for CD8+ (AUC: 0.66, P = 0.064) T-cells. At a cut-off value of 0.22% CMV-specific CD4+ T-cells, positive predictive value (PPV) for protection against CMV was 85% (95%CI 65-96%), and negative predictive value (NPV) was 67% (95%CI 41-87%). CONCLUSIONS: The CMV-TCIP, in particular %CMV-specific CD4+ T-cells, showed good diagnostic performance to predict CMV events. The CMV-TCIP may be a useful test in clinical practice, and merits further validation in larger prospective studies.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citomegalovirus/inmunología , Inmunoensayo/métodos , Adulto , Anciano , Área Bajo la Curva , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Citocinas/metabolismo , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/complicaciones , Infecciones por Citomegalovirus/diagnóstico , Femenino , Citometría de Flujo , Neoplasias Hematológicas/complicaciones , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/virología , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Trasplante de Órganos , Curva ROC , Estudios Retrospectivos
11.
Life Sci ; 245: 117347, 2020 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-31981628

RESUMEN

AIM: Oxidative stress plays an important role in myocardial ischemia-reperfusion injury. Pleckstrin homology-like domain, family A, member 1 (PHLDA1) was first identified in apoptosis induced by T cell receptor activation, and was shown to play a different role in different cell types and under different stimuli. The role and mechanism of PHLDA1 in oxidative stress-induced cardiomyocyte injury and cardiac ischemia-reperfusion were therefore determined. MAIN METHODS: Cell viability and apoptotic rate were measured by Cell Counting Kit-8 and flow cytometry, respectively. Mitochondrial membrane potential was measured using JC-1 test kit. Reactive oxygen species (ROS) production was detected using ROS kit. HE staining was used to detect histological morphology, 2,3,5-triphenyltetrazolium chloride staining to detect infarct size, terminal deoxynucleotidyl transferase dUTP nick end labeling staining to detect the apoptotic rate, and immunohistochemistry and western blot analysis to detect protein expression. The binding of PHLDA1 to Bcl-2 associated X (Bax) was detected by immunoprecipitation. KEY FINDINGS: The results indicated that PHLDA1 is highly expressed in oxidative stress-induced cardiomyocyte and myocardial ischemia-reperfusion injuries. PHLDA1 overexpression in cardiomyocytes promoted oxidative stress-induced cardiomyocyte injury. At the same time, PHLDA1 knockdown improved oxidative stress-induced cardiomyocyte and myocardial ischemia-reperfusion injuries. In addition, PHLDA1 binds to Bax and the interaction is enhanced under H2O2 stimulation. SIGNIFICANCE: The present results indicated that PHLDA1 interacts with Bax to participate in oxidative stress-induced cardiomyocyte injury and myocardial ischemia reperfusion injury.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Estrés Oxidativo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Western Blotting , Línea Celular , Citometría de Flujo , Inmunoprecipitación , Etiquetado Corte-Fin in Situ , Masculino , Potencial de la Membrana Mitocondrial , Miocitos Cardíacos/fisiología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa
12.
Einstein (Sao Paulo) ; 18: eAO4966, 2020.
Artículo en Inglés, Portugués | MEDLINE | ID: mdl-31994605

RESUMEN

OBJECTIVE: To validate multilineage score system correlating results of flow cytometry, cytogenetics, cytomorphology and histology from samples of patients with suspected myelodysplastic syndrome or cytopenia of unknown origin. METHODS: A retrospective study analyzing laboratory data of 49 patients with suspected myelodysplastic syndrome or cytopenia of unknown origin, carried out between May and September 2017. The inclusion criteria were availability of flow cytometry results, and at least one more method, such as morphology, histology or cytogenetics. Thirty-eight patients were classified as diagnosis of myelodysplastic syndromes, whereas 11 were classified as normal. Patients were evaluated based on score systems, Ogata score and flow cytometry multilineage score. RESULTS: Comparing the scores obtained in the Ogata score and the multilineage score, it was observed that in four cases the Ogata score was zero or 1 point, while the multilineage score was higher than 3 points. In addition, in 12 cases with Ogata score of 2, the multilineage score was greater than 3. CONCLUSION: The flow cytometry multilineage score system demonstrated to be more effective in dysplasia analysis, by assessing the erythroid, monocytic, granulocytic and precursor cell lineages, apart from the parameters evaluated by the Ogata score.


Asunto(s)
Citometría de Flujo/normas , Síndromes Mielodisplásicos/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Células de la Médula Ósea/patología , Niño , Preescolar , Análisis Citogenético/métodos , Análisis Citogenético/normas , Células Eritroides/patología , Femenino , Citometría de Flujo/métodos , Granulocitos/patología , Humanos , Lactante , Masculino , Persona de Mediana Edad , Monocitos/patología , Estándares de Referencia , Reproducibilidad de los Resultados , Estudios Retrospectivos , Adulto Joven
13.
Chemosphere ; 243: 125453, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31995893

RESUMEN

Fungal contamination in drinking water has been becoming a hot topic. The routine enumeration method of fungal spores is heterotrophic plate counts (HPC). However, this method is time-consuming and labor-intensive and there is also the difficulty of enumerating viable but non-culturable cells. In this study, a rapid, simple and accurate method for quantifying fungal spores and discriminating their viability in water was established using flow cytometry (FCM) combined with fluorescence dyes. The optimal staining conditions are as follows: spores suspensions are sonicated at 495 W for 5 min as pretreatment, and then 10 µL of SYBR Green I (100×) and 30 mM Ethylene diamine tetraacetic acid are added to a 500 µL water sample, which incubate at 35 °C for 20 min in dark. The concentration of fungal spores measured by FCM was highly correlated with HPC results and microscope observations, with correlation coefficient of 0.996 and 0.988, respectively. This staining method can be widely applied to the enumeration and viability evaluation of fungal spores. In addition, chlorine-based inactivation of three genera of fungal spores was assessed by plating and FCM. The result showed that all three genera of fungal spores lost culturability firstly and then membrane integrity decreased, preliminarily revealing the inactivation mechanism. The inactivation rate constants of membrane damage varied in the following order: chlorine dioxide > chlorine > chloramine. This study concluded that FCM is an appropriate and alternative tool to detect fungal spores' viability and can be used for evaluating the fungal inactivation by disinfectants.


Asunto(s)
Desinfección/métodos , Citometría de Flujo/métodos , Esporas Fúngicas/crecimiento & desarrollo , Cloraminas/farmacología , Cloro/farmacología , Compuestos de Cloro/farmacología , Desinfectantes/farmacología , Colorantes Fluorescentes , Hongos/citología , Hongos/efectos de los fármacos , Compuestos Orgánicos , Óxidos/farmacología , Esporas Fúngicas/citología , Coloración y Etiquetado/métodos
14.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 51(1): 67-73, 2020 Jan.
Artículo en Chino | MEDLINE | ID: mdl-31950792

RESUMEN

Objective: To develop and verify a flow cytometric measurement of reticulocytes (RETs) micronucleus in rat bone marrow. Methods: In our flow cytometric protocol, reticulocytes, leukocytes and DNA were labeled by anti-CD71-fluorescein isothiocyanate (FITC), anti-CD45-phycoerythrin (PE) and DRAQ5, respectively. Sprague-Dawley (SD) rats were assigned to four treatment groups randomly, and were exposed to ethyl methanesulfonate (EMS), cyclophosphamide (CP), ethyl nitrosourea (ENU) and colchicine (COL) respectively. Each treatment group was divided into four subgroups (5 rats per subgroup) according to different exposure dosage. A exposure dose of 0 was used as vehicle control for each group. Rats were administered with testing mutagens by gavage twice with a 24 h interval. Bone marrow from both femurs were collected 24 h after the last administration. The frequency of micronucleated reticulocytes (MN-RETs) and the percentage of reticulocytes (RETs%) were determined by flow cytometric measurement established in this study. And the manual counting method with microscope (by Giemsa staining) was conducted at the same time. Results: A method for detection of reticulocyte micronucleus in bone marrow based on flow cytometry was successfully established. The MN-RETs in rat bone marrow of 20 SD rats treated by vehicle (i.e., background value of MN-RETs) was 0.83‰±0.12‰ by this method. The background value of MN-RETs in manual enumeration method was 1.43‰±0.44‰. It was obvious that the flow cytometric method had lower background value and more stable results. The trend, in which MN-RETs ascended and RETs% descended with increasing dose, can be detected by both methods in rats that exposed to EMS, CP, ENU and COL. Both methods were good to detect the correlation of induced-MN-RETs with four testing mutagens (the correlation coefficients were ranged from 0.834 3 to 0.913 7). Conclusion: With its sensitivity, rapidity, easy operation and low background value, the three-color flow cytometric enumerative protocol established in our laboratory can be used as a good substitute for manual micronucleus counting method and used in genotoxicity assessment of chemical substances.


Asunto(s)
Médula Ósea , Citometría de Flujo , Reticulocitos , Animales , Pruebas de Micronúcleos , Ratas , Ratas Sprague-Dawley , Reticulocitos/citología
16.
Medicine (Baltimore) ; 99(2): e18367, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31914015

RESUMEN

Little is known about the decay kinetics of interferon (IFN)-γ response and its influencing factors in tuberculous pleurisy. We enrolled thirty-two patients with tuberculous pleurisy prospectively and followed up at month 0, 6, and 9, at which time peripheral venous blood was drawn for interferon gamma release assay (IGRA) by means of QuantiFERON-TB Gold In-Tube (QFT-GIT). Demographic and clinical data were captured. To identify significant predictive factors influencing the IFN-γ response, multiple linear regression analyses were performed. Percentage of CD4+, CD8+, Vγ2Vδ2 T cells and Treg cells were measured by flow cytometry. The percentage of QFT-GIT-positive patients at baseline, month 6 and month 9 were 96.9% (30/32), 90.6% (29/32) and 84.4% (27/32), respectively. Quantitative IFN-γ response at baseline were significantly correlated with symptom duration (P = .003, R = 0.261) and age (P = .041, R = 0.132). Besides, the decreases of the IFN-γ response at month 6 and month 9 were positively correlated with the IFN-γ level at baseline. The dynamic tendency of the percentages of Treg cells was similar to the IFN-γ responses at each time-point. Quantitative IFN-γ response could be influenced by host immune status, instead of disease burden and anti-tuberculosis treatment. IGRA is probably not a useful biomarker of treatment efficacy in tuberculous pleurisy.


Asunto(s)
Ensayos de Liberación de Interferón gamma/métodos , Interferón gamma/inmunología , Tuberculosis Pleural/sangre , Adulto , Antituberculosos/administración & dosificación , Antituberculosos/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Femenino , Citometría de Flujo/métodos , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Linfocitos T/inmunología , Tuberculosis Pleural/diagnóstico , Tuberculosis Pleural/tratamiento farmacológico , Tuberculosis Pleural/metabolismo
17.
Life Sci ; 242: 117248, 2020 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-31899224

RESUMEN

Diabetic nephropathy is the most common long-term complication of diabetes mellitus. The Methylglyoxal (MGO) production is mainly by metabolic pathways, such as lipolysis and glycolysis, its increases in the DM enhances oxidative stress and plays a crucial role in the diabetic nephrotic pathogenesis. Phosphocreatine (PCr) can improve lipopolysaccharide, ox-LDL-induced atherosclerosis, and alleviate vascular endothelial cell injury in diabetes. The aim of our present study is to examine the potential role of phosphocreatine (PCr) as a molecule protects against diabetes-induced Kidney Injury in-vitro and in-vivo through ERK/Nrf2/HO-1 signaling pathway. NRK-52E cells treatment with PCr obviously suppressed MGO-induced change of viability, apoptosis, coupled with decreased Bax/Bcl-2ratio, casapse-9 and caspase-3expressions. We determined the generation of reactive oxygen species (ROS) using membrane permeable fluorescent probe DCFH-DA as well as intracellular calcium by flow cytometry. ERK, Nrf2 and HO-1 expressions were determined by Western blot. PCr pretreatment significantly returned the oxidative stress enzymes to normal condition in-vitro and in-vivo. PCr pretreatment significantly reduced apoptosis, calcium and ROS production, induced by MGO, in NRK-52E cells. Moreover, pretreatment with PCr significantly inhibited cleaved caspase-3, cleaved caspase-9 and p-ERK expressions, while increased Nrf-2 and HO-1 expressions. Furthermore, PCr pretreatment significantly decreased p-ERK expression of MGO-induced injury in NRK-52E cells transfected with p-ERK cDNA. In conclusion, the renal protective effect of PCr in-vitro and in-vivo depends on suppressing apoptosis and ROS generation through ERK mediated Nrf-2/HO-1 pathway, suggesting that PCr may be a novel therapeutic candidate for the diabetic nephropathy treatment.


Asunto(s)
Nefropatías Diabéticas/prevención & control , Hemo Oxigenasa (Desciclizante)/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Fosfocreatina/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Calcio/metabolismo , Línea Celular , Diabetes Mellitus Experimental/complicaciones , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo
18.
J Appl Oral Sci ; 28: e20190215, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31939521

RESUMEN

OBJECTIVE: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). METHODOLOGY: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. RESULTS: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). CONCLUSION: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.


Asunto(s)
Inductores de la Angiogénesis/farmacología , Compuestos de Calcio/farmacología , Cerámica/farmacología , Materiales de Obturación del Conducto Radicular/farmacología , Silicatos/farmacología , Células Madre/efectos de los fármacos , Materiales Biocompatibles/farmacología , Supervivencia Celular/efectos de los fármacos , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Factor 2 de Crecimiento de Fibroblastos/análisis , Factor 2 de Crecimiento de Fibroblastos/efectos de los fármacos , Citometría de Flujo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Ensayo de Materiales , Neovascularización Fisiológica/efectos de los fármacos , Ligamento Periodontal/citología , Ligamento Periodontal/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/análisis , Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Reproducibilidad de los Resultados , Estadísticas no Paramétricas , Germen Dentario/citología , Germen Dentario/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos
19.
Anticancer Res ; 40(1): 169-176, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31892565

RESUMEN

BACKGROUND/AIM: Cancer stem cells (CSCs) are considered to be one of the causes of tumor recurrence after chemotherapy. The purpose of our study was to isolate CSCs from human colorectal cancer cell (CRC) lines. MATERIALS AND METHODS: Nine CRC lines were screened based on the expression level of potential CSC markers to identify putative CSCs. Tumor formation capacity in immunodeficient mice was compared with that of their counterparts. Stemness, differentiation potency and sensitivity to 5-fluorouracil (5-FU), in vitro, were also assessed. Microarray analysis was used to characterize the features of the putative CSCs. RESULTS: COLO 201 cells were separated into two populations based on CD44 expression. CD44 positive (CD44+) cells showed significantly higher tumor formation capacity than CD44- cells in immunodeficient mice. CD44+ cells also possessed stemness properties and lower sensitivity to 5-FU in vitro. Moreover, cancer stemness and chemoresistance-related genes were highly up-regulated in CD44+ cells. CONCLUSION: CD44+ COLO 201 cells possessed the features of CSCs; therefore, the present CSC model could serve as a valuable tool to accelerate CSC research.


Asunto(s)
Receptores de Hialuranos/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Biomarcadores , Biomarcadores de Tumor , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Fluorouracilo/farmacología , Xenoinjertos , Humanos , Receptores de Hialuranos/genética , Ratones
20.
Int J Oral Sci ; 12(1): 1, 2020 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-31900382

RESUMEN

Odontogenic tumours are a heterogeneous group of lesions that develop in the oral cavity region and are characterized by the formation of tumoural structures that differentiate as teeth. Due to the diversity of their histopathological characteristics and clinical behaviour, the classification of these tumours is still under debate. Alterations in morphogenesis pathways such as the Hedgehog, MAPK and WNT/ß-catenin pathways are implicated in the formation of odontogenic lesions, but the molecular bases of many of these lesions are still unknown. In this study, we used genetically modified mice to study the role of IKKß (a fundamental regulator of NF-κB activity and many other proteins) in oral epithelial cells and odontogenic tissues. Transgenic mice overexpressing IKKß in oral epithelial cells show a significant increase in immune cells in both the oral epithelia and oral submucosa. They also show changes in the expression of several proteins and miRNAs that are important for cancer development. Interestingly, we found that overactivity of IKKß in oral epithelia and odontogenic tissues, in conjunction with the loss of tumour suppressor proteins (p53, or p16 and p19), leads to the appearance of odontogenic tumours that can be classified as ameloblastic odontomas, sometimes accompanied by foci of secondary ameloblastic carcinomas. These tumours show NF-κB activation and increased ß-catenin activity. These findings may help to elucidate the molecular determinants of odontogenic tumourigenesis and the role of IKKß in the homoeostasis and tumoural transformation of oral and odontogenic epithelia.


Asunto(s)
Células Epiteliales/metabolismo , Genes Supresores de Tumor , Quinasa I-kappa B/biosíntesis , Mucosa Bucal/patología , Tumores Odontogénicos/patología , Odontoma/patología , ARN Mensajero/genética , Animales , Western Blotting , Células Epiteliales/patología , Citometría de Flujo , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Ratones , Ratones Transgénicos , Mucosa Bucal/metabolismo , Tumores Odontogénicos/metabolismo , Odontoma/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa
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