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1.
Mol Biol (Mosk) ; 53(5): 838-848, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31661482

RESUMO

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a small transmembrane protein that is found in lymphocytes and is tightly associated with the phosphatase CD45. The function of LPAP is still unknown. Studies of the LPAP interactome may reveal new details of how C45 and lymphocyte signaling in general are regulated. LPAP binding partners were sought using coimmunoprecipitation coupled with mass spectrometry, stabilization of protein complexes with chemical crosslinkers, and Blue Native electrophoresis. In addition to CD45, several proteins were identified as LPAP partners, including CD71, CD98, cytoskeletal proteins, the amino acid transporter SLC1A4, and the cell signaling component HS1. It was confirmed that more than 70% of LPAP molecules were bound with CD45 in a 1 : 1 complex. The effect of CD45 on LPAP was studied in CEM and Jurkat cells with a CD45 knockout. The LPAP levels in the cells were 10% of the level in wild-type cells. In the absence of CD45, LPAP phosphorylation at Ser-153 and Ser-163 was not affected, whereas phosphorylation at Ser-99 and Ser-172 decreased significantly. Based on the results, CD45 was assumed to play a role in regulating LPAP expression and phosphorylation status.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária , Proteínas de Membrana/metabolismo , Mapas de Interação de Proteínas , Sistema ASC de Transporte de Aminoácidos/metabolismo , Antígenos CD/metabolismo , Proteína-1 Reguladora de Fusão/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Antígenos Comuns de Leucócito/metabolismo , Proteínas de Membrana/química , Fosforilação , Ligação Proteica , Receptores da Transferrina/metabolismo
2.
Zhonghua Xue Ye Xue Za Zhi ; 40(9): 744-749, 2019 Sep 14.
Artigo em Chinês | MEDLINE | ID: mdl-31648475

RESUMO

Objective: To explore the expression of CD45 in newly diagnosed multiple myeloma (MM) and its relationship with clinical efficacy and prognosis. Methods: This study retrospectively analyzed expression and distribution of CD45 in 130 cases of newly diagnosed MM, comparing clinical efficacy and prognosis in CD45(+)/CD45(-) groups. Results: ①The CD45(+) group was 33 cases (25.38%) , and CD45(-) group was 97 cases (74.62%) . ②The objective remission rate (ORR) of CD45(+) and CD45(-)group was 33.33% and 64.95%, respectively. The difference was statistically significant (P=0.002) . For patients in Bortezomib regimen, the ORR of CD45(+) and CD45(-) group was 35.71% and 66.25%, respectively. The difference was statistically significant (P=0.005) . ③The median progress free survival (PFS) of CD45(+) group and CD45(-) group was 29.8 (95%CI 10.0-59.0) months vs 34.5 (95%CI 6.0-69.0) months (χ(2)=14.59, P<0.001) and the median overall survival (OS) was 32.5 (95%CI 10.0-68.0) months vs 37.6 (95%CI 6.0-78.0) months (χ(2)=11.42, P=0.001) , respectively. Among the patients in bortezomib regimen, The median PFS and median OS of CD45 (+) group and CD45(-) group were 30.3 (95%CI 10.0-59.0) months vs 36.3 (95%CI 6.0-69.0) months (χ(2)=14.75, P=0.001) and 34.0 (95%CI 10.0-68.0) months vs 39.5 (95%CI 6.0-78.0) months (χ(2)=10.62, P=0.001) . ④Cox risk regression model analysis showed that serum creatinine≥176.8 µmol/L (HR=5.078, 95%CI 1.744-14.723, P=0.001) , CD45 positive (HR=14.504, 95%CI 0.168-0.42, P=0.001) , LDH≥220 IU/L (HR=1.308, 95%CI 1.16-2.417, P=0.015) were independent risk prognostic factors. Conclusion: CD45 expression is a risk prognostic factor of MM patients. Bortezomib did not improve the poor prognosis of CD45(+) MM patients.


Assuntos
Antígenos Comuns de Leucócito/análise , Mieloma Múltiplo , Protocolos de Quimioterapia Combinada Antineoplásica , Intervalo Livre de Doença , Humanos , Antígenos Comuns de Leucócito/metabolismo , Mieloma Múltiplo/diagnóstico , Prognóstico , Estudos Retrospectivos
3.
Zhonghua Kou Qiang Yi Xue Za Zhi ; 54(7): 445-449, 2019 Jul 09.
Artigo em Chinês | MEDLINE | ID: mdl-31288323

RESUMO

Objective: To investigate the proportion and role of CD45+ erythroid progenitor cells (EPC) in patients with tongue cancer metastasis. Methods: The initial treatment of tongue cancer patients (n=40) from January 2017 to June 2018 in He'nan Provincial People's Hospital was included in this study. According to the presence or absence of lymph node metastasis, they were divided into tumor group (no lymph node metastasis was found in imaging and pathology) and metastasis group (both imaging and pathology confirmed lymph node metastasis). The expression of Ki-67 was detected by immunohistochemistry and the proportion of CD45+CD71+TER119+EPC was detected by flow cytometry. EPC was sorted by flow cytometry, interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß) were detected by enzyme-linked immunosorbent assay (ELISA), and reactive oxygen species (ROS) was detected by flow cytometry. Transwell was used for tumor invasion test; methyl thiazolyltetrazolium (MTT) assay was used to detect proliferation level. Results: There were 20 cases in the tumor group and metastasis group. There was no significant difference between the two groups in terms of age, sex, time of onset and size of tumors. Flow cytometry showed that the ratio of CD45+EPC in peripheral blood of tumor group and metastasis group was (1.2±0.2)% and (3.1±0.2)% (t=7.823, P<0.001). Correlation analysis showed that the ratio of CD45+EPC was positively correlated with the proliferation index of Ki-67 cells (r=0.592, P=0.006). The results of flow cytometry showed that the mean fluorescence intensity (MFI) of ROS in EPC was 102.1±22.9 in tumor group and 530.0±67.2 in metastasis group (t=6.025,P<0.001). The results of ELISA showed that the mass concentrations of IL-10 and TGF-ß in EPC supernatant of tumor group were (10.8±1.6) and (3.2±0.8) µg/L, respectively. The mass concentrations of IL-10 and TGF-beta in EPC supernatant of metastasis group were (26.9±3.7) and (6.4±0.9) µg/L, respectively (t=3.956, P=0.003; t=2.595, P=0.027). Transwell results showed that the proportion of invasive cells in the CD45+EPC group [(40.3±4.4)%] was higher than that in the control group [(17.5±2.2)%] (t=4.607, P=0.001). MTT proliferation experiment showed that the proliferation rate of the CD45+EPC group [(52.0±3.3)%] was higher than that of the control group [(30.5±1.9)%] (t=5.656, P<0.001). Conclusions: The proportion of CD45+EPC in patients with tongue cancer metastasis is significantly increased. CD45+EPC can promote the proliferation and metastasis of tongue cancer by secreting immunosuppressive molecules and ROS.


Assuntos
Células Precursoras Eritroides , Antígenos Comuns de Leucócito , Neoplasias da Língua , Contagem de Células , Proliferação de Células , Células Precursoras Eritroides/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Antígenos Comuns de Leucócito/metabolismo , Linfonodos/patologia , Masculino , Metástase Neoplásica , Neoplasias da Língua/patologia
4.
Gut ; 68(11): 2019-2031, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31227589

RESUMO

OBJECTIVE: Hepatocellular carcinoma (HCC) is heterogeneous, especially in multifocal tumours, which decreases the efficacy of clinical treatments. Understanding tumour heterogeneity is critical when developing novel treatment strategies. However, a comprehensive investigation of tumour heterogeneity in HCC is lacking, and the available evidence regarding tumour heterogeneity has not led to improvements in clinical practice. DESIGN: We harvested 42 samples from eight HCC patients and evaluated tumour heterogeneity using whole-exome sequencing, RNA sequencing, mass spectrometry-based proteomics and metabolomics, cytometry by time-of-flight, and single-cell analysis. Immunohistochemistry and quantitative polymerase chain reactions were performed to confirm the expression levels of genes. Three independent cohorts were further used to validate the findings. RESULTS: Tumour heterogeneity is considerable with regard to the genomes, transcriptomes, proteomes, and metabolomes of lesions and tumours. The immune status of the HCC microenvironment was relatively less heterogenous. Targeting local immunity could be a suitable intervention with balanced precision and practicability. By clustering immune cells in the HCC microenvironment, we identified three distinctive HCC subtypes with immunocompetent, immunodeficient, and immunosuppressive features. We further revealed the specific metabolic features and cytokine/chemokine expression levels of the different subtypes. Determining the expression levels of CD45 and Foxp3 using immunohistochemistry facilitated the correct classification of HCC patients and the prediction of their prognosis. CONCLUSION: There is comprehensive intratumoral and intertumoral heterogeneity in all dimensions of HCC. Based on the results, we propose a novel immunophenotypic classification of HCCs that facilitates prognostic prediction and may support decision making with regard to the choice of therapy.


Assuntos
Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunofenotipagem , Antígenos Comuns de Leucócito/metabolismo , Neoplasias Hepáticas/metabolismo , Microambiente Tumoral
5.
J Cancer Res Ther ; 15(3): 625-630, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169231

RESUMO

Background: The most fundamental trait of cancer cells involves their ability to sustain chronic proliferation. Tumors have a complex cellular ecology that establishes the malignant potential of the tumor. In these ecosystems, innate immune cells are highly represented. Many contradictory reports have been published regarding the impact of tumor-infiltrating immune cells on proliferation of the tumors. Aim: This study aims to assess the impact of CD45RO+ve immune cells on proliferation and dedifferentiation of node-negative squamous cell carcinomas of cheek mucosa (SCC-CM). Materials and Methods: Thirty formalin-fixed paraffin-embedded tissue blocks of previously diagnosed node-negative SCC-CM subclassified as Grade I SCC - 10 cases; Grade II SCC - 10 cases; and Grade III SCC - 10 cases (Broders' classification - 1927). Immunohistochemistry performed on each selected tissue section using anti-p53 and anti-CD45RO as primary antibodies. Semi-quantitative analyses performed for all the tissue sections to assess the p53 and CD45RO expression. p53:CD45RO expression ratio calculated. The data were statistically analyzed using GraphPad Prism 5 for Windows. Results: Our results showed statistically significant increase (P = 0.0006) in p53 expression and decrease (P = 0.0044) in CD45RO+ immune cell response with the decrease in differentiation of SCC-CMs using Fisher's exact test and statistically significant increase (P < 0.001) in p53:CD45RO expression ratio with decrease in differentiation using one-way ANOVA. Conclusion: Based on all these findings from the present study, we perceive the following findings. In node-negative SCC-CMs, CD45RO+ immune cells play a possible role in controlling the dedifferentiation of the tumor and in limiting the proliferative potential of the tumor cells which are tumor antagonistic in nature.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Antígenos Comuns de Leucócito/metabolismo , Mucosa Bucal/imunologia , Mucosa Bucal/patologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Células Estromais/metabolismo , Biomarcadores Tumorais , Carcinoma de Células Escamosas/genética , Desdiferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Bochecha/patologia , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Neoplasias Bucais/genética , Gradação de Tumores , Estadiamento de Neoplasias , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
6.
Breast Cancer Res Treat ; 176(2): 387-394, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31041685

RESUMO

PURPOSE: Circulating adipose stromal cells (CASC) are thought to be increased in obesity and facilitate angiogenesis, and tumor metastases. METHODS: CASC were identified from buffy coat peripheral blood mononuclear cells (PBMCs) by flow cytometry as CD34brightCD31- CD45- and CASC frequency was compared to adiposity measures in 33 women at increased risk for breast cancer. Feasibility of CASC as a response biomarker for a diet and exercise intervention in ten breast cancer survivors was then explored. RESULTS: For 33 high-risk women, median CASC frequency was 9.7 per million PBMCs and trended positively with body mass index, fat mass index (FMI), and percent android fat. Correlation was significant when BMI was dichotomized at > versus < 35 kg/m2 (p = 0.02). For ten breast cancer survivors with a median BMI of 37 kg/m2, median CASC frequency was 16.4 per million PBMCs. In univariate analyses, change in BMI, total fat and visceral fat were significantly correlated with change in CASC frequency. On multivariate analysis, change in visceral adipose had the strongest association with change in CASC frequency (p < 0.00078). CONCLUSIONS: The association between the reduction in visceral adipose tissue and the decrease in frequency of circulating adipose stromal cells suggests that the latter might be a useful biomarker in clinical trials of obese breast cancer survivors undergoing a weight loss intervention.


Assuntos
Tecido Adiposo/imunologia , Biomarcadores/sangue , Neoplasias da Mama/sangue , Obesidade/terapia , Tecido Adiposo/citologia , Idoso , Antígenos CD34/metabolismo , Neoplasias da Mama/imunologia , Sobreviventes de Câncer , Estudos Transversais , Dietoterapia , Terapia por Exercício , Feminino , Humanos , Antígenos Comuns de Leucócito/metabolismo , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Pós-Menopausa , Pré-Menopausa , Células Estromais/citologia , Células Estromais/imunologia
7.
Int J Pediatr Otorhinolaryngol ; 122: 111-116, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30999159

RESUMO

AIMS: Immunohistochemical analysis of retraction pocket pars tensa of tympanic membrane in children. Identification of signs typical for cholesteatoma and support of retraction theory of cholesteatoma. STUDY DESIGN: a prospective study analysing 31 surgically removed retraction pockets. DEPARTMENT: University Hospital, Children's Medical Centre Methods: Retraction pockets processed by a standard process for immunohistochemical analysis. The observed findings were specified using antibodies CD45 LCA (leukocyte common antigen), CD31 (platelet endothelial cell adhesion molecule), D2-40 (marker of lymphatic endothelium), MMP9 (marker of degradation of connective tissue extracellular matrix) and Ki67 (cellular marker of proliferation). RESULTS: All observed parameters except for MMP9 had a significantly higher incidence in retraction pocket stage III compared to stage II according to Charachon. CONCLUSION: We described immunohistochemical signs of retraction pocket pars tensa of tympanic membrane in children resulting in cholesteatoma. All the observed signs occur in the structure of matrix and perimatrix of cholesteatoma. A significantly higher incidence of all observed parameters except from MMP9 was proved in retraction pocket stage III, unlike in stage II. This observation proves the fact that retraction pocket is a progressive disease and is a procholesteatoma stage.


Assuntos
Colesteatoma da Orelha Média/metabolismo , Antígeno Ki-67/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Membrana Timpânica/metabolismo , Biomarcadores/metabolismo , Criança , Humanos , Imuno-Histoquímica , Estudos Prospectivos
8.
Eur J Clin Microbiol Infect Dis ; 38(6): 1123-1128, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31011855

RESUMO

To evaluate the early absolute CD64/CD15/CD45 neutrophil count as a marker of prognosis of sepsis outcome the absolute CD64/CD15/CD45 count was measured by flow cytometry in 65 patients with confirmed or suspected Gram-negative sepsis and organ dysfunction. Serum interleukin(IL)-8 and interferon-gamma (IFNγ) were measured by an enzyme immunoassay. An absolute count lower than 2500 cells/mm3 could early discriminate non-survivors with sensitivity 82.9% (OR 3.46, 95%CIs 1.10-10.95, p 0.042). After forward step-wise Cox- regression analysis, it was found that acute coagulopathy, acute renal injury, and an early absolute CD64/CD15/CD45 count lower than 2500/mm3 were independently associated with unfavorable outcome. The OR for death among patients with an absolute CD64/CD15/CD45 neutrophil count greater than 2500/mm3 and circulating IL-8 greater than 95 pg/ml was 0.44; this was significantly increased to 7.44 among patients with an absolute CD64/CD15/CD45 neutrophil count lower than 2500/mm3 (p 0.045 by the Breslow-Day's test; p 0.046 by the Tarone's test). An absolute CD64/CD15/CD45 count below 2500/mm3 can be a useful prognosticator of sepsis outcome and a probable indicator of sepsis immunosuppression.


Assuntos
Infecções por Bactérias Gram-Negativas/diagnóstico , Neutrófilos/metabolismo , Receptores de IgG/sangue , Sepse/diagnóstico , Idoso , Biomarcadores/sangue , Feminino , Citometria de Fluxo , Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/sangue , Infecções por Bactérias Gram-Negativas/mortalidade , Humanos , Interleucina-8/sangue , Antígenos Comuns de Leucócito/sangue , Antígenos Comuns de Leucócito/metabolismo , Contagem de Leucócitos , Antígenos CD15/sangue , Antígenos CD15/metabolismo , Masculino , Razão de Chances , Escores de Disfunção Orgânica , Prognóstico , Estudos Prospectivos , Receptores de IgG/metabolismo , Sepse/sangue , Sepse/mortalidade , Análise de Sobrevida
9.
EBioMedicine ; 43: 150-158, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30975542

RESUMO

BACKGROUND: Systemic mastocytosis (SM) is a haematological disease characterised by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution and autoactivation of the receptor. Mast cells and CD34+ haematopoietic progenitors can carry the mutation; however, in which progenitor cell subset the mutation arises is unknown. We aimed to investigate the distribution of the D816V mutation in single mast cells and single haematopoietic stem and progenitor cells. METHODS: Fluorescence-activated single-cell index sorting and KIT D816V mutation assessment were applied to analyse mast cells and >10,000 CD34+ bone marrow progenitors across 10 haematopoietic progenitor subsets. In vitro assays verified cell-forming potential. FINDINGS: We found that in SM 60-99% of the mast cells harboured the KIT D816V mutation. Despite increased frequencies of mast cells in SM patients compared with control subjects, the haematopoietic progenitor subset frequencies were comparable. Nevertheless, the mutation could be detected throughout the haematopoietic landscape of SM patients, from haematopoietic stem cells to more lineage-primed progenitors. In addition, we demonstrate that FcεRI+ bone marrow progenitors exhibit mast cell-forming potential, and we describe aberrant CD45RA expression on SM mast cells for the first time. INTERPRETATION: The KIT D816V mutation arises in early haematopoietic stem and progenitor cells and the mutation frequency is approaching 100% in mature mast cells, which express the aberrant marker CD45RA.


Assuntos
Substituição de Aminoácidos , Predisposição Genética para Doença , Células-Tronco Hematopoéticas/metabolismo , Mastocitose Sistêmica/etiologia , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Antígenos CD34/metabolismo , Biomarcadores , Células da Medula Óssea/metabolismo , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Estudos de Associação Genética , Humanos , Imunofenotipagem , Antígenos Comuns de Leucócito/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Mastocitose Sistêmica/diagnóstico , Mastocitose Sistêmica/metabolismo , Análise de Célula Única
10.
Int J Mol Sci ; 20(7)2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30987036

RESUMO

Treatment with extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have been suggested as novel therapeutic option in acute inflammation-associated disorders due to their immune-modulatory capacities. As we have previously observed differences in the cytokine profile of independent MSC-EV preparations, functional differences of MSC-EV preparations have to be considered. To evaluate the immune-modulatory capabilities of specific MSC-EV preparations, reliable assays are required to characterize the functionality of MSC-EV preparations prior to administration to a patient. To this end, we established an in vitro assay evaluating the immune-modulatory capacities of MSC-EV preparations. Here, we compared the efficacy of four independent MSC-EV preparations to modulate the induction of T cell differentiation and cytokine production after phorbol 12-myristate 13-acetate (PMA)/Ionomycin stimulation of peripheral blood mononuclear cells (PBMC) derived from six healthy donors. Flow cytometric analyses revealed that the four MSC-EV preparations differentially modulate the expression of surface markers, such as CD45RA, on CD4+ and CD8+ T cells, resulting in shifts in the frequencies of effector and effector memory T cells. Moreover, cytokine profile in T cell subsets was affected in a MSC-EV-specific manner exclusively in CD8+ naïve T cells. Strikingly, hierarchical clustering revealed that the T cell response towards the MSC-EV preparations largely varied among the different PBMC donors. Thus, besides defining functional activity of MSC-EV preparations, it will be crucial to test whether patients intended for treatment with MSC-EV preparations are in principal competent to respond to the envisioned MSC-EV therapy.


Assuntos
Vesículas Extracelulares/metabolismo , Imunomodulação , Células-Tronco Mesenquimais/metabolismo , Diferenciação Celular/efeitos dos fármacos , Análise por Conglomerados , Citocinas/biossíntese , Vesículas Extracelulares/efeitos dos fármacos , Humanos , Imunomodulação/efeitos dos fármacos , Ionomicina/farmacologia , Antígenos Comuns de Leucócito/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
11.
J Neuroinflammation ; 16(1): 77, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971272

RESUMO

BACKGROUND: During the acute stroke phase, neutrophils from the peripheral blood are first to arrive in the ischemic brain, which then attracts other immune cells that exacerbate neuroinflammation in the ischemic tissue. Myosin1f was reported to specifically mediate neutrophil migration in the peripheral tissues, but whether it plays a critical role in the neuroinflammatory response after ischemic stroke remains unknown. In this study, we aim to test the hypothesis that myosin1f-mediated neutrophil migration is critical in acute neuroinflammation induced by ischemic stroke. METHODS: Myosin1f -/- and wild type (WT) mice were subjected to transient middle cerebral artery occlusion (MCAO). To determine which cells determine myosin1f's transmigration ability, bone marrow transplantation, neutrophil depletion, and adoptive neutrophil transfer were performed. The myosin1f RNA level was assessed in peripheral neutrophils by reverse transcription polymerase chain reaction (RT-PCR) at 1 day and 3 days after stroke. The infiltrating neutrophils were quantified by immunofluorescence staining and FACS at 72 h after reperfusion. RESULTS: The myosin1f -/- mice had significantly smaller infarctions than the myosin1f +/+ mice. Bone marrow transplantation from myosin1f -/- mice to recipient mice also had smaller infarctions compared to animals receiving bone marrow from myosin1f +/+ mice. By performing neutrophil depletion and adoptive transfer, we confirmed that myosin1f acts mainly in circulating neutrophils. RT-PCR showed that myosin1f gene expression was increased in the circulating blood neutrophils at 3 days after ischemia. The confocal immunostaining and FACS results confirmed that fewer neutrophils infiltrated into the ischemic brain in myosin1f -/- mice compared to WT mice. CONCLUSIONS: Myosin1f determines neutrophil migration into the ischemic hemisphere, which directly affects stroke outcome.


Assuntos
Lesões Encefálicas/etiologia , Movimento Celular/genética , Encefalite/etiologia , Infarto da Artéria Cerebral Média/complicações , Miosinas/metabolismo , Neutrófilos/fisiologia , Animais , Transplante de Medula Óssea/métodos , Lesões Encefálicas/patologia , Lesões Encefálicas/cirurgia , Antígeno CD11b/metabolismo , Modelos Animais de Doenças , Encefalite/cirurgia , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miosinas/genética , Peroxidase/metabolismo , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença , Fatores de Tempo
12.
Mediators Inflamm ; 2019: 1648614, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31015794

RESUMO

Chronic inflammation is evident in the adipose tissue and periphery of patients with obesity, as well as mouse models of obesity. T cell subsets in obese adipose tissue are skewed towards Th1- and Th17-associated phenotypes and their secreted cytokines contribute to obesity-associated inflammation. Our lab recently identified a novel, myeloid-derived CD45+DDR2+ cell subset that modulates T cell activity. The current study sought to determine how these myeloid-derived CD45+DDR2+ cells are altered in the adipose tissue and peripheral blood of preobese mice and how this population modulates T cell activity. C57BL/6 mice were fed with a diet high in milkfat (60%·kcal, HFD) ad libitum until a 20% increase in total body weight was reached, and myeloid-derived CD45+DDR2+ cells and CD4+ T cells in visceral adipose tissue (VAT), mammary gland-associated adipose tissue (MGAT), and peripheral blood (PB) were phenotypically analyzed. Also analyzed was whether mediators from MGAT-primed myeloid-derived CD45+DDR2+ cells stimulate normal CD4+ T cell cytokine production. A higher percentage of myeloid-derived CD45+DDR2+ cells expressed the activation markers MHC II and CD80 in both VAT and MGAT of preobese mice. CD4+ T cells were preferentially skewed towards Th1- and Th17-associated phenotypes in the adipose tissue and periphery of preobese mice. In vitro, MGAT from HFD-fed mice triggered myeloid-derived CD45+DDR2+ cells to induce CD4+ T cell IFN-γ and TNF-α production. Taken together, this study shows that myeloid-derived CD45+DDR2+ cells express markers of immune activation and suggests that they play an immune modulatory role in the adipose tissue of preobese mice.


Assuntos
Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Dieta Hiperlipídica/efeitos adversos , Receptor com Domínio Discoidina 2/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Animais , Células Cultivadas , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL
13.
Int J Mol Sci ; 20(6)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897843

RESUMO

Viral infections and reactivations remain a serious obstacle to successful hematopoietic stem cell transplantation (HSCT). When antiviral drug treatment fails, adoptive virus-specific T-cell transfer provides an effective alternative. Assuming that naive T cells (TN) are mainly responsible for GvHD, methods were developed to generate naive T-cell-depleted products while preserving immune memory against viral infections. We compared two major strategies to deplete potentially alloreactive T cells: CD45RA and CD62L depletion and analyzed phenotype and functionality of the resulting CD45RA-/CD62L- naive T-cell-depleted as well as CD45RA⁺/CD62L⁺ naive T-cell-enriched fractions in the CMV pp65 and IE1 antigen model. CD45RA depletion resulted in loss of terminally differentiated effector memory T cells re-expressing CD45RA (TEMRA), and CD62L depletion in loss of central memory T cells (TCM). Based on these differences in target cell-dependent and target cell-independent assays, antigen-specific T-cell responses in CD62L-depleted fraction were consistently 3⁻5 fold higher than those in CD45RA-depleted fraction. Interestingly, we also observed high donor variability in the CD45RA-depleted fraction, resulting in a substantial loss of immune memory. Accordingly, we identified donors with expected response (DER) and unexpected response (DUR). Taken together, our results showed that a naive T-cell depletion method should be chosen individually, based on the immunophenotypic composition of the T-cell populations present.


Assuntos
Imunoterapia/métodos , Selectina L/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citomegalovirus/imunologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Humanos , Imunofenotipagem , Microesferas , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo
14.
J Nutr Sci Vitaminol (Tokyo) ; 65(1): 31-37, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30814409

RESUMO

Crohn's disease is a type of inflammatory bowel disease of unknown etiology. Administration of indomethacin (Indo) to rats induces acute mucosal lesions similar to those observed in Crohn's disease patients, but the damage can be prevented by feeding the animals an elemental diet (ED). In this study, we examined changes in intestinal macroscopic appearance, permeability, and immunoglobulin production after administration of Indo to male Sprague-Dawley rats fed normal lab chow or an ED. Intestinal damage was induced by subcutaneous injection of Indo on two successive days. Mucosal permeability, as measured by urinary excretion of phenolsulfonphthalein, peaked on day 2 after Indo injection, whereas the most severe intestinal damage, as scored by macroscopic inflammatory changes, was observed on day 3. Flow cytometric analysis of mesenteric lymph node cells revealed that the proportion of CD45RA+ cells was increased after Indo treatment. Furthermore, in vitro-cultured mesenteric lymph node and spleen lymphocytes from Indo-treated rats produced higher levels of IgA and IgG than did cells from vehicle-treated rats. In contrast, IgG and albumin concentrations in plasma were significantly decreased by Indo administration. Notably, none of the Indo-induced changes was observed in ED-fed rats. These findings suggest that an ED may prevent the appearance of Indo-induced mucosal lesions, at least in part, by modulating intestinal permeability and antibody production.


Assuntos
Doença de Crohn/dietoterapia , Doença de Crohn/metabolismo , Alimentos Formulados , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Animais , Formação de Anticorpos , Doença de Crohn/induzido quimicamente , Modelos Animais de Doenças , Indometacina , Antígenos Comuns de Leucócito/metabolismo , Masculino , Permeabilidade , Fenolsulfonaftaleína/metabolismo , Ratos , Ratos Sprague-Dawley
15.
Cell Oncol (Dordr) ; 42(3): 343-356, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30825183

RESUMO

PURPOSE: In recent years, the prognostic/predictive significance of tumor infiltrating lymphocytes (TILs) has become a topic of interest. Here, we aimed to evaluate the prognostic significance of CD3+, CD8+, CD45RO+ and Foxp3+ TILs in breast cancer, as well as the relation of these markers to other clinicopathological features of this disease. METHODS: FFPE tumor samples from 94 females with invasive ductal carcinoma of the breast were retrospectively selected and immunohistochemically assessed for CD3, CD8, CD45RO and Foxp3 expression. Digital photos were acquired from the center (CT) and invasive margins (IM) of the tumors, after which positive cells were counted using ImageJ software. RESULTS: We found that greater infiltrations of target lymphocyte subpopulations were associated with TNM stage III, lymph node metastasis, high histological grade, ER negativity and HER2 positivity. The ratios of CD8+ cytotoxic T cells to CD3+, CD45RO+ and Foxp3+ TILs were found to be relatively higher in tumors exhibiting the aforementioned characteristics. In univariate survival analyses, CD8+ TILs in the IM and total CD45RO+ TILs were found to be significantly associated with overall survival (OS). Infiltration of CD45RO+ TILs in the CT and lymph node status were variables that significantly correlated with disease-free survival (DFS). Multiple Cox regression analyses revealed independent significant prognostic effects of total CD45RO+ TILs and lymph node status (HR of 3.24 and 3.19, respectively) in predicting OS. Infiltration of CD45RO+ TILs in the CT (HR 3.12) and lymph node status (HR 3.15) also exhibited significant prognostic effects on DFS. CONCLUSION: From our data we conclude that CD45RO+ TILs serve as prognostic factors for predicting OS and DFS of breast cancer patients.


Assuntos
Neoplasias da Mama/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Estimativa de Kaplan-Meier , Mastectomia , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos
16.
Invest Ophthalmol Vis Sci ; 60(4): 1010-1020, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30884531

RESUMO

Purpose: The purpose of this study was to evaluate the effect of removal of Descemet's basement membrane and endothelium compared with removal of the endothelium alone on posterior corneal fibrosis. Methods: Twelve New Zealand White rabbits were included in the study. Six eyes had removal of the Descemet's membrane-endothelial complex over the central 8 mm of the cornea. Six eyes had endothelial removal with an olive-tipped cannula over the central 8 mm of the cornea. All corneas developed stromal edema. Corneas in both groups were cryofixed in optimum cutting temperature (OCT) formula at 1 month after surgery. Immunohistochemistry (IHC) was performed for α-smooth muscle actin (SMA), keratocan, CD45, nidogen-1, vimentin, and Ki-67, and a TUNEL assay was performed to detect apoptosis. Results: Six of six corneas that had Descemet's membrane-endothelial removal developed posterior stromal fibrosis populated with SMA+ myofibroblasts, whereas zero of six corneas that had endothelial removal alone developed fibrosis or SMA+ myofibroblasts (P < 0.01). Myofibroblasts in the fibrotic zone of corneas that had Descemet's membrane-endothelial removal were undergoing both mitosis and apoptosis at 1 month after surgery. A zone between keratocan+ keratocytes and SMA+ myofibroblasts contained keratocan-SMA-vimentin+ cells that were likely CD45- corneal fibroblasts and CD45+ fibrocytes. Conclusions: Descemet's basement membrane has an important role in modulating posterior corneal fibrosis after injury that is analogous to the role of the epithelial basement membrane in modulating anterior corneal fibrosis after injury. Fibrotic areas had myofibroblasts undergoing mitosis and apoptosis, indicating that fibrosis is in dynamic flux.


Assuntos
Substância Própria/patologia , Lâmina Limitante Posterior/fisiologia , Actinas/metabolismo , Animais , Apoptose/fisiologia , Edema da Córnea/etiologia , Substância Própria/metabolismo , Lâmina Limitante Posterior/cirurgia , Feminino , Fibrose/metabolismo , Fibrose/patologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Antígeno Ki-67/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitose/fisiologia , Miofibroblastos/citologia , Proteoglicanas/metabolismo , Coelhos , Vimentina/metabolismo
17.
Methods Mol Biol ; 1940: 267-273, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788832

RESUMO

The liver performs a number of physiologically important functions. Hepatocytes are the liver parenchymal cells performing most of those functions. Therefore, it is important to recover functional hepatocytes after hepatic injury and prepare a mass of hepatocytes for regenerative medicine. We have found that mature hepatocytes dedifferentiate to hepatocyte progenitors in chronically injured mouse liver. Those hepatocyte progenitors can be isolated as CD24+EpCAM- cells from the CD31-CD45- fraction, which clonally proliferate and efficiently re-differentiate to functional hepatocytes both in vitro and in vivo. Here, I describe the methods to isolate hepatocyte progenitors from chronically injured liver, to expand them in vitro, and to induce differentiation into functional hepatocytes.


Assuntos
Hepatócitos/citologia , Fígado/citologia , Fígado/lesões , Células-Tronco/citologia , Animais , Antígeno CD24/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Molécula de Adesão da Célula Epitelial/metabolismo , Citometria de Fluxo , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Piridinas/farmacologia , Fatores de Transcrição SOX9/metabolismo
18.
Int Immunopharmacol ; 69: 194-201, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30735938

RESUMO

CD4+ regulatory T cells (Tregs) play an important role in inducing immune tolerance in organ transplantation, which can be divided into CD45RA+Tregs (resting Tregs, rTregs) and CD45RO+Tregs (activated Tregs, aTregs). Currently, the expressions and phenotypic changes of Tregs in recipients after liver transplantation (LT) is unknown. We therefore investigated the expression and transformation of rTregs and aTregs in 83 cases of recipients with normal status post-LT. The percentages of CD45RA, CD45RO, CD31 in CD4+Tregs were detected by flow cytometry and the effective factors were analyzed. In LT recipients, the percentage of CD45RO+Tregs in CD4+Tregs was higher than that of CD45RA+Tregs. There was significant difference in the ratio of positive Foxp3 between CD45RA+Tregs and CD45RO+Tregs. Percentage of CD45RA+Tregs was higher in pediatric group than that in adult group, whereas percentage of CD45RO+Tregs was lower in the pediatric group. However, it was different only in CD45RO+Tregs in various survival periods post-LT. In conclusion, Tregs pool in human was heterogeneous post-LT and contained different subsets in phenotypes. Upon stimulation by donor graft, percentages of CD4+Tregs and CD45RO+Tregs were increased post-LT and most of rTregs was transformed into aTregs in peripheral blood, and rTregs and aTregs were both related to recipients' ages.


Assuntos
Rejeição de Enxerto/imunologia , Transplante de Fígado , Linfócitos T Reguladores/imunologia , Adulto , Fatores Etários , Antígenos CD4/metabolismo , Separação Celular , Células Cultivadas , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Antígenos HLA/imunologia , Humanos , Tolerância Imunológica , Imunofenotipagem , Lactente , Isoantígenos/imunologia , Antígenos Comuns de Leucócito/metabolismo , Masculino , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Transplantados
19.
Ann Lab Med ; 39(4): 388-395, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30809985

RESUMO

BACKGROUND: It is very important to accurately enumerate CD34-positive (CD34+) cells for successful hematopoietic stem cell transplantation (HSCT). We evaluated the ability of the newly developed image based-immunofluorescence cell counter ADAMII (NanoEntek, Seoul, Korea) to enumerate CD34+ cells, which was improved through simultaneous CD45 analysis. METHODS: We enumerated CD34+ cells with ADAMII using 19 peripheral blood (PB) and 91 leukapheresis samples from HSCT donors. Analytical performance, including precision and linearity, was analyzed, and sample stability during storage was evaluated. Viable CD34+ cell count (vCD34) and viable CD45+ cell count (vCD45) and the percentage of viable CD34+ cells among viable CD45+ cells (CD34/CD45) as measured by ADAMII were compared with the corresponding values from two flow cytometry assays, using regression analysis. RESULTS: ADAMII demonstrated acceptable precision, as CV values of vCD34 from six samples with different counts were all <10% (range: 3.49-9.51%). CV values of the vCD45 and CD34/45 ranged from 4.03% to 9.67% and from 2.48% to 10.07%, respectively. The linearity of vCD34 showed an excellent R² value (0.99) when analyzed using the intended count and flow cytometry data. The ADAMII and two flow cytometry-based assays generated very similar data for the PB and leukapheresis samples. CONCLUSIONS: ADAMII demonstrated excellent performance for use as a routine clinical assay in terms of CD34+ cell enumeration from PB and leukapheresis samples. Moreover, it could be used as a point-of-care-test for determining mobilization time and predicting an adequate apheresis stem cell product.


Assuntos
Antígenos CD34/metabolismo , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Antígenos Comuns de Leucócito/metabolismo , Testes Imediatos , Kit de Reagentes para Diagnóstico , Análise de Regressão
20.
Thromb Haemost ; 119(2): 274-284, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30609443

RESUMO

Endothelial progenitor cells (EPCs) have been suggested to contribute to the neovascularization of infantile haemangioma (IH). There is strong evidence of the efficacy of propranolol in the treatment of IH, possibly by inhibiting both vasculogenesis and angiogenesis in the tumour. We evaluate the frequency of circulating endothelial colony forming cells (ECFCs), as the best EPC surrogate, in patients with IH at diagnosis and while receiving propranolol by an ex vivo 12-month longitudinal study. Biological aspects of the ECFCs, such as their in vitro angiogenic potential, membrane CXCR4 expression and Ca2+ signalling, were investigated. Circulating ECFCs were isolated by in vitro culture and expanded for 2 to 3 passages in 23 patients with IH (median age: 5.5 months, range: 5.5 weeks-11 months) before and 3, 6, 9 and 12 months after receiving propranolol. Twenty-four healthy subjects comparable for age were also assessed (CTRLs). Untreated patients with IH had a circulating ECFC frequency lower (p = 0.001) than CTRLs; nevertheless, in in vitro starving conditions, ECFCs showed enhanced capacity to form tube-like structures than those of CTRLs. Patients with IH following the therapy with propranolol had a significantly increased (p = 0.022) circulating ECFC frequency, that showed a diminished tube-like formation capacity in vitro, and an altered constitutive store-operated Ca2+ entry. ECFCs play a role in IH pathogenesis; the response to propranolol therapy is associated with their increased frequency in the peripheral blood and a reduction of their vasculogenic activity.


Assuntos
Células Endoteliais/citologia , Hemangioma/tratamento farmacológico , Hemangioma/metabolismo , Neovascularização Patológica , Propranolol/uso terapêutico , Antagonistas Adrenérgicos beta/uso terapêutico , Antígenos CD34/metabolismo , Cálcio/química , Sinalização do Cálcio , Movimento Celular , Quimiocina CXCL12/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Progenitoras Endoteliais/citologia , Feminino , Citometria de Fluxo , Humanos , Lactente , Recém-Nascido , Cinética , Antígenos Comuns de Leucócito/metabolismo , Estudos Longitudinais , Masculino , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Fenótipo , Receptores CXCR4/metabolismo
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