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1.
Rev Esp Patol ; 53(3): 182-187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32650969
2.
Anticancer Res ; 40(5): 2583-2589, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366403

RESUMO

BACKGROUND/AIM: Certain integrins including integrin ß3 facilitate movement and survival of metastatic cancer cells. We examined whether benzoxazine dimer analogue N,N-bis(5-ethyl-2-hydroxybenzyl) methylamine (HM) has anti-metastatic effects. MATERIALS AND METHODS: Cell viability was examined by the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Wound healing and phalloidin-rhodamine assays were performed to evaluate the migration and filopodia formation, respectively. Anoikis resistance was studied by anchorage-independent growth assay. The expression of proteins regulating migration were examined by western blot. RESULTS: HM treatment significantly inhibited growth and survival of detached lung cancer cells as indicated by the reduced colony number and size of anchorage-independent growth analysis. HM inhibited cell migration and suppressed filopodia formation. Protein analysis indicated that the compound dramatically decreased integrin ß3 and its related downstream proteins including active focal adhesion kinase (FAK) and active protein kinase B (AKT); however, integrin ß1 and α5 were found to be unaltered. CONCLUSION: HM shows a potential in targeting integrin ß3 and could be a good candidate for further developed as an anti-metastatic therapy.


Assuntos
Anoikis/efeitos dos fármacos , Antineoplásicos/farmacologia , Benzoxazinas/farmacologia , Movimento Celular/efeitos dos fármacos , Integrina beta3/metabolismo , Antineoplásicos/química , Benzoxazinas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neoplasias Pulmonares , Modelos Biológicos , Cicatrização/efeitos dos fármacos
3.
Environ Sci Pollut Res Int ; 27(23): 29530-29538, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32440878

RESUMO

Chlorpyrifos (CPF), as a worldwide pesticide, can effect on the integrins αv and ß3 which play a main role in the implantation window. Therefore, the aim of this study was to consider CPF effects on integrin alpha v and beta 3 in implantation window phase. Thirty female NMRI mice were separated into groups of CPF, sham, and control. After 6 weeks, each group was mated, and on the 5th day of gestation, all mice were euthanized. Estradiol and progesterone levels were detected by the enzyme-linked immunosorbent assay (ELISA) test; two subunits of integrins (αv and ß3) genes and proteins of endometrium were analyzed by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry method, respectively. Fibrosis of the liver which evaluated by Masson's trichrome stain was increased in the CPF group compared with the others. But estradiol and progesterone levels were significantly decreased in CPF groups. Based on the findings, the proportion of genes' expressions of integrin subunits declined by the effect of CPF, while there was not any notable consequence on mice in the sham group. Alpha v and beta 3 integrin proteins expressed in all groups, but the concentration of these proteins in CPF groups was lower than in other groups. This study has shown that the decline of estradiol and progesterone downregulates the expression of αv and ß3 integrins which were influenced by CPF exposure. Changing these patterns of proteins could have numerous influences on unsuccessful implantation. Therefore, this experimental study recommends that inclusive consideration of the effects of insecticides may be crucial to women's unrecognized cause of infertility.


Assuntos
Clorpirifos , Inseticidas , Integrina alfaV , Integrina beta3 , Animais , Clorpirifos/toxicidade , Implantação do Embrião , Endométrio , Feminino , Humanos , Camundongos , Progesterona
4.
Wiad Lek ; 73(3): 471-477, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32285816

RESUMO

OBJECTIVE: The aim is the analysis of the relationship between the polymorphism of thrombophilic genes, in particular Serpin 1 (PAI-1), F2-prothrombin and ITGB3-α integrin, and the incidence of stroke, as well as the study of factor effects of this polymorphism in association with controlled risk factors (hypertonic disease, smoking, alcohol consumption, diabetes mellitus, obesity, atrial fibrillation). PATIENTS AND METHODS: Materials and methods: A total of 134 patients were examined (men accounted for 44.8%, women 55.2%, average age 62.5 ± 2.1). The statistical analysis was carried out using the following criteria: χ2-Pearson, Fisher's exact criterion (reversible), Chuprov's coefficient of conjugation and dispersion analysis (alternative complex). RESULTS: Results: The relationship between the frequency of a specific allele of thrombophilia and the incidence of stroke is absent. The reason for such results can be a significant effect of random factors (hypertension, diabetes ...), a significant variability of risk factors, their different frequency in groups (inter- and intra-group differences), a significant (95%) total effect of these factors. CONCLUSION: Conclusions:Identification of biochemical or genetic markers of thrombophilic conditions, including polymorphism of the hemostasis system genes, will significantly increase the possibility of adequate pathogenetic treatment and timely prevention of acute cerebrovascular disorders, especially persons of working age, which has great medical and social importance.


Assuntos
Acidente Vascular Cerebral , Trombofilia , Fator V , Feminino , Humanos , Integrina beta3 , Masculino , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio , Protrombina , Fatores de Risco , Serpinas
5.
PLoS One ; 15(4): e0230507, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32255777

RESUMO

The efficiency of in vitro platelet production is considerably low compared with physiological activity due to the lack of pivotal factors that are essential in vivo. We developed an ex vivo platelet production system, introducing human megakaryocytes into an isolated porcine thighbone and culturing in closed circuit. The efficiency of the ex vivo platelet production system was compared to those in vivo and in vitro. CD61+ platelet-like cells were counted by immunostaining and flow cytometry. Results showed that 4.41 ± 0.27 × 103 CD61+ platelet-like cells were produced by 1 × 103 megakaryocytes in the ex vivo system, while 3.80 ± 0.87 × 103 and 0.12 ± 0.02 × 103 were produced in the in vivo and in vitro systems, respectively. Notably, ex vivo and in vitro production systems generated cells that responded well to thrombin stimulation and expressed functional molecules, such as CD62P. Overall, our ex vivo production system was comparable to in vivo production system and produced platelet-like cells that were functionally superior to those produced in vitro. In future, the present ex vivo production system implementing xenogeneic bone marrow would offer a promising alternative for industrial-scale production of platelet-like cells.


Assuntos
Plaquetas/metabolismo , Células da Medula Óssea/citologia , Animais , Antígenos CD34/metabolismo , Plaquetas/citologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Integrina beta3/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Suínos , Trombina/farmacologia
6.
Arterioscler Thromb Vasc Biol ; 40(5): 1296-1310, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32237906

RESUMO

OBJECTIVE: Integrin ß3 is implicated in numerous biological processes such as its relevance to blood triglyceride, yet whether ß3 deficiency affects this metabolic process remains unknown. Approach and Results: We showed that the Chinese patients with ß3-deficient Glanzmann thrombasthenia had a 2-fold higher serum triglyceride level together with a lower serum LPL (lipoprotein lipase) level than those with an αIIb deficiency or healthy subjects. The ß3 knockout mice recapitulated these phenotypic features. The elevated plasma triglyceride level was due to impaired LPL-mediated triglyceride clearance caused by a disrupted LPL secretion. Further analysis revealed that ß3 directly bound LPL via a juxtamembrane TIH (threonine isoleucine histidine)720-722 motif in its cytoplasmic domain and functioned as an adaptor protein by interacting with LPL and PKD (protein kinase D) to form the PKD/ß3/LPL complex that is required for ß3-mediated LPL secretion. Furthermore, the impaired triglyceride clearance in ß3 knockout mice could be corrected by adeno-associated virus serotype 9 (AAV9)-mediated delivery of wild-type but not TIH720-722-mutated ß3 genes. CONCLUSIONS: This study reveals a hypertriglyceridemia in both ß3-deficient Chinese patients and mice and provides novel insights into the molecular mechanisms of the significant roles of ß3 in LPL secretion and triglyceride metabolism, drawing attention to the metabolic consequences in patients with ß3-deficient Glanzmann thrombasthenia.


Assuntos
Hipertrigliceridemia/etiologia , Cadeias beta de Integrinas/metabolismo , Integrina beta3/metabolismo , Lipase Lipoproteica/sangue , Trombastenia/complicações , Triglicerídeos/sangue , Adolescente , Animais , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , China , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Humanos , Hipertrigliceridemia/sangue , Hipertrigliceridemia/diagnóstico , Hipertrigliceridemia/enzimologia , Cadeias beta de Integrinas/genética , Integrina beta3/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/metabolismo , Fatores de Risco , Trombastenia/sangue , Trombastenia/diagnóstico , Trombastenia/genética
7.
Am J Med Sci ; 359(5): 296-302, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32265009

RESUMO

Thrombotic microangiopathy (TMA) is characterized by microangiopathic hemolytic anemia with thrombocytopenia. In addition to the primary TMA syndromes, microangiopathic hemolytic anemia with thrombocytopenia can be seen in many systemic diseases. Transplant associated TMA (TA-TMA) affects patients following stem cell or solid organ transplant. A 48-year-old male who underwent autologous stem cell transplant for nonsecretory multiple myeloma was admitted to our hospital with worsening anemia, thrombocytopenia, renal dysfunction and hepatosplenomegaly. Initial blood work revealed rare schistocytes and normal lactate dehydrogenase and haptoglobin levels. He underwent an extensive workup looking for an infectious, inflammatory or malignant etiology but a definitive diagnosis could not be reached. Over his prolonged stay at the hospital, he suffered from multiorgan failure and eventually passed away. An autopsy revealed TMA involving all clinically affected organ systems and was deemed to be the cause of his demise. The absence of typical blood work suggestive of hemolysis does not rule out a diagnosis of TA-TMA. Knowledge of this rare disease entity will help physicians identify and treat this life-threatening condition early and effectively.


Assuntos
Eritrócitos Anormais , Hemólise , Microangiopatias Trombóticas/complicações , Biópsia , Evolução Fatal , Hepatomegalia/complicações , Humanos , Inflamação , Integrina beta3/metabolismo , L-Lactato Desidrogenase/metabolismo , Fígado/patologia , Pulmão/patologia , Masculino , Microcirculação , Pessoa de Meia-Idade , Esplenomegalia/complicações , Transplante de Células-Tronco , Trombocitopenia/complicações , Trombose/metabolismo , Microangiopatias Trombóticas/terapia , Transplante Autólogo
8.
Chem Commun (Camb) ; 56(12): 1788-1791, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-31960841

RESUMO

In this study, αvß3 integrin in U87 tumor cells was imaged with a 64Cu-peptidic probe, in which the linear peptide GHRGDHG is used as a pre-ligand, while 64Cu bears three functional roles that include generation of the PET signal, coordination with two GH moieties of the pre-ligand, and cyclizing the linear pre-ligand into an active cyclic-RGD form (termed as 64Cu-Cyclo-RGD) for αvß3 integrin.


Assuntos
Radioisótopos de Cobre/química , Corantes Fluorescentes/química , Integrina beta3/análise , Peptídeos Cíclicos/química , Tomografia por Emissão de Pósitrons , Linhagem Celular Tumoral , Humanos , Imagem Óptica
9.
Arterioscler Thromb Vasc Biol ; 40(3): 624-637, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31969014

RESUMO

OBJECTIVE: The αIIbß3 antagonist antiplatelet drug abciximab is the chimeric antigen-binding fragment comprising the variable regions of murine monoclonal antibody 7E3 and the constant domains of human IgG1 and light chain κ. Previous mutagenesis studies suggested that abciximab binds to the ß3 C177-C184 specificity-determining loop (SDL) and Trp129 on the adjacent ß1-α1 helix. These studies could not, however, assess whether 7E3 or abciximab prevents fibrinogen binding by steric interference, disruption of either the αIIbß3-binding pocket for fibrinogen or the ß3 SDL (which is not part of the binding pocket but affects fibrinogen binding), or some combination of these effects. To address this gap, we used cryo-electron microscopy to determine the structure of the αIIbß3-abciximab complex at 2.8 Å resolution. Approach and Results: The interacting surface of abciximab is comprised of residues from all 3 complementarity-determining regions of both the light and heavy chains, with high representation of aromatic residues. Binding is primarily to the ß3 SDL and neighboring residues, the ß1-α1 helix, and ß3 residues Ser211, Val212 and Met335. Unexpectedly, the structure also indicated several interactions with αIIb. As judged by the cryo-electron microscopy model, molecular-dynamics simulations, and mutagenesis, the binding of abciximab does not appear to rely on the interaction with the αIIb residues and does not result in disruption of the fibrinogen-binding pocket; it does, however, compress and reduce the flexibility of the SDL. CONCLUSIONS: We deduce that abciximab prevents ligand binding by steric interference, with a potential contribution via displacement of the SDL and limitation of the flexibility of the SDL residues.


Assuntos
Abciximab/ultraestrutura , Microscopia Crioeletrônica , Integrina alfa2/ultraestrutura , Integrina beta3/ultraestrutura , Inibidores da Agregação de Plaquetas , Abciximab/metabolismo , Sítios de Ligação , Ligação Competitiva , Células HEK293 , Humanos , Integrina alfa2/genética , Integrina alfa2/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Inibidores da Agregação de Plaquetas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/ultraestrutura , Relação Estrutura-Atividade
10.
Reprod Biol Endocrinol ; 17(1): 94, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31729993

RESUMO

BACKGROUND: Although thyroid dysfunction caused by Hashimoto's thyroiditis (HT) is believed to be related to implantation failure due to the underdevelopment of the receptive uterus, it is unknown whether HT itself, even in the euthyroid state, impairs embryo implantation associated with endometrial receptivity defects. To address whether HT itself can affect endometrial receptivity accompanied by implantation alterations, a euthyroid HT model was established in mice. METHODS: Female NOD mice were immunized twice with thyroglobulin and adjuvant to induce the experimental HT model. Four weeks after the second treatment, the mice were normally mated, and pregnant ones were sacrificed in implantation window for thyroid-related parameter and steroid hormones measurements by electrochemiluminescence immunoassay and enzyme-linked immunosorbent assay and implantation site number calculation by uptake of Chicago Blue dye. In addition, certain morphological features of endometrial receptivity were observed by hematoxylin-eosin staining and scanning electron microscopy, and the expression of other receptivity markers were analyzed by immunohistochemistry, RT-qPCR or Western Blot. RESULTS: HT mice displayed intrathyroidal monocyte infiltration and elevated serum thyroid autoantibody levels without thyroid dysfunction, defined as euthyroid HT in humans. Euthyroid HT resulted in implantation failure, fewer pinopodes, retarded pinopode maturation, and inhibited expression of receptivity markers: estrogen receptor α (ERα), integrin ß3, leukemia inhibitory factor (LIF), and cell adhesion molecule-1 (ICAM-1). Interestingly, despite this compromised endometrial receptivity response, no statistical differences in serum estradiol or progesterone level between groups were found. CONCLUSIONS: These findings are the first to indicate that HT induces a nonreceptive endometrial milieu in the euthyroid state, which may underlie the detrimental effects of HT itself on embryo implantation.


Assuntos
Biomarcadores/metabolismo , Implantação do Embrião , Endométrio/fisiopatologia , Doença de Hashimoto/fisiopatologia , Animais , Endométrio/metabolismo , Endométrio/ultraestrutura , Estradiol/sangue , Feminino , Expressão Gênica , Doença de Hashimoto/sangue , Humanos , Integrina beta3/genética , Integrina beta3/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Masculino , Camundongos Endogâmicos NOD , Microscopia Eletrônica de Varredura , Gravidez , Testosterona/sangue , Tireotropina/sangue
11.
J Exp Clin Cancer Res ; 38(1): 449, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31684995

RESUMO

BACKGROUND: Interleukin-8 (IL-8) plays a vital role in the invasion and metastasis of hepatocellular carcinoma (HCC), and is closely associated with poor prognosis of HCC patients. Integrin αvß3, a member of the integrin family, has been reported to be overexpressed in cancer tissues and mediate the invasion and metastasis of HCC cells. However, the relationship between IL-8 and integrin αvß3 in HCC and the underlying mechanism of IL-8 and integrin αvß3 in the invasion of HCC remains unclear. METHODS: The expression of IL-8, integrin αv and integrin ß3 in HCC cells and tissues was detected by quantitative real-time PCR, Western blot and immunohistochemistry. Transwell assay and Western blot was used to detect the invasiveness, the expression of integrin ß3 and the activation of PI3K/Akt pathway of HCC cells pretreated with IL-8 knockdown or exogenous IL-8. RESULTS: IL-8, integrin αv and integrin ß3 were overexpressed in highly metastatic HCC cell lines compared with low metastatic cell lines. There was a positive correlation between integrin ß3 and IL-8 expression in HCC tissues. IL-8 siRNA transfection reduced HCC cell invasion and the levels of integrin ß3, p-PI3K and p-Akt. IL-8 induced HCC cell invasion and integrin ß3 expression was significantly inhibited by transfection with CXCR1 siRNA or CXCR2 siRNA. When we stimulated HCC cells with exogenous IL-8, cell invasion and the levels of integrin ß3, p-PI3K, and p-Akt increased, which could be effectively reversed by adding PI3K inhibitor LY294002. CONCLUSIONS: Our results suggest that IL-8 promotes integrin ß3 upregulation and the invasion of HCC cells through activation of the PI3K/Akt pathway. The IL-8/CXCR1/CXCR2/PI3K/Akt/integrin ß3 axis may serve as a potential treatment target for patients with HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Integrinas/genética , Integrinas/metabolismo , Neoplasias Hepáticas/genética , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Regulação para Cima
12.
Anal Chim Acta ; 1091: 160-168, 2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31679569

RESUMO

A new, fast and selective immunoaffinity chromatographic method including a methacrylate-based convective interaction media (CIM®) disk monolithic column, immobilized with anti-human CD61 antibody, was developed for the isolation of CD61-containing platelet-derived extracellular vesicles (EVs) from plasma. The isolated EVs were detected and size characterized by asymmetrical flow field-flow fractionation (AsFlFFF) with multi-angle light-scattering (MALS) and dynamic light-scattering (DLS) detection, and further confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The mean size of platelet-derived EV isolates from the anti-CD61 CIM® disk monolithic column were 174 nm (SD 60 nm) based on the NTA results. These results indicated a successful isolation of platelet-derived EVs, which was confirmed by Western blotting the isolates against the EV-specific markers CD9 and TSG101 together with transmission electron microscopy. Additional elucidation of MALS and DLS data provided detailed information of the size distribution of the isolated fractions, confirming the successful isolation of also small platelet-derived EVs ranging from 30 to 130 nm based on the hydrodynamic radii. The isolation procedure took only 19 min and the time can be even further decreased by increasing the flow rate. The same immunoaffinity chromatographic procedure, following AsFlFFF allowed also the isolation and characterization of platelet-derived EVs from plasma in under 60 min. Since it is possible to regenerate the anti-CD61 disk for multiple uses, the methodology developed in this study provides a viable substitution and addition to the conventional EV isolation procedures.


Assuntos
Plaquetas/citologia , Cromatografia de Afinidade/métodos , Vesículas Extracelulares , Animais , Anticorpos Imobilizados/imunologia , Difusão Dinâmica da Luz , Vesículas Extracelulares/química , Vesículas Extracelulares/imunologia , Fracionamento por Campo e Fluxo , Humanos , Integrina beta3/imunologia , Camundongos , Tamanho da Partícula , Ácidos Polimetacrílicos/química
13.
Mar Drugs ; 17(12)2019 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-31771240

RESUMO

Chitosan is sensitive to environmental pH values due to its electric property. This study investigates whether the pH-responsive chitosan assay can provide a simple method to evaluate the aggressive behavior of cancer cells with cell detachment ratio. The epithelial-mesenchymal transition (EMT) is induced with transforming growth factor-ß1 (TGF-ß1) in the human non-small cell lung cancer cell line (A549). EMT-induced cells and untreated cells are cultured on chitosan substrates at pH 6.99 for 24 h, followed by pH 7.65 for 1 h. The cell detachment ratio (CDR) on pH-responsive chitosan rises with an increasing of the TGF-ß1 concentration. The protein array reveals that the expression levels of the α2, α3, α5, ß2, and ß3 integrins are higher in EMT-induced A549 cells than in untreated cells. A further inhibition assay shows that adding ß3 integrin blocking antibodies significantly decreases the CDR of EMT-induced cells from 32.7 ± 5.7% to 17.8 ± 2.1%. The CDR of mesenchymal-type lung cancer cells increases on pH-responsive chitosan through the ß3 integrin. Notably, the CDR can be theoretically predicted according to the individual CDR on the pH-responsive chitosan surface, irrespective of heterogeneous cell mixture. The pH-responsive chitosan assay serves as a simple in vitro model to investigate the aggressive behavior of lung cancer including the heterogeneous cell population.


Assuntos
Bioensaio/métodos , Carcinoma Pulmonar de Células não Pequenas/patologia , Quitosana/química , Neoplasias Pulmonares/patologia , Células A549 , Adesão Celular , Movimento Celular , Transição Epitelial-Mesenquimal , Humanos , Concentração de Íons de Hidrogênio , Integrina beta3/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo
14.
Mediators Inflamm ; 2019: 4567106, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31772502

RESUMO

Aim: Aware that Down Syndrome patients present among their clinical characteristics impaired immunity, the aim of this study is to identify the statistically significant differences in inflammation-related gene expression by comparing Down Syndrome patients with Periodontal Disease (DS+PD+) with Down Syndrome patients without Periodontal Disease (DS+PD-), and their relationship with periodontitis as a chronic oral inflammatory clinical feature. Materials and Methods: Case study and controls on eleven Down Syndrome patients (DS+PD+ vs. DS+PD-). RNA was extracted from peripheral blood using a Qiagen PAXgene Blood miRNA Kit when performing an oral examination. A search for candidate genes (92 selected) was undertaken on the total genes obtained using a Scientific GeneChip® Scanner 3000 (Thermo Fisher Scientific) and Clariom S solutions for human, mouse, and rat chips, with more than 20,000 genes annotated for measuring expression levels. Results: Of the 92 inflammation-related genes taken initially, four genes showed a differential expression across both groups with a p value of <0.05 from the data obtained using RNA processing of the patient sample. Said genes were TNFSF13B (p = 0.0448), ITGB2 (p = 0.0033), ANXA3 (p = 0.0479), and ANXA5 (p = 0.016). Conclusions: There are differences in inflammation-related gene expression in Down Syndrome patients when comparing patients who present a state of chronic oral inflammation with patients with negative rates of periodontal disease.


Assuntos
Síndrome de Down/imunologia , Síndrome de Down/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Anexina A3/genética , Anexina A3/metabolismo , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Feminino , Humanos , Integrina beta3/genética , Integrina beta3/metabolismo , Masculino , Doenças Periodontais/imunologia , Doenças Periodontais/metabolismo , Periodontite/imunologia , Periodontite/metabolismo
15.
Thromb Haemost ; 119(11): 1807-1815, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31587244

RESUMO

BACKGROUND: Fetal/neonatal alloimmune thrombocytopenia (FNAIT) results from maternal alloantibodies (abs) reacting with fetal platelets expressing paternal human platelet antigens (HPAs), mostly HPA-1a. Anti-HPA-1a abs, are the most frequent cause of severe thrombocytopenia and intracranial hemorrhage (ICH). OBJECTIVES: Titration of anti-HPA-1a in maternal serum using standard National Institute for Biological Standards and Control (NIBSC) 03/152 is one diagnostic approach to predict the severity of FNAIT. Recently, we found three anti-HPA-1a subtypes reacting with the ß3 subunit independently or dependently from complexes with αIIb and αv. Endothelial cell-reactive anti-αvß3 abs were found predominantly in cases with ICH. Our aim was to assess whether available standard material represents all anti-HPA-1a subtypes. MATERIALS AND METHODS: In this study, anti-HPA-1a sera (NIBSC 03/152) and human monoclonal antibodies (moabs) against HPA-1a (moabs 26.4 and 813) were evaluated using transfected cell lines expressing αIIbß3, αvß3 or monomeric cß3. RESULTS: Flow cytometry analyses with well-characterized murine moabs recognizing αIIbß3, αvß3, or ß3 alone demonstrated that AP3 reacts compound-independently, whereas compound-dependent moabs Gi5 and 23C6 reacted only with complexes. NIBSC 03/152, moabs 26.4, and 813 against HPA-1a reacted like AP3, same results were obtained with monomeric cß3 in immunoblotting. Antigen capture assay targeting endothelial cells showed anti-HPA-1a reactivity disappearance after cß3 beads adsorption. Furthermore, in contrast to anti-HPA-1a abs from ICH cases, none of NIBSC 03/152, 26.4, and 813 inhibited tube formation. CONCLUSION: These results suggest that current anti-HPA-1a standard material contains only the anti-ß3 subtype. The absence of anti-αvß3 makes NIBSC 03/152 less suitable as standard to predict the severity of FNAIT.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos de Plaquetas Humanas/imunologia , Testes Imunológicos , Integrina alfaVbeta3/imunologia , Integrina beta3/imunologia , Isoanticorpos/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Trombocitopenia Neonatal Aloimune/diagnóstico , Células Endoteliais/imunologia , Células HEK293 , Humanos , Isoanticorpos/sangue , Neovascularização Fisiológica , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Trombocitopenia Neonatal Aloimune/sangue , Trombocitopenia Neonatal Aloimune/imunologia
17.
Mol Vis ; 25: 237-254, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31516309

RESUMO

Purpose: The purpose of this study is to examine the expression profile of genes related to integrin-mediated phagocytosis that are altered by dexamethasone (DEX) and/or αvß3 integrin signaling to gain a better understanding of the molecular basis of phagocytosis and the pathophysiology of glucocorticoid-induced ocular hypertension. Methods: RNA and cell lysates were obtained from human trabecular meshwork (HTM) cells incubated with and without DEX for 4-5 d. The relative level of gene expression was evaluated using the Affymetrix Gene Chip® human gene microarray and quantitative PCR (qPCR). Changes in protein expression were validated using western blots or FACS analyses. The involvement of proteins in phagocytosis was determined using siRNA to knock down the expression of these proteins in an immortalized TM-1 cell line. Changes in the phagocytic activity were measured using pHrodo™-labeled S. aureus bioparticles followed by immunofluorescence microscopy. The effect of αvß3 integrin expression and activity on GULP1 mRNA levels was measured using qPCR in TM-1 cells overexpressing wild type or constitutively active αvß3 integrin. Results: Gene microarrays revealed statistically significant differences (>2 fold) in the expression of seven genes known to be involved in phagocytosis. Three genes (CD36, ABR, and GULP1) were downregulated, while four genes (ITGB3, CHN1, PIK3R1, and MFGE8) were upregulated. The genes were either associated with modulating RAC1 activity (ABR and CHN1) or integrin signaling (CD36, GULP1, ITGB3, PIK3R1, and MFGE8). Another gene, SIRPA, was also downregulated (1.6 fold) but only in one cell strain. qPCR and western blot analyses verified that DEX caused a decrease in SIRPA and GULP1 mRNA and their protein levels, while levels of CHN1 mRNA and its protein were upregulated by DEX. qPCR showed that although ABR mRNA was downregulated compared to non-treated controls after 5 d of treatment with DEX, no change at the protein level was detected. qPCR analysis also revealed that DEX caused an increase in MFGE8 mRNA levels. The levels of CD36 mRNA and protein varied between cell strains treated with DEX and were not statistically different compared to controls. The knockdown of GULP1 and ABR using siRNAs decreased phagocytosis by 40%. Interestingly, GULP1 mRNA levels were also decreased by 60% when αvß3 integrin was overexpressed in TM-1 cells. Conclusion: The DEX-induced inhibition of phagocytosis may be caused by the downregulation of ABR and GULP1 disrupting the αvß5 integrin/RAC1-mediated engulfment pathway. The downregulation of GULP1 by αvß3 integrin further suggests that this integrin may be a negative regulator of phagocytosis by transcriptionally downregulating proteins needed for phagocytosis. In summary, these results represent new insights into the effects of glucocorticoids and integrin signaling on the phagocytic process in the TM.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dexametasona/farmacologia , Proteínas Ativadoras de GTPase/metabolismo , Fagocitose , Proteômica , Malha Trabecular/citologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Antígenos CD/metabolismo , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Linhagem Celular , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Integrina beta3/metabolismo , Ligantes , Masculino , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Fagocitose/efeitos dos fármacos , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores de Vitronectina/metabolismo , Staphylococcus aureus/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
18.
Int J Med Sci ; 16(8): 1157-1170, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31523179

RESUMO

Background: Current opinion suggests that expansion of cancer stem cells (CSCs) and activation of pro-tumoral inflammation cascade correlate with cancer progression. Materials and methods: We explored the possible contributions of MRC-5 cancer-associated fibroblasts to the expression profiles of CSC markers and inflammation-associated cell surface molecules. The liver cancer cell lines Bel-7402, SMMC-7721, MHCC-LM3, and HepG2 cultured in conditioned medium (CM) from MRC-5 served as test groups, whereas the liver cancer cell lines cultured in normal medium served as control groups. Results: Flow cytometry revealed that the proportions of CD90+ cells were significantly higher in MHCC-LM3-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, and moderately higher in Bel-7402-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, than in controls. The CD90+/CD45- proportions were elevated in Bel-7402-(MRC-5)-CM and MHCC-LM3-(MRC-5)-CM cells, but reduced in HepG2-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, as compared to controls. Western blotting indicated that Nanog was downregulated in MHCC-LM3-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, compared to controls; that POU5F1 (OCT4/3) was downregulated in MHCC-LM3-(MRC-5)-CM, but upregulated in Bel-7402-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, compared to controls, and that CK19 was upregulated in Bel-7402-(MRC-5)-CM and MHCC-LM3-(MRC-5)-CM cells, compared to controls. Proportions of cells expressing Toll-like receptor-1+ (TLR1) and TLR4 were significantly higher in MHCC-LM3-(MRC-5)-CM cells, and moderately higher in HepG2-(MRC-5)-CM cells, than controls. However, the TLR1+ and TLR4+ proportions were lower in Bel-7402-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells than controls. Proportions of CD25+ cells were reduced in HepG2-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, but elevated in MHCC-LM3-(MRC-5)-CM and Bel-7402-(MRC-5)-CM cells, compared to controls. Proportion of CD61+ cells was higher in liver cancer cells cultured in MRC-5-CM than in controls. Proportion of CD14+ cells was lower in HCC cells cultured in MRC-5-CM than in controls. Conclusion: MRC-5 extensively affected the production of CSC markers and inflammation-associated cell surface molecules. Tumor-targeting molecular therapies should consider these findings.


Assuntos
Biomarcadores Tumorais/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Inflamação/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Citometria de Fluxo , Células Hep G2 , Humanos , Integrina beta3/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 1 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Microambiente Tumoral , Ensaio Tumoral de Célula-Tronco
20.
Int J Mol Sci ; 20(16)2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31426484

RESUMO

Estrogen and hypoxia promote an aggressive phenotype in prostate cancer (PCa), driving transcription of progression-associated genes. Here, we molecularly dissect the contribution of long non-coding RNA H19 to PCa metastatic potential under combined stimuli, a topic largely uncovered. The effects of estrogen and hypoxia on H19 and cell adhesion molecules' expression were investigated in PCa cells and PCa-derived organotypic slice cultures (OSCs) by qPCR and Western blot. The molecular mechanism was addressed by chromatin immunoprecipitations, overexpression, and silencing assays. PCa cells' metastatic potential was analyzed by in vitro cell-cell adhesion, motility test, and trans-well invasion assay. We found that combined treatment caused a significant H19 down-regulation as compared with hypoxia. In turn, H19 acts as a transcriptional repressor of cell adhesion molecules, as revealed by up-regulation of both ß3 and ß4 integrins and E-cadherin upon H19 silencing or combined treatment. Importantly, H19 down-regulation and ß integrins induction were also observed in treated OSCs. Combined treatment increased both cell motility and invasion of PCa cells. Lastly, reduction of ß integrins and invasion was achieved through epigenetic modulation of H19-dependent transcription. Our study revealed that estrogen and hypoxia transcriptionally regulate, via H19, cell adhesion molecules redirecting metastatic dissemination from EMT to a ß integrin-mediated invasion.


Assuntos
Regulação Neoplásica da Expressão Gênica , Integrina beta3/genética , Integrina beta4/genética , Neoplasias da Próstata/genética , RNA Longo não Codificante/metabolismo , Animais , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Estrogênios/metabolismo , Estrogênios/farmacologia , Humanos , Hipóxia , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/fisiopatologia , Ratos , Fatores de Transcrição/metabolismo , Transcrição Genética
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