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1.
J Hematol Oncol ; 13(1): 89, 2020 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-32653014

RESUMO

BACKGROUND: The oncolytic viruses have shown promising results for the treatment of multiple myeloma. However, the use of human viruses is limited by the patients' antiviral immune response. In this study, we investigated an alternative oncolytic strategy using non-human pathogen viruses as the bovine viral diarrhea virus (BVDV) that were able to interact with CD46. METHODS: We treated several human myeloma cell lines and non-myeloma cell lines with BVDV to evaluate the expression of CD46 and to study the effect on cell viability by flow cytometry. The possible synergistic effect of bortezomib in combination with BVDV was also tested. Moreover, we infected the bone marrow mononuclear cells obtained from myeloma patients and we checked the BVDV effect on different cell populations, defined by CD138, CD14, CD3, CD19, and CD56 expression evaluated by flow cytometry. Finally, the in vivo BVDV effect was tested in NOD-SCID mice injected subcutaneously with myeloma cell lines. RESULTS: Human myeloma cells were selectively sensitive to BVDV treatment with an increase of cell death and, consequently, of apoptotic markers. Consistently, bone marrow mononuclear cells isolated from myeloma patients treated with BVDV, showed a significant selective decrease of the percentage of viable CD138+ cells. Interestingly, bortezomib pre-treatment significantly increased the cytotoxic effect of BVDV in myeloma cell lines with a synergistic effect. Finally, the in vitro data were confirmed in an in vivo myeloma mouse model showing that BVDV treatment significantly reduced the tumoral burden compared to the vehicle. CONCLUSIONS: Overall, our data indicate, for the first time, a direct oncolytic effect of the BVDV in human myeloma cells suggesting its possible use as novel alternative anti-myeloma virotherapy strategy.


Assuntos
Vírus da Diarreia Viral Bovina , Mieloma Múltiplo/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos CD/análise , Apoptose , Células da Medula Óssea/química , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/virologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Vírus da Diarreia Viral Bovina/fisiologia , Feminino , Herpesvirus Bovino 4 , Humanos , Masculino , Proteína Cofatora de Membrana/biossíntese , Proteína Cofatora de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Vírus Oncolíticos/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Organismos Livres de Patógenos Específicos
2.
Mol Immunol ; 96: 83-87, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29525453

RESUMO

During infection and budding, human immunodeficiency virus-1 (HIV-1) acquires regulators of Complement Activation (RCAs) along with the host cell membrane on the viral envelope. Activation of host complement system results in opsonization of virus by complement fragments, however the virus evades complement mediated lysis (CoML) by virtue of the RCAs on the viral envelope. The RCAs on HIV-1 envelope process complement protein C3 into various fragments that promote viral entry and infection of cells through different complement receptors. Complement opsonized HIV-1 has been shown in vitro to infect dendritic cells (DCs) in a CR3 dependent manner, although the role of CR3 and CD46 in natural HIV-1 infection is not clear. Surface expression of CR3 and CD46 on DC subsets of 30 antiretroviral naïve, 31 treated (cART) HIV-1 infected individuals and 30 seronegative controls was measured by flow cytometry and plasma levels of cytokines and complement activity (C3c levels) were quantitated by sandwich ELISA. Significantly lower surface expression of CR3 and CD46 was observed on DC subsets in naïve and treated HIV-1 infected individuals compared to controls. Significantly higher complement activation and plasma levels of IL-4, IL-8, IL-10 and IFN-γ were observed in treatment naïve HIV-1 infected individuals than controls. Significantly lower plasma levels of IL-4, IL-6, IL-8 and IL-10 were observed in treated vs. naïve HIV-1 infected individuals. Our findings suggest that alterations in expression of CR3 and CD46 on DCs along with complement activity could be factors that influence viral persistence and HIV-1 disease progression and need to be further evaluated.


Assuntos
Células Dendríticas/imunologia , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Antígeno de Macrófago 1/biossíntese , Proteína Cofatora de Membrana/biossíntese , Adulto , Fármacos Anti-HIV/uso terapêutico , Células Dendríticas/metabolismo , Feminino , Infecções por HIV/tratamento farmacológico , HIV-1 , Humanos , Masculino
3.
Blood ; 128(18): 2206-2217, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27554082

RESUMO

Current protocols for hematopoietic stem/progenitor cell (HSPC) gene therapy, involving the transplantation of ex vivo genetically modified HSPCs are complex and not without risk for the patient. We developed a new approach for in vivo HSPC transduction that does not require myeloablation and transplantation. It involves subcutaneous injections of granulocyte-colony-stimulating factor/AMD3100 to mobilize HSPCs from the bone marrow (BM) into the peripheral blood stream and the IV injection of an integrating, helper-dependent adenovirus (HD-Ad5/35++) vector system. These vectors target CD46, a receptor that is uniformly expressed on HSPCs. We demonstrated in human CD46 transgenic mice and immunodeficient mice with engrafted human CD34+ cells that HSPCs transduced in the periphery home back to the BM where they stably express the transgene. In hCD46 transgenic mice, we showed that our in vivo HSPC transduction approach allows for the stable transduction of primitive HSPCs. Twenty weeks after in vivo transduction, green fluorescent protein (GFP) marking in BM HSPCs (Lin-Sca1+Kit- cells) in most of the mice was in the range of 5% to 10%. The percentage of GFP-expressing primitive HSPCs capable of forming multilineage progenitor colonies (colony-forming units [CFUs]) increased from 4% of all CFUs at week 4 to 16% at week 12, indicating transduction and expansion of long-term surviving HSPCs. Our approach was well tolerated, did not result in significant transduction of nonhematopoietic tissues, and was not associated with genotoxicty. The ability to stably genetically modify HSPCs without the need of myeloablative conditioning is relevant for a broader clinical application of gene therapy.


Assuntos
Terapia Genética/métodos , Mobilização de Células-Tronco Hematopoéticas/métodos , Proteína Cofatora de Membrana/biossíntese , Transdução Genética/métodos , Adenoviridae , Animais , Vetores Genéticos/administração & dosagem , Células-Tronco Hematopoéticas , Xenoenxertos , Humanos , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos C57BL
4.
Oncotarget ; 7(25): 38210-38223, 2016 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-27203670

RESUMO

CD46 is a complement inhibitor membrane cofactor which also acts as a receptor for various microbes, including species B adenoviruses (Ads). While most Ad gene therapy vectors are derived from species C and infect cells through coxsackie-adenovirus receptor (CAR), CAR expression is downregulated in many cancer cells, resulting inefficient Ad-based therapeutics. Despite a limited knowledge on the expression status of many cancer cells, an increasing number of cancer gene therapy studies include fiber-modified Ad vectors redirected to the more ubiquitously expressed CD46. Since our finding from tumor microarray indicate that CD46 was overexpressed in cancers of the prostate and colon, fiber chimeric Ad5/35 vectors that have infection tropism for CD46 were employed to demonstrate its efficacy in colorectal cancers (CRC). CD46-overexpressed cells showed a significantly higher response to Ad5/35-GFP and to Ad5/35-tk/GCV. While CRC cells express variable levels of CD46, CD46 expression was positively correlated with Ad5/35-mediated GFP fluorescence and accordingly its cell killing. Injection of Ad5/35-tk/GCV caused much greater tumor-suppression in mice bearing CD46-overexpressed cancer xenograft compared to mock group. Analysis of CRC samples revealed that patients with positive CD46 expression had a higher survival rate (p=0.031), carried tumors that were well-differentiated, but less invasive and metastatic, and with a low T stage (all p<0.05). Taken together, our study demonstrated that species B-based adenoviral gene therapy is a suitable approach for generally CD46-overexpressed CRC but would require careful consideration preceding CD46 analysis and categorizing CRC patients.


Assuntos
Adenoviridae/genética , Neoplasias Colorretais/terapia , Terapia Genética/métodos , Proteína Cofatora de Membrana/biossíntese , Idoso , Animais , Células CACO-2 , Quimerismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/virologia , Feminino , Vetores Genéticos/genética , Células HCT116 , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Invest Ophthalmol Vis Sci ; 56(8): 4231-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26161984

RESUMO

PURPOSE: Loss of CD46 has recently been implicated in choroidal neovascularization in mice. Herein we investigated the effect of nitrite modification of the extracellular matrix (ECM) as an in vitro model of "aging" and its effect on CD46 expression and vascular endothelial growth factor (VEGF) release in cocultured human retinal pigment epithelium (RPE). METHODS: ARPE-19 cells were plated onto RPE-derived ECM conditions (untreated; nitrite modified; nitrite modified followed by washing with Triton X-100; or nitrite modified followed by washing with Triton X-100 and coated with extracellular matrix ligands). Cells were cultured for 7 days and CD46 expression was analyzed by immunohistochemistry and Western blot. Additionally, CD46 short interfering RNA (siRNA) was transfected into ARPE-19 cells, and VEGF levels were determined by ELISA. Finally, in the same ECM conditions, ARPE-19 cells were challenged with normal human serum and VEGF levels determined by ELISA. RESULTS: CD46 is expressed on the basolateral surface of ARPE-19 cells on RPE-derived ECM. Nitrite modification of ECM reduced the expression of CD46 on ARPE-19 cells by 0.5-fold (P = 0.003) and increased VEGF release in ARPE-19 cells by 1.7-fold (P < 0.001). CD46 knockdown also increased release of VEGF on the apical and basal sides of ARPE-19 cells in culture by 1.3- (P = 0.012) and 1.2-fold (P = 0.017), respectively. CONCLUSIONS: Nitrite modification of the ECM decreased CD46 expression and increased the release of VEGF from ARPE-19 cells. Changes in CD46 expression may lead to changes in VEGF and play a pathologic role in the development of age-related macular degeneration.


Assuntos
Neovascularização de Coroide/genética , DNA/genética , Regulação da Expressão Gênica , Proteína Cofatora de Membrana/genética , Nitritos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Western Blotting , Células Cultivadas , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/metabolismo , Humanos , Proteína Cofatora de Membrana/biossíntese , Camundongos , Camundongos Knockout , Microscopia Confocal , Epitélio Pigmentado da Retina/patologia
6.
PLoS One ; 10(2): e0114856, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25658605

RESUMO

OBJECTIVE: The role of complement system in the pathogenesis of systemic sclerosis (SSc) has been debated during the last decade but an evident implication in this disease has never been found. We carried out an explorative study on SSc patients to evaluate the expression of soluble and local C5b-9 complement complex and its relation with a complement regulator, the Membrane Cofactor Protein (MCP, CD46) on skin vascular bed as target distinctive of SSc disease. We also analyzed two polymorphic variants in the complement activation gene cluster involving the MCP region. METHODS: C5b-9 plasma levels of SSc patients and healthy subjects were analyzed by ELISA assay. Archival skin biopsies of SSc patients and controls were subjected to immunofluorescence analysis to detect C5b-9 and MCP on vascular endothelial cells. The expression of MCP was validated by immunoblot analysis with specific antibody. Polymorphic variants in the MCP gene promoter were tested by a quantitative PCR technique-based allelic discrimination method. RESULTS: Even though circulating levels of C5b-9 did not differ between SSc and controls, C5b-9 deposition was detected in skin biopsies of SSc patients but not in healthy subjects. MCP was significantly lower in skin vessels of SSc patients than in healthy controls and was associated with the over-expression of two polymorphic variants in the MCP gene promoter, which has been related to more aggressive phenotypes in other immune-mediated diseases. CONCLUSIONS: Our results firsty document the local complement activation with an abnormal expression of MCP in skin vessels of SSc patients, suggesting that a subset of SSc patients might be exposed to more severe organ complications and clinical evolution due to abnormal local complement activation.


Assuntos
Ativação do Complemento/genética , Células Endoteliais , Regulação da Expressão Gênica , Proteína Cofatora de Membrana , Polimorfismo Genético , Regiões Promotoras Genéticas , Escleroderma Sistêmico , Idoso , Complexo de Ataque à Membrana do Sistema Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Feminino , Humanos , Masculino , Proteína Cofatora de Membrana/biossíntese , Proteína Cofatora de Membrana/genética , Pessoa de Meia-Idade , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Pele/irrigação sanguínea , Pele/patologia
7.
Oncol Rep ; 32(6): 2619-27, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25241923

RESUMO

Complement-dependent cytotoxicity (CDC) is an important antitumor mechanism of monoclonal antibodies (mAbs). However, trastuzumab, an anti-HER2 mAb, exerts only minor CDC. Overexpression of membrane-bound complement regulatory proteins (mCRPs), which suppress CDC, have been implicated in various malignant tumors. Here, we explored the predictive role of the expression levels of three mCRPs (CD55, CD59 and CD46) in the prognosis of breast cancer cases that underwent adjuvant trastuzumab treatment. We also studied the effect of mCRP downregulation on trastuzumab-induced CDC in vitro. Sixty-five HER2-positive breast cancer patients who received adjuvant therapy containing trastuzumab, were retrospectively analyzed. Levels of CD55, CD59 and CD46 expression were detected by immunohistochemistry. Chi-square test, Kaplan­Meier survival analysis and a Cox proportional hazards model were used to analyze the association between CD55, CD59 and CD46 expression and prognosis. HER2-positive SK-Br3 and BT-474 breast cancer cells were pretreated with various drugs to reduce mCRP expression. Afterwards, trastuzumab­mediated cytolytic effects were measured. Among the 65 patients, 46.2% had high expression of CD55, 44.6% had high expression of CD59 and 44.6% had high expression of CD46. The median follow-up duration was 47 months (range from 24 to 75 months). Patients with CD55 or CD59 overexpression had a higher relapse rate than those with low expression of CD55 (33.3 vs. 8.6%; P=0.013) or CD59 (31.0 vs. 11.1%; P=0.046). Similarly, mean disease-free survival of patients with CD55 or CD59 overexpression was significantly shorter than those with a low expression of CD55 (56 vs. 70 months; log-rank test, P=0.008) or CD59 (56 vs. 69 months; log-rank test, P=0.033). Multivariate analysis confirmed that CD55, but not CD59, was an independent risk factor of recurrence (HR=4.757; 95% CI, 0.985-22.974; P=0.05). In vitro, we found that tamoxifen inhibited both the protein and mRNA expression levels of CD55, but not CD59 or CD46 in SK-Br3 and BT-474 cells. After pretreatment of tamoxifen, trastuzumab-induced cytolysis was enhanced through CD55 downregulation. In conclusion, CD55 overexpression is an independent risk factor for recurrence in breast cancer patients receiving postoperative adjuvant therapy containing trastuzumab. Combined use of tamoxifen and trastuzumab for HER2-positive breast cancer treatment may enhance the antitumor effects of trastuzumab by elevated CDC, which warrants further study.


Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Proteínas do Sistema Complemento/biossíntese , Recidiva Local de Neoplasia/tratamento farmacológico , Prognóstico , Adulto , Idoso , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Antígenos CD55/biossíntese , Antígenos CD55/genética , Antígenos CD59/biossíntese , Proteínas do Sistema Complemento/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína Cofatora de Membrana/biossíntese , Proteína Cofatora de Membrana/genética , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Estudos Retrospectivos , Trastuzumab
8.
PLoS One ; 9(8): e104293, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25101906

RESUMO

Gene expression is a dynamic and coordinated process coupling transcription with pre-mRNA processing. This regulation enables tissue-specific transcription factors to induce expression of specific transcripts that are subsequently amplified by alternative splicing allowing for increased proteome complexity and functional diversity. The intestine-specific transcription factor CDX2 regulates development and maintenance of the intestinal epithelium by inducing expression of genes characteristic of the mature enterocyte phenotype. Here, sequence analysis of CDX2 mRNA from colonic mucosa-derived tissues revealed an alternatively spliced transcript (CDX2/AS) that encodes a protein with a truncated homeodomain and a novel carboxy-terminal domain enriched in serine and arginine residues (RS domain). CDX2 and CDX2/AS exhibited distinct nuclear expression patterns with minimal areas of co-localization. CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2. Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35. CDX2/AS altered splicing patterns of CD44v5 and Tra2-ß1 minigenes in Lovo colon cancer cells independent of CDX2 expression. These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2) and pre-mRNA processing (CDX2/AS).


Assuntos
Processamento Alternativo/fisiologia , Colo/metabolismo , Enterócitos/metabolismo , Proteínas de Homeodomínio/biossíntese , Animais , Fator de Transcrição CDX2 , Linhagem Celular Tumoral , Colo/citologia , Enterócitos/citologia , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/genética , Proteína Cofatora de Membrana/biossíntese , Proteína Cofatora de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo
9.
J Immunol Methods ; 408: 24-34, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24810583

RESUMO

We here report the production and purification of the extracellular domains of two Fcγ receptors, namely CD16a and CD64, by transient transfection in mammalian cells. The use of these two receptor ectodomains for the development of quantitative assays aiming at controlling the quality of monoclonal antibody production lots is then discussed. More specifically, the development of surface plasmon resonance-based biosensor assays for the evaluation of the glycosylation pattern and the aggregation state of monoclonal antibodies is presented. Our biosensor approach allows discriminating between antibodies harboring different galactosylation profiles as well as to detect low levels (i.e., less than 2%) of monoclonal antibody aggregates.


Assuntos
Anticorpos Monoclonais/metabolismo , Técnicas Biossensoriais , Proteína Cofatora de Membrana/imunologia , Processamento de Proteína Pós-Traducional , Receptores de IgG/imunologia , Ressonância de Plasmônio de Superfície , Animais , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Glicosilação , Células HEK293 , Humanos , Cinética , Proteína Cofatora de Membrana/biossíntese , Proteína Cofatora de Membrana/genética , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de IgG/biossíntese , Receptores de IgG/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transfecção
10.
Mol Immunol ; 60(1): 14-22, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24732065

RESUMO

Inferior tendon healing can lead to scarring and tendinopathy. The role of complement in tendon healing is still unclear. The aim of this study was to understand tenocytes response to mechanical injury and whether complement is regulated by injury. Tenocytes were injured using an optimized automated scratch assay model. Using a self-assembled plotter system, 50 parallel lines of injury were created in a 6 cm diameter tenocyte cell layer. Tenocytes mitotic activity and survival post injury was assessed using FDA/ethidiumbromide assay. Furthermore, this injury model was combined with stimulation of the tenocytes with the complement split fragment C3a. Gene expression of C3aR, C5aR (CD88), CD46, CD55, tumor necrosis factor (TNF)α, interleukin (IL)-1ß, matrix metalloproteinase (MMP)-1 was analyzed. Immunolabeling for C5aR and CD55 was performed. An enhanced mitotic activity and some dead cells were detected in the vicinity of the scratches. Gene expression of the C3aR was suppressed after 4 h but induced after 24 h post injury. C5aR was down-regulated at 24 h, CD46 and CD55 were induced at 24 h in response to injury and CD55 was also elevated at 4 h. MMP-1 was upregulated by injury but both proinflammatory cytokines remained mainly unaffected. Combination of injury with C3a stimulation led to an enhanced C3aR, CD55 and TNFα gene expression. According to the gene expression data, the protein expression of C5aR was reduced and that of CD55 induced. In summary, a specific response of complement regulation was found in mechanically injured tenocytes which may be involved in healing responses.


Assuntos
Proteínas do Sistema Complemento/imunologia , Traumatismos dos Tendões/imunologia , Tendões/imunologia , Cicatrização/imunologia , Antígenos CD55/biossíntese , Proliferação de Células , Sobrevivência Celular/imunologia , Células Cultivadas , Complemento C3a/farmacologia , Expressão Gênica , Humanos , Interleucina-1beta/biossíntese , Metaloproteinase 1 da Matriz/biossíntese , Proteína Cofatora de Membrana/biossíntese , RNA Mensageiro/biossíntese , Receptor da Anafilatoxina C5a/biossíntese , Receptores de Complemento/biossíntese , Tendões/citologia , Fator de Necrose Tumoral alfa/biossíntese
11.
Oncol Rep ; 31(2): 557-64, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24297460

RESUMO

The therapeutic potential of membrane complement regulatory protein (mCRP)-neutralizing antibodies is unsatisfactory, which perhaps lies in the complex role of mCRPs in tumor occurrence and development. As a member of the mCRPs, CD46 is a transmembrane protein with a cytoplasmic domain and is implicated more in the control of the alternative complement pathway than of the classical complement pathway. Growing evidence has revealed that both the CD46 signaling pathway and microRNAs (miRNAs) play an important role in the development and progression of hepatocellular carcinoma (HCC). In the present study, we analyzed mCRP expression in different tumor tissues by employing western blotting and qPCR. To address the potential role of miRNAs in CD46 signaling, we set out to profile miRNA expression in CD46-overexpressed and -silenced HepG2 cell lines. Furthermore, bioinformatic analysis was performed to identify downstream targets of CD46 signaling. We found that the levels of CD46 expression in HCC tissues were significantly higher compared to that in the adjacent normal tissues. After complement-related gene expression profiling and unsupervised hierarchical clustering analysis of 10 HCC tissues, a total of 37 miRNAs showed significantly different expression levels before and after CD46 expression change. By bioinformatic analysis, we identified let-7b and miR-17 as downstream targets of CD46 signaling, and that the expression levels of let-7b and miR-17 were negatively correlated with that of CD46 in HepG2 cells. The present study suggests that CD46 plays an important role in HCC carcinogenesis by regulating let-7b and miR-17.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteína Cofatora de Membrana/genética , MicroRNAs/genética , Linhagem Celular Tumoral , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Proteína Cofatora de Membrana/biossíntese , MicroRNAs/biossíntese , Interferência de RNA , RNA Interferente Pequeno
12.
J Immunol ; 191(8): 4431-9, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24043901

RESUMO

Obliterative bronchiolitis (OB) post-lung transplantation involves IL-17-regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB is unknown. The current study examines the role of complement activation in OB. Complement-regulatory protein (CRP) (CD55, CD46, complement receptor 1-related protein y/CD46) expression was downregulated in human and murine OB; and C3a, a marker of complement activation, was upregulated locally. IL-17 differentially suppressed complement receptor 1-related protein y expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen- or autoantigen (type V collagen)-reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17-mediated downregulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed-forward loop that may enhance CRP downregulation, suggesting that complement blockade could be a therapeutic strategy for OB.


Assuntos
Bronquiolite Obliterante/imunologia , Ativação do Complemento , Rejeição de Enxerto/imunologia , Interleucina-17/metabolismo , Transplante de Pulmão/efeitos adversos , Animais , Autoimunidade , Líquido da Lavagem Broncoalveolar , Antígenos CD55/biossíntese , Colágeno Tipo V/imunologia , Complemento C3a/biossíntese , Complemento C5 , Regulação para Baixo , Humanos , Interleucina-17/biossíntese , Interleucina-17/imunologia , Interleucina-6/biossíntese , Teste de Cultura Mista de Linfócitos , Proteína Cofatora de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Complemento/biossíntese , Receptores de Complemento 3b
13.
Int J Oncol ; 42(4): 1337-48, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23403990

RESUMO

To overcome the poor tumor transduction efficiency of adenovirus serotype 5 (Ad5) observed in several types of cancer, the fiber region of Ad5, apart from its tail, was replaced by adenovirus serotype 35 (Ad35). The chimeric Ad5/F35 adenoviral vector did not exhibit any significant enhancement of transduction efficiency. CD46, a receptor for Ad35, was expressed in relatively small amounts in most of the cancer cells examined. Therefore, we investigated the pivotal factor(s) that render cancer cells susceptible to transduction. We discovered that the tumor transduction efficiency of Ad5/F35 was enhanced in the presence of rapamycin, an autophagy inducer, in some cancer cells. Analysis of survival potential and cell proliferation rates revealed that Ad5/F35 exerted a more pronounced oncolytic effect in cancer cells with higher survival potential in the presence of rapamycin.


Assuntos
Adenoviridae/genética , Autofagia , Sobrevivência Celular , Vírus Oncolíticos/genética , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Citomegalovirus/genética , Vetores Genéticos , Humanos , Proteína Cofatora de Membrana/biossíntese , Proteína Cofatora de Membrana/genética , Terapia Viral Oncolítica , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Recoverina/biossíntese , Recoverina/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Transdução Genética , Proteína bcl-X/metabolismo
14.
Anticancer Res ; 32(9): 3743-7, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22993314

RESUMO

BACKGROUND: Adenovirus vectors have been utilized for cancer gene therapies. The present study examined the oncolytic effects of adenovirus type 5 (Ad5) and fiber-substituted conditionally replicating adenovirus (CRAD) Ad5/F35 vectors on the human malignant mesothelioma cells MSTO-211H, NCI-H28, NCI-H2052, and NCI-H2452 cells. MATERIALS AND METHOD: For the adenovirus, the first mRNA/protein to be made (~1 h after infection) is E1A. Ad5F35 and Ad5 CRAD vectors containing the E1 gene controlled by the human midkine promoter (Ad5F35/MKp-E1 and Ad5/MKp-E1, respectively) were constructed. Western blotting and cell viability assays were carried out in cells transfected with Ad5/MKp-E1 and Ad5F35/MKp-E1. RESULTS: Coxsackie and adenovirus receptor (CAR), a cell surface target of Ad5, and CD46, a cell surface target of Ad35, were expressed in all the malignant mesothelioma cell lines examined here, as much as in HEK293 cells, with no significant differences in the expression levels among cells. Both Ad5/MKp-E1 and Ad5F35/MKp-E1 induced oncolysis of malignant mesothelioma cells in a viral particle-dependent manner, with similar efficacy. CONCLUSION: The results of the present study suggest that both Ad5/MKp-E1 and Ad5F35/MKp-E1 are useful for the gene therapy of human malignant mesothelioma.


Assuntos
Adenovírus Humanos/fisiologia , Terapia Genética/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Mesotelioma/genética , Mesotelioma/terapia , Terapia Viral Oncolítica/métodos , Proteínas E1 de Adenovirus/genética , Adenovírus Humanos/genética , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/biossíntese , Células HEK293 , Humanos , Neoplasias Pulmonares/virologia , Proteína Cofatora de Membrana/biossíntese , Mesotelioma/virologia , Mesotelioma Maligno , Midkina , Fatores de Crescimento Neural/genética , Regiões Promotoras Genéticas , Transfecção , Replicação Viral
15.
Anticancer Res ; 32(7): 2985-9, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22753762

RESUMO

BACKGROUND: Adenovirus vectors have lately been highlighted in gene therapies. We investigated the oncolytic effects of a chimeric adenovirus type 5 (Ad5) with replacement of Ad5 fiber knob with adenovirus type 35 (Ad35) fiber knob (Ad5F35) on human renal cell carcinoma (RCC). MATERIALS AND METHODS: The conditionally replicating Ad5F35 vector was constructed and infected into RCC cell lines 786-O, ACHN, and RCC4-VHL. For these cells, reverse transcription-polymerase chain reaction and western blotting were carried out and the cell viability was assayed. RESULTS: In all RCC cell lines, it was found that CD46, a cell surface target of Ad35, was well-expressed, while coxsackie and adenovirus receptor (CAR), a cell surface target of Ad5, was considerably less expressed. The Ad5F35 vector induced oncolysis of RCC cells, with significantly higher efficacy as compared with that for the Ad5 vector. CONCLUSION: Ad5F35 vector could be a candidate for promising gene therapy of human RCC.


Assuntos
Adenoviridae/fisiologia , Carcinoma de Células Renais/terapia , Carcinoma de Células Renais/virologia , Neoplasias Renais/terapia , Neoplasias Renais/virologia , Terapia Viral Oncolítica/métodos , Adenoviridae/genética , Proteínas E1 de Adenovirus/genética , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Citocinas/genética , Terapia Genética/métodos , Vetores Genéticos , Células HEK293 , Humanos , Proteína Cofatora de Membrana/biossíntese , Proteína Cofatora de Membrana/genética , Midkina , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Virais/biossíntese , Receptores Virais/genética , Replicação Viral
16.
J Immunol ; 188(11): 5303-10, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22544928

RESUMO

The ubiquitous protein CD46, a regulator of complement activity, promotes T cell activation and differentiation toward a regulatory Tr1-like phenotype. The CD46-mediated differentiation pathway is defective in several chronic inflammatory diseases, underlying the importance of CD46 in controlling T cell function and the need to understand its regulatory mechanisms. Using an RNA interference-based screening approach in primary T cells, we have identified that two members of the G protein-coupled receptor kinases were involved in regulating CD46 expression at the surface of activated cells. We have investigated the role of PGE(2), which binds to the E-prostanoid family of G protein-coupled receptors through four subtypes of receptors called EP 1-4, in the regulation of CD46 expression and function. Conflicting roles of PGE(2) in T cell functions have been reported, and the reasons for these apparent discrepancies are not well understood. We show that addition of PGE(2) strongly downregulates CD46 expression in activated T cells. Moreover, PGE(2) differentially affects T cell activation, cytokine production, and phenotype depending on the activation signals received by the T cells. This was correlated with a distinct pattern of the PGE(2) receptors expressed, with EP4 being preferentially induced by CD46 activation. Indeed, addition of an EP4 antagonist could reverse the effects observed on cytokine production after CD46 costimulation. These data demonstrate a novel role of the PGE(2)-EP4 axis in CD46 functions, which might at least partly explain the diverse roles of PGE(2) in T cell functions.


Assuntos
Dinoprostona/fisiologia , Ativação Linfocitária/imunologia , Proteína Cofatora de Membrana/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Proliferação de Células , Células Cultivadas , Dinoprostona/metabolismo , Receptor Quinase 1 Acoplada a Proteína G/fisiologia , Regulação da Expressão Gênica/imunologia , Humanos , Proteína Cofatora de Membrana/antagonistas & inibidores , Proteína Cofatora de Membrana/biossíntese , Interferência de RNA/imunologia , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo
17.
Cancer Gene Ther ; 19(2): 118-25, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22076042

RESUMO

Carrier cells delivering a conditionally replicating adenovirus (CRAd), which selectively replicates in tumor cells and induces tumor cell lysis, have promising potential for treatment of cancer because CRAd-loaded carrier cells evade inhibition by neutralizing anti-adenovirus (Ad) antibodies and because the carrier cells are locally retained at the injection point after local injection. A previous study by Hamada et al. demonstrated that carrier cells (CRAd-containing cell fragments derived from the carrier cells) are engulfed into the target cells, probably through a pathway independent of the primary receptor for Ad, the coxsackievirus and Ad receptor (CAR) (Mol Ther, 15: 1121-1128; 2007); however, it remains to be elucidated whether carrier cells infected with a conventional CRAd, which is composed of subgroup-C Ad serotype-5 (Ad5), mediate antitumor effects on CAR-negative cells. In order to examine whether carrier cells delivering a conventional CRAd (Carrier-F5) induce lysis of CAR-negative tumor cells, CAR-positive and CAR-negative tumor cells were incubated with Carrier-F5. Carrier-F5 mediated efficient killing of CAR-positive tumor cells; however, CAR-negative tumor cells were almost refractory to Carrier-F5. On the other hand, carrier cells loaded with a fiber-substituted CRAd containing fiber proteins of Ad serotype-35 (Ad35) (CRAd-F35), which binds to human CD46 for infection, showed efficient killing of both CAR-positive and CAR-negative tumor cells. Intra-tumoral injection of carrier cells loaded with CRAd-F35 (Carrier-F35) also resulted in efficient regression of both CAR-positive and CAR-negative tumors. These results demonstrated that the expression levels of receptors for Ad are an important factor for CRAd-loaded carrier cell-mediated cancer therapy, and that Carrier-F35 would have potential as a cancer treatment for not only CAR-positive tumors but also CAR-negative tumors.


Assuntos
Adenocarcinoma/terapia , Adenocarcinoma/virologia , Adenoviridae/fisiologia , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/virologia , Terapia Viral Oncolítica/métodos , Receptores Virais/deficiência , Neoplasias da Bexiga Urinária/terapia , Neoplasias da Bexiga Urinária/virologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Adenoviridae/genética , Animais , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Vetores Genéticos , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteína Cofatora de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Receptores Virais/biossíntese , Transdução Genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Replicação Viral , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Life Sci ; 90(1-2): 47-53, 2012 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-22056374

RESUMO

AIMS: Endothelial dysfunction is a common manifestation of chronic kidney disease (CKD). The protein-bound uremic toxins have emerged as important factors associated with cardiovascular disease and the outcome of CKD. The effect of indoxyl sulfate (IS) on endothelial cells remains unclear. MAIN METHODS: Human umbilical endothelial cells (HUVEC) were incubated using IS at two concentrations: 100 µM and 1000 µM over two periods of time: 16 and 48 h. HUVEC were also pre-treated with simvastatin to examine its effect. RT-PCR was used to assess changes in the gene expression of intracellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), Monocyte chemotactic protein-1 (MCP-1), E-selectin, and angiotensin receptor type 1 (AT1R). Protein abundance of the investigated molecules was assessed by immunoblotting. KEY FINDINGS: Treatment with 100 µM IS for 16 h induced a 2-fold increase in the expression of ICAM-1, VCAM-1, and MCP-1. At a concentration of 1000 µM, there was a 2-3-fold increase. An extended treatment period at low concentrations was associated with a 2-3 fold increase and the increase of ICAM-1 and VCAM-1 was more prominent under high concentration. Results of immunoblotting confirmed an increase in the abundance of ICAM-1, VCAM-1 and MCP-1. No significant change was noted in E-selectin and AT1R according to concentration or treatment duration. Pre-treatment with simvastatin did not alter IS-induced changes. SIGNIFICANCE: IS increased the expression of adhesion molecules of endothelial cells exhibiting a concentration and duration dependent pattern. Simvastatin did not demonstrate any effect on IS-associated endothelial activation.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Indicã/toxicidade , Sinvastatina , Células Cultivadas , Endotélio Vascular/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Indicã/metabolismo , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Proteína Cofatora de Membrana/biossíntese , Proteína Cofatora de Membrana/genética , Sinvastatina/farmacologia , Molécula 1 de Adesão de Célula Vascular/biossíntese , Molécula 1 de Adesão de Célula Vascular/genética
19.
Rev Bras Reumatol ; 51(5): 503-10, 2011.
Artigo em Inglês, Português | MEDLINE | ID: mdl-21953001

RESUMO

Rheumatoid arthritis (RA) is an autoimmune disease associated with polyarticular inflammatory synovitis affecting mainly peripheral joints. It affects approximately 1% of the world population, being two to three times more prevalent in women. Rheumatoid arthritis has a complex and multifactorial pathogenesis. The synovium of the affected joints is infiltrated by T and B lymphocytes, macrophages, and granulocytes. The rheumatoid synovium has proliferative characteristics, forming the pannus, which invades cartilage and bone, leading to normal architecture destruction and function loss. The decreased expression of complement regulatory proteins (CRP) seems to play an important role in RA activity, and is associated with worsening of the clinical symptoms. In several models of autoimmune diseases, the overactivation of the complement system (CS) is the cause of disease exacerbation. This article aimed at reviewing the main aspects related to CS regulation in RA in order to provide a better understanding of the potential role of this system in the pathophysiology and activity of the disease.


Assuntos
Artrite Reumatoide/imunologia , Antígenos CD55/biossíntese , Antígenos CD59/biossíntese , Proteína Cofatora de Membrana/biossíntese , Receptores de Complemento 3b/biossíntese , Humanos
20.
Hum Reprod ; 26(11): 2944-9, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21857011

RESUMO

We here report a successful pregnancy and healthy childbirth obtained in a case of total globozoospermia after intracytoplasmic morphologically selected sperm injection (IMSI) without assisted oocyte activation (AOA). Two semen analyses showed 100% globozoospermia on classic spermocytogram. Motile sperm organelle morphology examination (MSOME) analysis at ×10,000 magnification confirmed the round-headed aspect for 100% of sperm cells, but 1% of the spermatozoa seemed to present a small bud of acrosome. This particular aspect was confirmed by transmission electron microscopy and anti-CD46 staining analysis. Results from sperm DNA fragmentation and fluorescence in situ hybridization analyses were normal. The karyotype was 46XY, and no mutations or deletions in SPATA16 and DPY19L2 genes were detected. Considering these results, a single IMSI cycle was performed, and spermatozoa were selected for the absence of vacuoles and the presence of a small bud of acrosome. A comparable fertilization rate with or without calcium-ionophore AOA was observed. Two fresh top-quality embryos obtained without AOA were transferred at Day 2 after IMSI, leading to pregnancy and birth of a healthy baby boy. This successful outcome suggests that MSOME may be useful in cases of globozoospermia in order to carefully evaluate sperm morphology and to maximize the benefit of ICSI/IMSI.


Assuntos
Oócitos/citologia , Injeções de Esperma Intracitoplásmicas/métodos , Espermatozoides/patologia , Reação Acrossômica , Adulto , Feminino , Humanos , Recém-Nascido , Ionóforos/farmacologia , Cariotipagem , Masculino , Proteína Cofatora de Membrana/biossíntese , Oligospermia/patologia , Gravidez , Resultado da Gravidez , Técnicas de Reprodução Assistida , Sêmen/metabolismo
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