RESUMO
Acute exacerbation of chronic obstructive pulmonary disease (COPD) is often induced by infection and often has a poor prognosis. Bacterial LPS activates innate immune receptor TLR4 followed by activation of a transcriptional factor IFN regulatory factor-3 (IRF3) as well as NF-κB, resulting in upregulation of various inflammatory mediators. To clarify the role of IRF3 in the pathogenesis of LPS-triggered COPD exacerbation, porcine pancreatic elastase (PPE) followed by LPS was administered intranasally to wild-type (WT) or IRF3-/- male mice. Sequential quantitative changes in emphysema were evaluated by microcomputed tomography, and lung histology was evaluated at the sixth week. WT mice treated with PPE and LPS exhibited enlarged alveolar spaces, whereas this feature was attenuated in similarly treated IRF3-/- mice. Moreover, LPS-induced emphysema aggravation was detected only in WT mice. Analysis of acute inflammation induced by PPE plus LPS revealed that the lungs of treated IRF3-/- mice had decreased mRNA transcripts for MCP-1, MIP-1α, TNF-α, and IFN-γ-inducible protein-10 but had increased neutrophils. IRF3 was involved in the production of mediators from macrophages, alveolar epithelial cells, and neutrophils. Furthermore, compared with isolated WT neutrophils from inflamed lung, those of IRF3-/- neutrophils exhibited impaired autophagic activation, phagocytosis, and apoptosis. These results suggest that IRF3 accelerated emphysema formation based on distinct profiles of mediators involved in LPS-induced COPD exacerbation. Regulation of the IRF3 pathway can affect multiple cell types and contribute to ameliorate pathogenesis of infection-triggered exacerbation of COPD.
Assuntos
Enfisema/imunologia , Fator Regulador 3 de Interferon/metabolismo , Pulmão/imunologia , Neutrófilos/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Animais , Apoptose/genética , Autofagia/genética , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Humanos , Fator Regulador 3 de Interferon/genética , Interferon gama/metabolismo , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Atrial fibrillation (AF) is associated with an increased risk of stroke and other thromboembolic events. Left atrial (LA) thrombus formation is closely related to LA dysfunction, particularly to decreased LA appendage flow velocity (LAA-FV) in patients with AF. We estimated LAA-FV using parameters noninvasively obtained by transthoracic echocardiography (TTE) in patients with paroxysmal AF.Echocardiographic and clinical parameters were assessed in 190 patients with nonvalvular paroxysmal AF showing sinus heart rhythm during transesophageal echocardiography (TEE) and TTE.LAA-FV (60 ± 22 cm/s) significantly correlated with the time interval between the initiation of the P-wave on ECG and that of the A-wave of transmitral flow on TTE (PA-TMF, correlation coefficient, -0.32; P < 0.001), LA dimension (LAD, -0.31; P < 0.001), septal a' velocity of tissue Doppler imaging (TDI, 0.35; P < 0.001), E/e' ratio (-0.28, P < 0.001), E velocity of transmitral flow (-0.20, P = 0.008), E/A ratio of transmitral flow (-0.18, P = 0.02), CHA2DS2-VASc score (-0.15, P = 0.04), and BNP plasma level (-0.32, P = 0.002). Multivariate analysis revealed that PA-TMF (standardized partial regression coefficient, -0.17; P = 0.03), a' velocity (0.24, P = 0.004), and LAD (-0.20, P = 0.01) were independent predictors of LAA-FV (multiple correlation coefficient R, 0.44; P < 0.001).Parameters of atrial remodeling, ie, decreased a' velocity, increased LAD, and PA-TMF during sinus rhythm may be useful predictors of LA blood stasis in patients with nonvalvular PAF. LAA-FV can be estimated using these TTE parameters instead of TEE.
Assuntos
Apêndice Atrial/fisiopatologia , Fibrilação Atrial/diagnóstico por imagem , Remodelamento Atrial , Velocidade do Fluxo Sanguíneo/fisiologia , Ecocardiografia Doppler/métodos , Taquicardia Paroxística/diagnóstico por imagem , Apêndice Atrial/diagnóstico por imagem , Fibrilação Atrial/fisiopatologia , Eletrocardiografia Ambulatorial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valva Mitral/diagnóstico por imagem , Valva Mitral/fisiopatologia , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Risco , Taquicardia Paroxística/fisiopatologiaRESUMO
Anticoagulants such as warfarin are recommended for patients with atrial fibrillation (AF) to decrease stroke risk associated with thrombus formation in the left atrium (LA). In a subgroup of patients, however, warfarin is unable to prevent LA thrombus formation at therapeutic doses. This study characterized the clinical and echocardiographic features of patients having warfarin-resistant LA thrombus.Of the 1364 nonvalvular AF patients examined by transesophageal echocardiography, 431 received warfarin. A total of 10 patients (2.3% of warfarin-treated patients) exhibited LA thrombus formation even during warfarin treatment at a dose and duration sufficient for increasing the prothrombin time-international normalized ratio (PT-INR) to the therapeutic range for ≥ 30 days. Categorical regression analysis revealed that decreased LA appendage (LAA) flow velocity, greater LA spontaneous echocardiographic contrast (LASEC), and lower left ventricular ejection fraction (LVEF) significantly contributed to residual LA thrombus (P < 0.05 for all). Receiver operating characteristic (ROC) curve analysis indicated that higher right ventricular systolic pressure, which suggests LA pressure (area under curve, 0.85), LV mass index (0.81), and LA dimension (0.68), as well as lower LAA flow velocity (0.92) and LVEF (0.91) predicted warfarin resistant LA thrombus formation (all P < 0.05).These results suggest that blood stasis secondary to heart failure contributes to the formation of warfarin-resistant LA thrombus. We propose that therapies to increase LVEF should be administered together with warfarin for AF patients with heart failure to decrease stroke risk.
Assuntos
Fibrilação Atrial/complicações , Insuficiência Cardíaca/complicações , Coração/fisiopatologia , Trombose/etiologia , Idoso , Anticoagulantes/uso terapêutico , Fibrilação Atrial/fisiopatologia , Ecocardiografia , Feminino , Insuficiência Cardíaca/fisiopatologia , Hemodinâmica , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Trombose/tratamento farmacológico , Varfarina/uso terapêuticoRESUMO
BACKGROUND: The pathogenesis of influenza A virus subtype H5N1 (hereafter, "H5N1") infection in humans is not completely understood, although hypercytokinemia is thought to play a role. We previously reported that most H5N1 viruses induce high cytokine responses in human macrophages, whereas some H5N1 viruses induce only a low level of cytokine production similar to that induced by seasonal viruses. METHODS: To identify the viral molecular determinants for cytokine induction of H5N1 viruses in human macrophages, we generated a series of reassortant viruses between the high cytokine inducer A/Vietnam/UT3028II/03 clone 2 (VN3028IIcl2) and the low inducer A/Indonesia/UT3006/05 (IDN3006) and evaluated cytokine expression in human macrophages. RESULTS: Viruses possessing the acidic polymerase (PA) gene of VN3028IIcl2 exhibited high levels of hypercytokinemia-related cytokine expression in human macrophages, compared with IDN3006, but showed no substantial differences in viral growth in these cells. Further, the PA gene of VN3028IIcl2 conferred enhanced virulence in mice. CONCLUSIONS: These results demonstrate that the PA gene of VN3028IIcl2 affects cytokine production in human macrophages and virulence in mice. These findings provide new insights into the cytokine-mediated pathogenesis of H5N1 infection in humans.
Assuntos
Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Humana/virologia , Macrófagos/imunologia , Infecções por Orthomyxoviridae/veterinária , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Animais , Citocinas/biossíntese , Citocinas/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Feminino , Células HEK293 , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Humana/imunologia , Macrófagos/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Vírus Reordenados/genética , Vírus Reordenados/metabolismo , Vírus Reordenados/patogenicidade , Virulência/genéticaRESUMO
Acetylcholine (ACh) exerts various anti-inflammatory effects through α7 nicotinic ACh receptors (nAChRs). We have previously shown that secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of α7 nAChR signaling, is down-regulated both in an animal model of asthma and in human epithelial cells treated with an inflammatory cytokine related to asthma. Our aim of this study was to explore the effect of SLURP-1, signal through α7 nAChR, in the pathophysiology of airway inflammation. Cytokine production was examined using human epithelial cells. Ciliary beat frequency of murine trachea was measured using a high speed camera. The IL-6 and TNF-α production by human epithelial cells was augmented by siRNA of SLURP-1 and α7 nicotinic ACh receptor. The cytokine production was also dose-dependently suppressed by human recombinant SLURP-1 (rSLURP-1). The ciliary beat frequency and amplitude of murine epithelial cells were augmented by PNU282987, a selective α7 nAChR agonist. Those findings suggested that SLURP-1 and stimulus through α7 nicotinic ACh receptors actively controlled asthmatic condition by stimulating ciliary beating and also by suppressing airway inflammation.
Assuntos
Células Epiteliais/imunologia , Depuração Mucociliar/imunologia , Mucosa Respiratória/imunologia , Antígenos Ly/farmacologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Humanos , Depuração Mucociliar/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
House dust mite (HDM), the most common allergen, activate both the IgE-associated and innate immune responses. To clarify the process of sensitization, we investigated the role of the CCL21, CCL19, and CCR7 axis in a mouse model of HDM-induced allergic asthma. HDM inhalation without systemic immunization resulted in a HDM-specific IgE response. CCR7-knockout (CCR7KO) mice exhibited greater airway inflammation and IgE responses compared to wild-type mice. We examined FoxP3 expression in these mice to clarify the contribution of regulatory cells to the responses. FoxP3 expression was higher in the lungs but not in the lymph nodes of CCR7KO mice compared to wild-type mice. In CCR7KO mice, FoxP3-positive cells were found in lung, but we observed higher release of IL-13, IL-5, TGF-ß, IL-17, and HMGB1 in bronchoalveolar lavage fluid. We demonstrate here that immuno-regulation through CCR7 expression in T cells plays a role in HDM-specific sensitization in the airway.
Assuntos
Asma/imunologia , Pyroglyphidae/imunologia , Receptores CCR7/imunologia , Animais , Asma/genética , Asma/patologia , Citocinas/biossíntese , Citocinas/imunologia , Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR7/deficiênciaRESUMO
OBJECTIVES: Cord blood (CB) transplantation has advantages in terms of incidence and severity of acute graft-versus-host disease (GVHD), while it has disadvantages in terms of infection. Our aim is to elucidate the molecular mechanism underlying the immune response of CB-derived cells during acute GVHD and infection following CB transplantation. METHODS: We examined expression of 69 immune-relating microRNAs in CD4(+), CD8(+), and CD14(+) cells of CB and adult peripheral blood (APB) upon interferon-γ or lipopolysaccharide (LPS) stimulation. RESULTS: Under basal condition, 20 microRNAs showed differential expression between CB and APB. Compared to APB counterparts, six microRNAs (miR-21, miR-22, miR-29a, miR-29b, miR-29c, and let-7c) were underexpressed in at least two cell lineages of CB, while five microRNAs (miR-15b, miR-181a, miR-181c, miR-363, and miR-424) were overexpressed in CD4(+) and CD8(+) CB cells. Upon interferon-γ stimulation, seven microRNAs (miR-29a, miR-29b, miR-34c-5p, miR-132, miR-146a, miR-146b-5p, and miR-155) changed in expression mainly in CD14(+) CB cells, while only two microRNAs (miR-18a and miR-155) changed in expression in CD14(+) CB cells upon LPS stimulation. These results suggest that the mechanisms regulating the expression of such immune-relating microRNAs in CD14(+) CB cells are much more sensitive to proinflammatory stimuli than those in APB CD14(+) cells, which might be related to the poor immunoreactivity of CD14(+) CB cells. CONCLUSIONS: Our results suggest essential roles of specific microRNAs in regulating immune function of CB cells, providing insight into the underlying molecular mechanism.
Assuntos
Antígenos CD/imunologia , Sangue Fetal/imunologia , Regulação da Expressão Gênica/imunologia , Doença Aguda , Adulto , Antígenos CD/metabolismo , Antivirais/farmacologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Feminino , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/metabolismo , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , MicroRNAs , Pessoa de Meia-Idade , Transplante HomólogoRESUMO
To advance the control of airway epithelial cell function and asthma, we investigated the effects of a new curcumin derivative, CNB001, which possesses improved pharmacological properties. Normal human bronchial epithelial (NHBE) cells were stimulated with synthetic double-stranded RNA, Poly(I:C). CNB001 significantly suppressed IL-6, TNF-α, and GM-CSF production by NHBE cells, and did so more effectively than did curcumin or dexamethasone (DEX). CNB001 significantly inhibited the decrease of E-cadherin mRNA expression and increase of vimentin mRNA expression observed in NHBE cells induced by a combination of TGF-ß1 and TNF-α, which are markers of airway remodeling. In NHBE cells stimulated by TGF-ß1, CNB001 significantly downregulated the level of active serine peptidase inhibitor clade E member (SERPINE) 1, which is also reported to be related to airway remodeling. Whereas DEX alone significantly increased the active SERPINE1 level, the combination of DEX and CNB001 significantly suppressed active SERPINE1. In addition, CNB001 significantly suppressed neutrophil infiltration, IL-6, TNF-α, IL-13 and active SERPINE1 production in bronchoalveolar lavage fluid of the murine asthma model, which was not observed in the case of DEX. In conclusion, the curcumin derivative, CNB001, is a promising candidate to treat asthma associated with neutrophilic airway inflammation and remodeling.
Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Asma/tratamento farmacológico , Brônquios/efeitos dos fármacos , Curcumina/análogos & derivados , Curcumina/farmacologia , Pirazóis/farmacologia , Animais , Caderinas/biossíntese , Curcumina/síntese química , Curcumina/química , Citocinas/biossíntese , Dexametasona/farmacologia , Feminino , Glucocorticoides/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Pirazóis/síntese química , Pirazóis/química , RNA de Cadeia Dupla/toxicidadeRESUMO
Highly pathogenic H5N1 avian influenza viruses have caused infection in humans, with a high mortality rate, since 1997. While the pathogenesis of this infection is not completely understood, hypercytokinaemia and alveolar macrophages are thought to play a role. To gain further insight into the cytokine-mediated pathogenesis of this infection in humans, we measured various cytokines produced by primary human macrophages infected with H5N1, pandemic H1N1 or seasonal influenza viruses. We found that many cytokines were produced at higher levels on infection with the H5N1 strains tested compared with seasonal influenza viruses. Interestingly, the extent of cytokine induction varied among the H5N1 strains and did not correlate with replicative ability in macrophages. Further, a pandemic H1N1 virus induced higher levels of several cytokines compared with seasonal viruses and some H5N1 strains. Our results demonstrate that high cytokine induction is not a universal feature of all H5N1 viruses.
Assuntos
Citocinas/imunologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Humana/imunologia , Macrófagos/imunologia , Pandemias , Células Cultivadas , Citocinas/genética , Expressão Gênica , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Humana/epidemiologia , Influenza Humana/virologia , Japão , Macrófagos/virologiaRESUMO
Chronic graft-versus-host disease (cGVHD) is a limiting factor in allogeneic hematopoietic stem cell transplantation (alloHSCT) for the treatment of leukemia and other malignancies. Relative to the process that initiates and promotes cGVHD, the regulation is poorly understood. In this study, we examined the role of naturally occurring regulatory dendritic cells (DC(regs)) in murine major histocompatibility complex (MHC)-compatible and multiple minor histocompatibility antigen (miHAg)-incompatible model of cGVHD in alloHSCT. DC(regs) generated from bone marrow in vitro (BM-DC(regs)) exclusively expressed CD200 receptor 3 (CD200R3), which exerted a suppressive function in the Ag-specific CD4(+) T-cell response. CD49(+)CD200R3(+) cells showed similarities in phenotype and function to BM-DC(regs), which formally distinguishes them from other leukocytes, suggesting that they are the natural counterpart of BM-DC(regs). Treatment of the recipient mice after alloHSCT with the recipient-type CD49(+)CD200R3(+) cells as well as BM-DC(regs) protected against cGVHD, and the protection was associated with the generation of Ag-specific anergic CD4(+) T cells as well as CD4(+)CD25(+)Foxp3(+) regulatory T cells (T(regs)) from donor-derived alloreactive CD4(+)CD25(-)Foxp3(-) T cells. In addition, the depletion of CD49(+)CD200R3(+) cells before alloHSCT enhanced the progression of cGVHD. In conclusion, CD49(+)CD200R3(+) cells act as naturally occurring DC(regs) to regulate the pathogenesis of cGVHD in alloHSCT mediated through the control of the transplanted alloreactive CD4(+) T cells.
Assuntos
Células Dendríticas/imunologia , Doença Enxerto-Hospedeiro/imunologia , Dermatopatias/prevenção & controle , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Medula Óssea , Transplante de Medula Óssea , Doença Crônica , Proteínas de Ligação a DNA/fisiologia , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas , Leucócitos/metabolismo , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dermatopatias/imunologia , Tolerância ao Transplante , Transplante HomólogoRESUMO
Whereas acetylcholine (ACh) acts as a bronchoconstrictor and stimulator of mucus secretion from bronchial epithelium, it acts via alpha7 nicotinic Ach receptors (nAChRs) on macrophages in the airways to exert anti-inflammatory effects by reducing synthesis of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha). Moreover, the effects of ACh are modified by secreted ly-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of alpha7 nAChR signaling. Our aim was to explore the roles played by SLURP-1 in the pathophysiology of asthma by assessing SLURP-1 expression in the OVA-sensitized murine asthma model and in cultured human bronchial epithelial cells. Using real-time PCR we found that expression of SLURP-1 mRNA is down-regulated in the lungs of asthmatic model mice, as compared to healthy mice. In addition, immunohistochemical studies confirmed the diminished expression of SLURP-1 in the bronchioles of asthmatic mice, and showed it was due to extensive metaplasia of mucus-secreting cells and the concomitant loss of ciliated epithelial cells. Expression of SLURP-1 mRNA and protein was also significantly down-regulated in human epithelial cells stimulated with the pro-inflammatory cytokine interleukin-13 (IL-13), which is related to asthmatic condition. Thus SLURP-1 appears to be down-regulated in both an animal model of asthma and human epithelial cells treated with an inflammatory cytokine related to asthma. Those findings suggest that diminished expression of SLURP-1 in asthma attenuates its negative regulation of airway inflammation, and that perhaps changes in SLURP-1 expression could serve as a marker of airway damage in asthma.
Assuntos
Antígenos Ly/metabolismo , Asma/metabolismo , Receptores Nicotínicos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Antígenos Ly/genética , Asma/patologia , Biomarcadores/metabolismo , Brônquios/metabolismo , Brônquios/patologia , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/genética , Receptor Nicotínico de Acetilcolina alfa7RESUMO
BACKGROUND: Dendritic cells (DCs) are crucial for the induction of immunity and tolerance. Despite an improved understanding of the DC-mediated control of T(H)1-biased immunity, little is known about how DCs regulate T(H)2-mediated immunity. OBJECTIVE: The effects of immunostimulatory mature DCs (maDCs) and regulatory DCs (DCregs) on T(H)2-driven allergic immunity involving IgE production were examined. METHODS: A murine model of airway hyperresponsiveness; the adoptive transfer of maDCs, DCregs, and T cells; and T-cell function were studied. RESULTS: Antigen-pulsed maDCs inhibited antigen-specific IgE production but enhanced the production of antigen-specific IgG1 and IgG2a. Analysis of Ifng-/- mice and Il21r-/- mice revealed that the inhibitory effect of antigen-pulsed maDCs on antigen-specific IgE production involved IL-21-producing T follicular helper cells but not IFN-gamma-producing T(H)1 cells. In contrast, antigen-pulsed DCregs impaired the production of antigen-specific IgE, IgG1, and IgG2a. In vivo blockade experiments showed that antigen-specific CD4+CD25+Foxp3+ regulatory T cells mainly mediated the suppressive effect of antigen-pulsed DCregs on the production of antigen-specific IgE. Antigen-pulsed maDCs promoted airway inflammation, whereas antigen-pulsed DCregs markedly suppressed the pathogenesis. CONCLUSION: DCregs abolish T(H)2-mediated IgE production and allergic inflammation based on antigen-specific dominant tolerance, whereas maDCs exacerbate the pathogenesis despite inhibiting the IgE response through the activation of diverse types of T(H) cell responses.
Assuntos
Asma/imunologia , Hiper-Reatividade Brônquica/imunologia , Células Dendríticas/imunologia , Células Dendríticas/transplante , Modelos Animais de Doenças , Hipersensibilidade/imunologia , Inflamação/imunologia , Transferência Adotiva , Animais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T ReguladoresRESUMO
OBJECTIVES: There are many direct-to-consumer (DTC)-type personal genomic testing (PGT) services commercially available to the public, providing the specific disease susceptibilities of individuals. While these services do not appear to stimulate disease-prevention behavior, few studies have addressed the methods to do so. We investigated the effectiveness of combining a consultation with a physician with the delivery of test results from a DTC-type PGT, as a preliminary study to identify the effective genomic testing for disease-prevention. A prepared physician disclosed the PGT results of twenty healthy subjects and provided a specific consultation on the high-risk diseases for each subject. The effects on the sense of health, understanding of possible future diseases, and preventive behaviors for each subject were examined pre-PGT, post-PGT, and 3, 6, and 12 months post-PGT. RESULTS: Significant increases between the pre- and post-PGT scores were observed for the awareness of lifestyle effects on developing those diseases (P < 0.05) and the awareness of the ability to influence disease onset (P < 0.01). The follow-up questionnaire results showed that over 60% of the subjects changed their lifestyles in favor of disease prevention. These results suggest that combining the DTC-PGT with a careful physician consultation may be effective at motivating people toward preventive behavior.
Assuntos
Triagem e Testes Direto ao Consumidor , Aconselhamento Genético , Testes Genéticos , Conhecimentos, Atitudes e Prática em Saúde , Prevenção Primária , Comportamento de Redução do Risco , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , MédicosRESUMO
OBJECTIVE: We assessed the feasibility and efficacy of dendritic cell (DC) therapy for advanced thyroid papillary and follicular cancer. DESIGN: Six Japanese patients (2 men and 4 women; aged 46-72 years, mean 60 years), who were diagnosed as advanced thyroid cancer with refractory distant metastases (papillary, n=5; follicular, n=1), were enrolled. Patients were first vaccinated weekly for 4 weeks with 10(7) autologous tumor lysate-pulsed monocyte-derived mature DCs followed by fortnightly vaccinations for 8 weeks (total=8 vaccinations). Lowdose (350 KIU) interleukin-2 was also administered for 3 days at each vaccination. Clinical response, adverse effects, delayed-type hypersensitivity skin testing (DTH), and IFN-( ) production by peripheral CD3(+) lymphocytes were evaluated. MAIN OUTCOME: Of the 6 patients, disease was assessed as stable in 2 and as progressive in 4. No adverse events were observed. Results of DTH and IFN-( ) production in peripheral lymphocytes did not correlate to the clinical response. CONCLUSIONS: DC immunotherapy could be administered to patients with thyroid papillary or follicular cancer without substantial side effects.
Assuntos
Vacinas Anticâncer/administração & dosagem , Carcinoma Papilar, Variante Folicular/terapia , Células Dendríticas/transplante , Neoplasias Pulmonares/terapia , Neoplasias da Glândula Tireoide/terapia , Idoso , Vacinas Anticâncer/efeitos adversos , Carcinoma Papilar, Variante Folicular/imunologia , Carcinoma Papilar, Variante Folicular/secundário , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Neoplasias da Glândula Tireoide/imunologia , Neoplasias da Glândula Tireoide/patologia , Resultado do TratamentoRESUMO
Biliary tract cancer (BTC) is one of the most devastating types of malignant neoplasms worldwide. However, the mechanisms underlying the development and progression of BTC remain unresolved. BTC includes extrahepatic bile duct carcinoma (EBDC), gallbladder carcinoma (GBC) and ampulla of Vater carcinoma (AVC), named according to the location of the tumor. Although genetic alterations of intrahepatic cholangiocarcinoma have been investigated, those of EBDC, GBC and AVC have not yet been fully understood. The present study analyzed somatic mutations of 50 cancer-associated genes in 27 Japanese BTC cells, including: 11 EBDC, 14 GBC and 2 AVC. Next-generation sequencing using an Ion AmpliSeq Cancer Panel identified a total of 44 somatic mutations across 14 cancer-associated genes. Among the 44 mutations, 42 were judged as pathological mutations. Frequent mutations were identified in tumor protein 53 (TP53) (14/27), SMAD family member 4 (SMAD4) (6/27), phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PIK3CA) (6/27), and Kirsten rat sarcoma (KRAS) (6/27); no significant differences were identified between EBDC and GBC tissues. Notably, the frequency of the PIK3CA mutation was higher when compared with previous reports. This result may suggest that the activation of the PIK3CA-protein kinase B signaling pathway, in addition to the abrogation of p53, SMAD4 and RAS mitogen-activated protein kinase may have a crucial role in the carcinogenesis of Japanese BTC. These findings may be useful for the development of personalized therapies for BTC.
RESUMO
PURPOSE: Identification of cancer/testis antigens useful for diagnosis or immunotherapy of cancers was attempted by cDNA expression cloning with patients' sera (SEREX). EXPERIMENTAL DESIGN: cDNA expression libraries made from testis or endometrial cancer cell lines were screened using sera from patients with endometrial cancer or melanoma patients immunized with dendritic cells pulsed with autologous tum or lysates. Tissue-specific expression by RT-PCR and immunogenicity by Western blotting of the bacterial recombinant antigen with sera from cancer patients were evaluated. RESULTS: A cancer/testis antigen, CAGE, was isolated by two independently performed SEREX. CAGE was expressed in various cancer cell lines including endometrial cancer, colon cancer, and melanoma in 7 of 10 endometrial cancer tissues and in 1 of 3 atypical endometrial hyperplasia, but not in normal tissues including the endometrium and testis. The protein expression on cancer cells was confirmed by Western blot analysis with the recombinant CAGE protein, anti-CAGE IgG antibody was detected in sera from 5 of 45 endometrial cancer, 2 of 24 melanoma, and 2 of 33 colon cancer patients, but not in sera from healthy individuals. By ELISA analysis, anti-CAGE antibody was detected in 12 of 45 endometrial cancer, 2 of 20 melanoma, and 4 of 33 colon cancer patients. Intriguingly, anti-CAGE antibody was highly positive in 7 of the 13 (53.8%) microsatellite instability (MSI)-H patients with endometrial cancer, but negative in 20 non-MSI-H patients (P = 0.001). CONCLUSION: CAGE may be useful for immunotherapy and diagnosis of various cancers particularly MSI-positive endometrial cancer.
Assuntos
Neoplasias do Endométrio/genética , Neoplasias do Endométrio/imunologia , Perfilação da Expressão Gênica , Repetições de Microssatélites , Proteínas Nucleares/biossíntese , Proteínas Nucleares/imunologia , Anticorpos Antineoplásicos/análise , Formação de Anticorpos , Antígenos Nucleares , RNA Helicases DEAD-box , DNA Complementar/biossíntese , Diagnóstico Diferencial , Neoplasias do Endométrio/diagnóstico , Ensaio de Imunoadsorção Enzimática , Feminino , Biblioteca Gênica , Humanos , Imunoterapia , Melanoma/genética , Melanoma/imunologia , Pessoa de Meia-Idade , Proteínas de Neoplasias , Proteínas Nucleares/análiseRESUMO
Tumor-associated antigens are promising candidates as target molecules for immunotherapy and a wide variety of tumor-associated antigens have been discovered through the presence of serum antibodies in cancer patients. We previously conducted dendritic cell therapy on 10 malignant melanoma patients and shrinkage or disappearance of metastatic tumors with massive necrosis occurred in two patients. In this study, we found a 29-kDa protein against which antibody was elicited by dendritic cell therapy in one of the two patients. Matrix-assisted laser desorption ionization-time of flight/mass spectrometry analysis of the protein isolated by two-dimensional electrophoresis combined with Western blots revealed that the 29-kDa protein was carbonic anhydrase II (CA-II). Immunohistochemistry of the tumors and normal tissues showed that CA-II was expressed in the tumor vessel but not in normal vessel endothelium. CA-II expression in tumor endothelium was observed as well in other cancers including esophageal, renal, and lung cancers. In an in vitro angiogenesis model, CA-II expression of normal human vein endothelial cells was significantly up-regulated when cells were cultured in the acidic and hypoxic conditions indicative of a tumor environment. These findings suggest that CA-II is a tumor vessel endothelium-associated antigen in melanoma and other cancers, and elicitation of serum anti-CA-II antibody by dendritic cell therapy may be associated with good clinical outcome including tumor reduction.
Assuntos
Anidrase Carbônica II/imunologia , Células Dendríticas/imunologia , Endotélio Vascular/imunologia , Imunoterapia Adotiva/métodos , Melanoma/terapia , Adulto , Idoso , Sequência de Aminoácidos , Anticorpos Antineoplásicos/sangue , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Anidrase Carbônica II/química , Anidrase Carbônica II/genética , Linhagem Celular , Linhagem Celular Tumoral , Células Dendríticas/transplante , Eletroforese em Gel Bidimensional , Células Endoteliais/enzimologia , Células Endoteliais/fisiologia , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peso Molecular , Neovascularização Fisiológica/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
OBJECTIVES: It has been demonstrated that 17beta-estradiol (E2) has an inhibitory effect on the proliferation of vascular smooth muscle cells (VSMCs) through an estrogen receptor (ER)-dependent pathway. Both ER subtypes, classical ER (ERalpha) and the newly identified ER subtype (ERbeta), are expressed in VSMCs. However, it remains unknown which receptor plays the critical role in the inhibitory effect on VSMC proliferation. METHODS AND RESULTS: We constructed replication-deficient adenoviruses bearing the coding region of human ERalpha, ERbeta, and the dominant-negative form of ERbeta (designated AxCAERalpha, AxCAERbeta, and AxCADNERbeta, respectively). Prior to infection with the adenoviruses, 100 nmol/l E2 attenuated DNA synthesis by up to 14% and transactivated the estrogen-induced expression of the desired mRNA in rat VSMCs. This was accompanied by increased transcriptional activity of estrogen responsive element in response to E2, and the increase was comparable between AxCAERalpha and AxCAERbeta. When VSMCs were infected with AxCAERbeta at a multiplicity of infection of 5 or higher, DNA synthesis as well as cell number decreased by 50% in response to E2, and the effect was abolished by co-infection with AxCADNERbeta. In contrast, when VSMCs were infected with AxCAERalpha, the reduction in DNA synthesis was minimal. CONCLUSIONS: Our results indicate that ERbeta is more potent than ERalpha in the inhibitory effect on VSMC proliferation.
Assuntos
Estradiol/farmacologia , Músculo Liso Vascular/metabolismo , Receptores de Estrogênio/metabolismo , Adenoviridae/genética , Animais , Northern Blotting/métodos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ciclina A/metabolismo , Depressão Química , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Vetores Genéticos/administração & dosagem , Luciferases/análise , Luciferases/genética , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de Estrogênio/análise , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução Genética/métodosRESUMO
Pseudomyxoma peritonei (PMP) is a rare disease exhibiting a distinct clinical feature caused by cancerous cells that produce mucinous fluid in the abdominal cavity. PMPs originate most frequently from the appendix and less frequently from the ovary. This disease can range from benign to malignant, and histologically, PMP is classified into two types: disseminated peritoneal adenomucinosis (DPAM) representing the milder phenotype, and peritoneal mucinous adenocarcinomas (PMCA) representing the aggressive phenotype. Although histological classification is clinically useful, the pathogenesis of PMP remains largely unknown. To elucidate the molecular mechanisms underlying PMP, we analyzed 18 PMP tumors comprising 10 DPAMs and 8 PMCAs. DNA was extracted from tumor and matched non-tumorous tissues, and was sequenced using Ion AmpliSeq Cancer Panel containing 50 cancer-related genes. Analysis of the data identified a total of 35 somatic mutations in 10 genes, and all mutations were judged as pathological mutations. Mutations were frequently identified in KRAS (14/18) and GNAS (8/18). Interestingly, TP53 mutations were found in three of the eight PMCAs, but not in the DPAMs. PIK3CA and AKT1 mutations were also identified in two PMCAs, but not in the DPAMs. These results suggested that KRAS and/or GNAS mutations are common genetic features of PMP, and that mutations in TP53 and/or genes related to the PI3K-AKT pathway may render malignant properties to PMP. These findings may be useful for the understanding of tumor characteristics, and facilitate the development of therapeutic strategies.
Assuntos
Perfilação da Expressão Gênica , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/patologia , Pseudomixoma Peritoneal/genética , Pseudomixoma Peritoneal/patologia , Transcriptoma , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Terapia Combinada , Análise Mutacional de DNA , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Neoplasias Peritoneais/terapia , Pseudomixoma Peritoneal/terapia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Proline-rich tyrosine kinase 2 (Pyk2), a member of the focal adhesion kinase family, is thought to act as a key component in vasculogenesis and angiogenesis. Therefore, we studied the effect of mutant Pyk2 expression on the migration and proliferation in endothelial cells (ECs). Two types of mutant Pyk2 were examined by adenovirus vectors AxCA-Pyk2K457A, expressing a kinase inactive mutant, and AxCA-Pyk2Y402F, expressing a tyrosine autophosphorylation site mutant, in addition to AxCA-Pyk2, expressing wild-type Pyk2. Migration of ECs infected with AxCA-Pyk2Y402F increased to a level similar to that of ECs infected with AxCA-Pyk2. The size of effect was dependent on the amount of applied adenoviruses within the range of 3-30 multiplicity of infection. In contrast, AxCA-Pyk2K457A infection did not show any significant effect on cell migration. Western blotting showed that both phosphorylation of Pyk2 Y(881) and association of p130(Cas) with Pyk2 were enhanced in ECs infected with AxCA-Pyk2Y402F as well as with AxCA-Pyk2, but not in ECs infected with AxCA-Pyk2K457A. Therefore, signaling mediated by Pyk2 Y(881) and p130(Cas) may be involved in the migration of ECs infected either with AxCA-Pyk2Y402F or with AxCA-Pyk2. In proliferation assay, AxCA-Pyk2 infection suppressed EC proliferation significantly; however, neither AxCA-Pyk2Y402F nor AxCA-Pyk2K457A showed such an inhibitory effect. Thus, the two Pyk2 mutants revealed that Pyk2 signaling differentially regulates cell migration and proliferation pathways.