RESUMO
The characterization of protein-protein interactions (PPI) often provides functional information about a target protein. Yeast-two-hybrid (Y2H) and luminescence/fluorescence-based detections, therefore, have been widely utilized for assessing PPI. In addition, a co-immunoprecipitation (co-IP) method has also been adopted with transient protein expression in Nicotiana benthamiana (N. benthamiana) infiltrated with Agrobacterium tumefaciens. Herein, we describe a co-IP procedure in which structural maintenance of chromosome 1 (SMC1), identified from a Y2H screening, was verified as an interacting partner for microchidia 1 (MORC1), a protein well known for its function in plant immunity and epigenetics. SMC1 and MORC1 were transiently expressed in N. benthamiana when infiltrated by Agrobacterium with the respective genes. From this approach, we identified a region of SMC1 responsible for interacting with MORC1. The co-IP method, of which outputs are mainly from immunoblot analysis, provided information about target protein expression as well, which is often useful for troubleshooting. Using this feature, we showcased a PPI confirmation from our SMC1-MORC1 study in which a full-length SMC1 protein was not detectable, and, therefore, a subsequent truncated mutant analysis had to be employed for PPI verification.
Assuntos
Nicotiana , Proteínas , Nicotiana/metabolismo , Proteínas/metabolismo , Agrobacterium tumefaciens/genética , Proteína Estafilocócica A/metabolismo , ImunoprecipitaçãoRESUMO
BACKGROUND: To investigate the cytotoxicities of the topical ocular dual-action anti-allergic agents (alcaftadine 0.25%, bepotastine besilate 1.5%, and olopatadine HCL 0.1%) on human corneal epithelial cells (HCECs) and their anti-allergic effects on cultured conjunctival epithelial cells. METHODS: A Methylthiazolyltetrazolium(MTT)-based calorimetric assay was used to assess cytotoxicities using HCECs at concentrations of 10, 20 or 30% for exposure durations of 30 min, 1 h, 2 h, 12 h or 24 h. Cellular morphologies were evaluated by inverted phase-contrast and electron microscopy. Wound widths were measured 2 h, 18 h, or 24 h after confluent HCECs monolayers were scratched. Realtime PCR was used to quantify anti-allergic effects on cultured human conjunctival cells, in which allergic reactions were induced by treating them with Aspergillus antigen. RESULTS: Cell viabilities decreased in time- and concentration-dependent manners. Cells were detached from dishes and showed microvilli loss, cytoplasmic vacuoles, and nuclear condensation when exposed to antiallergic agents; alcaftadine was found to be least cytotoxic. Alcaftadine treated HCECs monolayers showed the best wound healing followed by bepotastine and olopatadine (p < 0.0001). All agents significantly reduced the gene expressions of allergic cytokines (IL-5, IL-25, eotaxin, thymus and activation-regulated chemokine, and thymic stromal lymphopoietin) and alcaftadine had the greatest effect (p < 0.0001 in all cases). CONCLUSIONS: Alcaftadine seems to have less side effects and better therapeutic effects than the other two anti-allergic agents tested. It may be more beneficial to use less toxic agents for patients with ocular surface risk factors or presumed symptoms of toxicity.
Assuntos
Antialérgicos/toxicidade , Benzazepinas/toxicidade , Células Epiteliais/efeitos dos fármacos , Imidazóis/toxicidade , Cloridrato de Olopatadina/toxicidade , Piperidinas/toxicidade , Piridinas/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Túnica Conjuntiva/citologia , Córnea/citologia , HumanosAssuntos
Corpos Estranhos/diagnóstico por imagem , Corpos Estranhos/terapia , Trato Gastrointestinal/diagnóstico por imagem , Adolescente , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Endoscopia Gastrointestinal , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Radiografia , República da Coreia , Estudos Retrospectivos , Distribuição por Sexo , Adulto JovemRESUMO
Rice leaf blight, which is caused by the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), results in huge losses in grain yield. Here, we show that Xoo-induced rice leaf blight is effectively controlled by niclosamide, an oral antihelminthic drug and molluscicide, which also functions as an anti-tumor agent. Niclosamide directly inhibited the growth of the three Xoo strains PXO99, 10208 and K3a. Niclosamide moved long distances from the site of local application to distant rice tissues. Niclosamide also increased the levels of salicylate and induced the expression of defense-related genes such as OsPR1 and OsWRKY45, which suppressed Xoo-induced leaf wilting. Niclosamide had no detrimental effects on vegetative/reproductive growth and yield. These combined results indicate that niclosamide can be used to block bacterial leaf blight in rice with no negative side effects.
Assuntos
Niclosamida/farmacologia , Oryza/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/microbiologia , Xanthomonas/efeitos dos fármacos , Anti-Infecciosos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Oryza/genética , Doenças das Plantas/genética , Xanthomonas/crescimento & desenvolvimentoRESUMO
PURPOSE: Pattern electroretinogram (PERG) in idiopathic epiretinal membrane (ERM) was investigated to find the correlation of the ganglion cell function with postoperative visual acuity. DESIGN: This was a retrospective consecutive chart review. METHODS: Medical records of 24 eyes that underwent vitrectomy and membrane peeling for idiopathic ERM were reviewed retrospectively. The amplitude and implicit time of P50 and N95 in preoperative PERG were analyzed to find correlation with visual acuity and foveal thickness. The ratio of the parameters in involved eyes to those in healthy fellow eyes was calculated for analysis. RESULTS: Visual acuity (logarithm of the minimum angle of resolution) improved from 0.53 at baseline to 0.34 at 6 months (P = 0.003). Foveal thickness decreased significantly from 488.3 µm at baseline to 374.7 µm (P = 0.001). The preoperative N95 amplitude ratio was significantly correlated with visual acuity at 6 months after ERM removal (r = -0.423, P = 0.040), whereas the amplitude of P50 and implicit time of both waves showed no significant correlation with postoperative visual acuity. The implicit time ratio of P50 (r = 0.530, P = 0.008) and N95 (r = 0.436, P = 0.033) showed significant correlation with preoperative foveal thickness on optical coherence tomography. CONCLUSIONS: N95 amplitude in PERG was a predictor of visual outcomes after ERM surgery. These results suggest the correlation of postoperative visual acuity with the function of the ganglion cell layer, which is the closest cell layer to be affected by ERM.
Assuntos
Eletrorretinografia , Membrana Epirretiniana/cirurgia , Reconhecimento Visual de Modelos/fisiologia , Células Ganglionares da Retina/fisiologia , Acuidade Visual/fisiologia , Adulto , Idoso , Membrana Epirretiniana/fisiopatologia , Feminino , Fóvea Central/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Tomografia de Coerência Óptica , VitrectomiaRESUMO
Gibberellins affect various plant development processes including germination, cell division and elongation, and flowering. A large number of studies have been carried out to address the molecular mechanisms that mediate gibberellin signalling effects on plant growth. However, such studies have been limited to DELLA protein degradation; the regulatory mechanisms controlling how the stability and function of SLEEPY1 (SLY1), a protein that interacts with target DELLA proteins as components of the Skp, Cullin, F-box (SCF)(SLY1) complex, are modulated at the post-translational level have not been addressed. In the present study, we show that the E3 SUMO (small ubiquitin-related modifier) ligase AtSIZ1 regulates gibberellic acid signalling in Arabidopsis species by sumoylating SLY1. SLY1 was less abundant in siz1-2 mutants than in wild-type plants, but the DELLA protein repressor of ga1-3 (RGA) was more abundant in siz1-2 mutants than in wild-type plants. SLY1 also accumulated to a high level in the SUMO protease mutant esd4. Transgenic sly1-13 mutants over-expressing SLY1 were phenotypically similar to wild-type plants; however, sly1-13 plants over-expressing a mutated mSLY1 protein (K122R, a mutation at the sumoylation site) retained the mutant dwarfing phenotype. Over-expression of SLY1 in sly1-13 mutants resulted in a return of RGA levels to wild-type levels, but RGA accumulated to high levels in mutants over-expressing mSLY1. RGA was clearly detected in Arabidopsis co-expressing AtSIZ1 and mSLY1, but not in plants co-expressing AtSIZ1 and SLY1. In addition, sumoylated SLY1 interacted with RGA and SLY1 sumoylation was significantly increased by GA. Taken together, our results indicate that, in Arabidopsis, AtSIZ1 positively controls GA signalling through SLY1 sumoylation.
Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Giberelinas/metabolismo , Ligases/metabolismo , Transdução de Sinais/fisiologia , Sumoilação/fisiologia , Alquil e Aril Transferases/genética , Substituição de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Giberelinas/genética , Ligases/genética , Mutação de Sentido Incorreto , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: To evaluate the effect of reduced-fluence photodynamic therapy (PDT) on polypoidal choroidal vasculopathy (PCV) unresponsive to intravitreal ranibizumab. PATIENTS AND METHODS: Patients with PCV unresponsive to ranibizumab administered 3 months consecutively who then received reduced-fluence PDT were retrospectively surveyed. Nonresponders were defined as patients having no reduction in intraretinal and/or subretinal fluid after 3 consecutive treatments. RESULTS: In total, 22 of 104 eyes (21.2%) were non-responders, and 16 of 22 nonresponders received reduced-fluence PDT. Nine eyes achieved complete fluid resolution, and six had reduced but persistent fluid. In one eye, fluid persisted at 6 months despite an additional anti-vascular endothelial growth factor (anti-VEGF) injection after reduced-fluence PDT. Mean macular thickness decreased significantly at 3 and 6 months after PDT, but the mean visual acuity was worse than baseline. CONCLUSION: Reduced-fluence PDT in nonresponders gradually decreased intraretinal and/or subretinal fluid over several months but did not maintain visual acuity.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Neovascularização de Coroide/tratamento farmacológico , Fotoquimioterapia/métodos , Pólipos/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Neovascularização de Coroide/diagnóstico , Corantes , Resistência a Medicamentos , Feminino , Angiofluoresceinografia , Humanos , Verde de Indocianina , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/uso terapêutico , Ranibizumab , Estudos Retrospectivos , Tomografia de Coerência Óptica , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Verteporfina , Acuidade Visual/efeitos dos fármacosRESUMO
TGF-ß-activated kinase 1 (TAK1) is a key intermediate in signal transduction induced by TGF-ß or inflammatory cytokines, such as TNF-α and IL-1, which are potent inducers of podocyte injury responses that lead to proteinuria and glomerulosclerosis. Nevertheless, little is known about the physiologic and pathologic roles of TAK1 in podocytes. To examine the in vivo role of TAK1, we generated podocyte-specific Tak1 knockout mice (Nphs2-Cre(+):Tak1(fx/fx); Tak1(∆/∆)). Targeted deletion of Tak1 in podocytes resulted in perinatal lethality, with approximately 50% of animals dying soon after birth and 90% of animals dying within 1 week of birth. Tak1(∆/∆) mice developed proteinuria from P1 and exhibited delayed glomerulogenesis and reduced expression of Wilms' tumor suppressor 1 and nephrin in podocytes. Compared with Tak1(fx/fx) mice, Tak1(∆/∆) mice exhibited impaired formation of podocyte foot processes that caused disruption of the podocyte architecture with prominent foot process effacement. Intriguingly, Tak1(∆/∆) mice displayed increased expression of vascular endothelial growth factor within the glomerulus and abnormally enlarged glomerular capillaries. Furthermore, 4- and 7-week-old Tak1(∆/∆) mice with proteinuria had increased collagen deposition in the mesangium and the adjacent tubulointerstitial area. Thus, loss of Tak1 in podocytes is associated with the development of proteinuria and glomerulosclerosis. Taken together, our data show that TAK1 regulates the expression of Wilms' tumor suppressor 1, nephrin, and vascular endothelial growth factor and that TAK1 signaling has a crucial role in podocyte differentiation and attainment of normal glomerular microvasculature during kidney development and glomerular filtration barrier homeostasis.
Assuntos
Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/enzimologia , MAP Quinase Quinase Quinases/metabolismo , Podócitos/citologia , Podócitos/enzimologia , Animais , Animais Recém-Nascidos , Capilares/enzimologia , Capilares/crescimento & desenvolvimento , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Colágeno/metabolismo , Feminino , Barreira de Filtração Glomerular/irrigação sanguínea , Barreira de Filtração Glomerular/enzimologia , Barreira de Filtração Glomerular/crescimento & desenvolvimento , Glomérulos Renais/crescimento & desenvolvimento , MAP Quinase Quinase Quinases/deficiência , MAP Quinase Quinase Quinases/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Proteinúria/enzimologia , Proteinúria/etiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMO
Clinical guidelines regarding anti-viral prophylaxis for HBV surface antigen (HBsAg) carriers starting anti-TNFα agents are not yet fully established, even in endemic regions of HBV infection. We retrospectively collected the clinical data of 52 HBsAg carriers with rheumatoid arthritis (RA) or ankylosing spondylitis (AS) that had been administered anti-TNFα treatment at seven medical centers in South Korea. Periodic data of liver function tests and serum HBV DNA were both utilized to assess HBV reactivation. The YMDD motif mutation of HBV DNA polymerase was tested in lamivudine-treated patients with elevated HBV DNA. Three of the 52 patients were excluded from the analysis. Of the 49 analyzed patients, 20 patients received anti-viral prophylaxis (15 lamivudine, five entecavir) with anti-TNFα treatment. The remaining 29 patients were treated with anti-viral agents if needed at the discretion of the clinician and did not receive prophylaxis. Of the 29 patients who did not receive primary prophylaxis, two (6.9%) developed viral reactivation within a year of anti-TNFα treatment. In the prophylaxis group, one patient developed viral reactivation at week 64 of anti-TNFα therapy attributed to YMDD mutation caused by lamivudine. Patients with HBV reactivation all responded well to anti-viral therapy. In summary, anti-viral prophylaxis helped preventing HBV reactivation in HBsAg carriers with RA or AS starting anti-TNFα, yet mutation in the YMDD motif of HBV DNA polymerase could be detrimental to some patients under long-term lamivudine prophylaxis.
Assuntos
Artrite Reumatoide/virologia , Vírus da Hepatite B/metabolismo , Hepatite B/virologia , Espondilite Anquilosante/virologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Motivos de Aminoácidos , Antivirais/farmacologia , Artrite Reumatoide/complicações , DNA Polimerase Dirigida por DNA/metabolismo , Feminino , Hepatite B/complicações , Humanos , Lamivudina/farmacologia , Masculino , Pessoa de Meia-Idade , Mutação , Estudos Retrospectivos , Espondilite Anquilosante/complicações , Ativação ViralRESUMO
OBJECTIVE: Bone destruction is a critical pathology involved in the functional disability caused by rheumatoid arthritis (RA). Osteoclasts, which are specialized bone-resorbing cells regulated by cytokines such as RANKL, are implicated in bone destruction in RA. The aim of this study was to determine whether interleukin-21 (IL-21), a potent immunomodulatory 4-α-helical bundle type 1 cytokine, has osteoclastogenic activity in patients with RA and in mice with collagen-induced arthritis (CIA). METHODS: The expression of IL-21 in synovial tissue was examined using immunohistochemistry. The concentrations of IL-21 in serum and synovial fluid were determined by enzyme-linked immunosorbent assay. The levels of RANKL and osteoclastogenic markers were measured using real-time polymerase chain reaction. CD14+ monocytes from patients with RA or mouse bone marrow cells were cocultured with fibroblast-like synoviocytes (FLS) from patients with RA or CD4+ T cells from mice with CIA in the presence of IL-21 and subsequently stained for tartrate-resistant acid phosphatase activity to determine osteoclast formation. RESULTS: IL-21 was up-regulated in the synovium, synovial fluid, and serum of patients with RA and in the synovium and serum of mice with CIA. IL-21 induced RANKL expression in mixed joint cells and CD4+ T cells from mice with CIA and in CD4+ T cells and FLS from patients with RA. Moreover, IL-21 enhanced in vitro osteoclastogenesis without the presence of RANKL-providing cells and by inducing RANKL expression in CD4+ T cells and FLS. CONCLUSION: Our data suggest that IL-21 promotes osteoclastogenesis in RA. We believe that therapeutic strategies targeting IL-21 might be effective for the treatment of patients with RA, especially in preventing bone destruction.
Assuntos
Artrite Experimental/patologia , Artrite Reumatoide/patologia , Interleucinas/metabolismo , Osteoclastos/patologia , Membrana Sinovial/patologia , Fosfatase Ácida/metabolismo , Animais , Artrite Experimental/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Biomarcadores/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Monócitos/metabolismo , Monócitos/patologia , Osteoclastos/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Membrana Sinovial/metabolismoRESUMO
The aim of this study was to investigate the expressions of Toll-like receptor (TLR) 2, TLR4, TLR9, and their correlations with the expression of cytokines that are associated with activation of CD4(+) T cells and inflammation including interferon gamma (IFNgamma), interleukin 4 (IL4), interleukin 17 (IL17), and tumor necrosis factor alpha (TNFalpha) in muscle tissues of patients with dermatomyositis (DM) and polymyositis (PM). The expressions of TLR2, TLR4, TLR9, IFNgamma, IL4, IL17, and TNFalpha were measured by real-time reverse transcription-polymerase chain reaction in muscle tissues from 14 patients with DM and PM (nine patients with DM, five patients with PM) and three controls. The expressions of TLR2, TLR4, and TLR9 were also localized with immunohistochemistry. The expression levels of TLR2, TLR4, TLR9, IFNgamma, IL4, IL17, and TNFalpha were significantly high in patients with DM and PM compared with those in the controls, and the expression levels of TLR4 and TLR9 had significant positive correlations with the expressions of IFNgamma, IL4, IL17, and TNFalpha. Immunohistochemistry showed that TLR2, TLR4, and TLR9 were expressed by infiltrating cells of perimysium in DM, whereas they were expressed by infiltrating cells of endomysium in PM. These results suggest that the involvement of TLR4 and TLR9 in immunopathogenesis of DM and PM might be connected with activation of CD4(+) T cells.
Assuntos
Dermatomiosite , Polimiosite , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptor Toll-Like 9 , Adolescente , Adulto , Idoso , Biópsia , Linfócitos T CD4-Positivos/imunologia , Dermatomiosite/genética , Dermatomiosite/imunologia , Dermatomiosite/metabolismo , Feminino , Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/imunologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Polimiosite/genética , Polimiosite/imunologia , Polimiosite/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
To examine the effects of nuclear factor kappa B (NF-kappaB) inhibition on the secretion of macrophage migration inhibitory factor (MIF) in human CD4(+) T cells. Isolated human CD4(+) T cells were cultured for 24h with pharmacological inhibitors of NF-kappaB including parthenolide, pyrrolidine dithiocarbamate, BAY 11-7082, gliotoxin, oridonin, andrographolide, and NF-kappaB shRNA. MIF concentration was measured by intracellular flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction. The intracellular concentrations O(2)(-), H(2)O(2), and glutathione were measured using the oxidation-sensitive fluorescent dyes dihydroethidium, dichlorodihydrofluorescein diacetate, and monochlorobimane, respectively. The amount of phosphorylated c-Jun was measured by Western blotting. Treatment of CD4(+) T cells with NF-kappaB inhibitors significantly increased MIF concentration in culture supernatants, MIF gene expression, and O(2)(-) production, and decreased the intracellular concentrations of MIF, H(2)O(2), and glutathione. Treatment with LY294002 (PI3K inhibitor) and SP600125 (JNK inhibitor) suppressed NF-kappaB inhibitor induced MIF mRNA expression and MIF secretion. LY294002 and SP600125 inhibited the parthenolide-induced phosphorylation of c-Jun. Treatment with H(2)O(2) decreased the amount of intracellular MIF protein and increased MIF concentration in the culture supernatant. N-acetylcysteine, an antioxidant precursor of glutathione, inhibited the parthenolide-induced and H(2)O(2)-induced secretion of MIF. These results indicate that pharmacological inhibition of NF-kappaB causes the release of MIF through de novo synthesis of MIF and the secretion of preformed MIF in CD4(+) T cells through the production of reactive oxygen species.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , NF-kappa B/antagonistas & inibidores , Linfócitos T CD4-Positivos/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Glutationa/imunologia , Glutationa/metabolismo , Humanos , Oxirredutases Intramoleculares/biossíntese , Fatores Inibidores da Migração de Macrófagos/biossíntese , NF-kappa B/genética , NF-kappa B/metabolismo , Peroxidase/imunologia , Peroxidase/metabolismo , Peroxidase/farmacologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
The aim of this study was to determine whether stimulation of toll-like receptor 2 (TLR2), TLR4, and TLR6 by their specific ligands induces the production of tumor necrosis factor-alpha (TNF-alpha) in fibroblast-like synoviocytes (FLS) from interleukin-1 receptor antagonist (IL-1Ra)-deficient mice. FLS were isolated from synovial tissues from IL-1Ra-deficient mice and stimulated with various ligands of TLRs. The concentrations of TNF-alpha, interleukin (IL)-1beta, and IL-10 in the culture supernatants of spleen cells were measured by ELISA, and mRNA levels were assessed by real-time PCR. The expression of TLR2, TLR4, TLR6, and TNF-alpha in the synovial tissue was quantified by immunohistochemistry. Cytokine production and TLR expression were measured in FLS stimulated in the presence of the TLR2 ligand PAM3, the TLR4 ligand lipopolysaccharide (LPS), and the TLR6 ligand zymosan, with and without blocking antibody to TNF-alpha and IL-1beta. Stimulation of TLR2, TLR4, and TLR6 by their specific ligands increased the production of TNF-alpha in FLS from IL-1Ra-deficient mice. The stimulatory effect of these TLR ligands showed a dose-dependent pattern. The combination of TLR2, TLR4, and TLR6 synergistically increased the production of TNF-alpha, IL-1beta, TLR2, TLR4, and TLR6. Addition of blocking antibodies to TNF-alpha and IL-1beta abrogated the stimulatory effect of the ligands of TLR2, TLR4, and TLR6 on the production of TNF-alpha, IL-1beta, TLR2, TLR4, and TLR6. These data show that TLR2, TLR4, and TLR6 ligation synergistically stimulates the production of TNF-alpha and IL-1beta in IL-1Ra-deficient mice and suggest that TLRs contribute to the perpetuation of spontaneous arthritis in this animal model.
Assuntos
Artrite Experimental/imunologia , Receptor 2 Toll-Like/agonistas , Receptor 4 Toll-Like/agonistas , Receptor 6 Toll-Like/agonistas , Fator de Necrose Tumoral alfa/biossíntese , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Modelos Animais de Doenças , Fibroblastos/citologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-10/biossíntese , Interleucina-10/imunologia , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Articulação do Joelho/imunologia , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Ligantes , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Receptor 6 Toll-Like/imunologia , Receptor 6 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Zimosan/farmacologiaRESUMO
OBJECTIVE: To examine the effect of interleukin 17 (IL-17) on the expression of Toll-like receptor (TLR)-2, 4, and 9 in collagen-induced arthritis (CIA) in mice. METHODS: On Days 28 and 32 after induction of CIA in mice, phosphate-buffered saline (PBS group) or IL-17 (IL-17 group) was injected into both knee joints. On Day 35, mice were sacrificed. The severity of knee joint arthritis, synovial inflammation, and bone destruction was measured by a scoring system using macrography and histological analysis. Synovial expression of TLR-2, 4, 9, IL-17, IL-1beta, tumor necrosis factor-alpha (TNF-alpha), and IL-6 was determined by real-time PCR and immunohistochemistry. Synoviocytes of CIA mice were cultured with IL-17 and with neutralizing antibodies to cytokine, and the expression of TLR-2, 4, 9, IL-1beta, TNF-alpha, and IL-6 was determined by real-time RT-PCR. RESULTS: In CIA mice, knee arthritis scores, synovial inflammation, bone destruction scores, and expression of synovial TLR-2, 4, and 9, IL-17, IL-1beta, TNF-alpha and IL-6 were higher in the IL-17 and PBS groups than in normal DBA1 mice. These variables were also significantly higher in the IL-17 group than in the PBS group. In CIA synoviocytes, IL-17 increased the expression of TLR-2, 4, and 9, and this effect was significantly alleviated by neutralizing antibodies to IL-17, IL-1beta, and IL-6. CONCLUSION: IL-17 aggravates joint inflammation and destruction, and increases the synovial expression of TLR-2, 4, and 9 by increasing IL-1beta and IL-6. These results imply that the IL-17-induced increase in expression of TLR-2, 4, and 9, and IL-1beta and IL-6 production are involved in the IL-17-induced aggravation of arthritis.
Assuntos
Artrite Experimental/imunologia , Interleucina-17/imunologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Animais , Artrite Experimental/patologia , Células Cultivadas , Interleucina-17/genética , Interleucina-1beta/genética , Interleucina-6/genética , Articulação do Joelho/anatomia & histologia , Articulação do Joelho/imunologia , Articulação do Joelho/patologia , Camundongos , Membrana Sinovial/citologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor Toll-Like 9/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Tubulointerstitial fibrosis is a hallmark of chronic progressive kidney disease leading to end-stage renal failure. An endogenous product of heme oxygenase activity, carbon monoxide (CO), has been shown to exert cytoprotection against tissue injury. Here, we explored the effects of exogenous administration of low-dose CO in an in vivo model of renal fibrosis induced by unilateral ureteral obstruction (UUO) and examined whether CO can protect against kidney injury. UUO in mice leads to increased extracellular matrix (ECM) deposition and tubulointerstitial fibrosis within 4 to 7 days. Kidneys of mice exposed to low-dose CO, however, had markedly reduced ECM deposition after UUO. Moreover, low-dose CO treatment inhibited the induction of alpha-smooth muscle actin (alpha-SMA) and major ECM proteins, type 1 collagen and fibronectin, in kidneys after UUO. In contrast, these anti-fibrotic effects of CO treatment were abrogated in mice carrying null mutation of Mkk3, suggesting involvement of the MKK3 signaling pathway in mediating the CO effects. Additionally, in vitro CO exposure markedly inhibited TGF-beta(1)-induced expression of alpha-SMA, collagen, and fibronectin in renal proximal tubular epithelial cells. Our findings suggest that low-dose CO exerts protective effects, via the MKK3 pathway, to inhibit development of renal fibrosis in obstructive nephropathy.
Assuntos
Antimetabólitos/uso terapêutico , Monóxido de Carbono/uso terapêutico , Nefropatias/prevenção & controle , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Obstrução Ureteral/complicações , Actinas/metabolismo , Administração por Inalação , Animais , Antimetabólitos/administração & dosagem , Monóxido de Carbono/administração & dosagem , Colágeno Tipo I/metabolismo , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Fibrose/etiologia , Fibrose/prevenção & controle , Nefropatias/etiologia , Nefropatias/patologia , Túbulos Renais Proximais/metabolismo , MAP Quinase Quinase 3/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVES: Cross-hatching incisions have been considered mandatory for correcting cartilaginous septal deviation. We evaluated the clinical outcome of septoplasty without cross-hatching incisions to determine the necessity for making septal cartilage incisions. METHODS: THE RECONSTRUCTED SEPTAL COMPONENTS DURING SEPTOPLASTY WERE CATEGORIZED INTO FOUR ANATOMICAL AREAS: vomer, maxillary crest, perpendicular plate of ethmoid (PPE) and septal cartilage (the area for cross-hatching incisions). During septoplasty, we attempted to complete the surgery only by removing or fracturing the bony part of the septum without cross-hatching incisions on the cartilage. Only in the cases that the deviation was not immediately corrected, the cross-hatching incisions were made onto the cartilage at the end of the procedure. We analyzed the frequency of manipulating the septal components. The changes of symptoms were evaluated using a modified nasal obstruction symptom evaluation (NOSE) scale and a visual analog scale (VAS) preoperatively, 1 and 3 months after the surgery. RESULTS: Seventy five percents of the deviated septums were immediately corrected only by removing or fracturing of the bony septal components. In decreasing order of frequency, the sepal components for correcting septal deviation were the vomer (59%), maxillary crest (49%), septal cartilage (cross-hatching only: 25%) and PPE (15%). The modified NOSE scale and the VAS demonstrated significant improvement of the nasal symptoms postoperatively (P<0.05). CONCLUSION: Most of septal deviations could be corrected by manipulating only the bony septum. The results of this procedure were not different from conventional septoplasty with cross-hatching incisions. Our data suggest cross-hatching incisions during septoplasty might have been overemphasized and that the main cause for cartilaginous deviation may be the extrinsic forces that are generated by the neighboring bony structures.
RESUMO
BACKGROUND: Excision repair cross-complementing group 1 (ERCC1) is a major DNA repair protein. Recent studies have addressed the association between ERCC1 polymorphism and carcinogenesis. METHODS: We investigated a polymorphism of ERCC1 at nucleotide 19007 (C-->T, Asn118Asn) in 94 epithelial ovarian cancer patients, 102 endometrial cancer patients, and in 329 control subjects. RESULTS: We observed no evidence of associations between the C19007T ERCC1 polymorphism and risk of epithelial ovarian or endometrial cancer, which had the same adjusted odds ratios of 0.91 (p = 0.711, 0.691 respectively). CONCLUSION: Our findings suggest that the C19007T ERCC1 polymorphism is unlikely to play an important role in epithelial ovarian or endometrial cancer in Korean women.
Assuntos
Proteínas de Ligação a DNA/genética , Neoplasias do Endométrio/genética , Endonucleases/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Polimorfismo Genético , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Neoplasias do Endométrio/epidemiologia , Neoplasias do Endométrio/patologia , Feminino , Genótipo , Humanos , Coreia (Geográfico)/epidemiologia , Modelos Logísticos , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/epidemiologia , Neoplasias Epiteliais e Glandulares/patologia , Razão de Chances , Neoplasias Ovarianas/epidemiologia , Neoplasias Ovarianas/patologia , Estudos Prospectivos , Fatores de RiscoRESUMO
OBJECTIVE: To delineate the expression of transforming growth factor beta-inducible gene h3 (betaIG-H3) in rheumatoid synovitis and to determine the regulatory role of betaIG-H3 in the adhesion and migration of fibroblast-like synoviocytes (FLS). METHODS: Synovial tissue was obtained from patients with rheumatoid arthritis (RA) during joint replacement surgery, and FLS were isolated using enzymatic treatment. Immunohistochemical staining was performed to show the expression of betaIG-H3 within rheumatoid synovium. Synthesis of betaIG-H3 from FLS was determined by semiquantitative reverse transcription-polymerase chain reaction, Western blot analysis, and enzyme-linked immunosorbent assay. Cell adhesion and migration assays were performed using the YH18 peptide in the fourth fas-1 domain of betaIG-H3 and function-blocking antibodies to integrins. RESULTS: Expression of betaIG-H3 was up-regulated in RA synovial tissue compared with synovial tissue from patients with osteoarthritis. FLS isolated from RA synovial tissue constitutively produced betaIG-H3, which was up-regulated by transforming growth factor beta1, interleukin-1beta, and tumor necrosis factor alpha. Although FLS expressed a variety of integrins, betaIG-H3 mediated adhesion and migration of FLS through interaction with alpha v beta3 integrin. Cytokines failed to affect the betaIG-H3-mediated adhesion. However, migration of FLS guided by betaIG-H3 was enhanced by interferon-gamma and platelet-derived growth factor type BB. The YH18 peptide in the fourth fas-1 domain of betaIG-H3 inhibited adhesion and migration in a dose-dependent manner. CONCLUSION: The results suggest that betaIG-H3, which is abundantly expressed in RA synovial tissue, plays a regulatory role in chronic destructive inflammation through the modulation of the adhesion and migration of FLS. This finding indicates the relevance of betaIG-H3 and alpha v beta3 integrin-interacting motifs as potential therapeutic targets in this disease.
Assuntos
Artrite Reumatoide/genética , Proteínas da Matriz Extracelular/genética , Integrina alfaVbeta3/genética , Fator de Crescimento Transformador beta/fisiologia , Artrite Reumatoide/cirurgia , Artroplastia de Substituição , Adesão Celular , Movimento Celular , Citocinas/farmacologia , Primers do DNA , Regulação da Expressão Gênica , Humanos , Cinética , RNA/genética , RNA/isolamento & purificação , Membrana Sinovial/citologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Fator de Crescimento Transformador beta/genéticaRESUMO
OBJECTIVE: To determine whether CD40 ligation of rheumatoid arthritis synovial fibroblasts (RASFs) is able to induce RANKL expression and osteoclastogenesis in RASFs, and to identify its mechanism of action in patients with RA. METHODS: CD40 of RASFs was ligated with CD40 ligand (CD40L)-transfected L cells or activated T cells. The formation of osteoclasts in cocultures of CD40-ligated RASFs and T lymphocyte-depleted peripheral blood mononuclear cells was evaluated by tartrate-resistant acid phosphatase staining, detection of calcitonin receptor, and resorption pit formation assay. The expression of NF-kappaB, IkappaB alpha, ERK-1/2, phospho-ERK-1/2, p38, phospho-p38, and RANKL was examined by immunoblotting and/or semiquantitative reverse transcription-polymerase chain reaction. RESULTS: CD40 ligation of RASFs by CD40L-transfected L cells or activated T cells induced RANKL expression and enhanced osteoclastogenesis. CD40 ligation of RASFs also induced activation of ERK-1/2, p38 MAPK, and NF-kappaB and up-regulation of CD40 ligation-induced RANKL expression, whereas osteoclastogenesis was reduced in RASFs transfected with a dominant-negative mutant of IkappaB alpha or by an NF-kappaB inhibitor. However, specific inhibitors of MAPK/ERK-1/2 and p38 MAPK partially blocked the induction of RANKL expression and osteoclastogenesis. Monoclonal antibodies against interleukin-1 and tumor necrosis factor alpha partially inhibited CD40 ligation-mediated osteoclastogenesis. CONCLUSION: These results indicate that CD40 ligation of RASFs induces RANKL expression mainly via NF-kappaB activation and also results in enhanced osteoclast formation, both of which might play important roles in bone and cartilage destruction in RA. Inhibition of the CD40-CD40L interaction is a potential strategy for the prevention of bone damage in RA.
Assuntos
Artrite Reumatoide/fisiopatologia , Antígenos CD40/fisiologia , Proteínas de Transporte/fisiologia , Fibroblastos/fisiologia , Glicoproteínas de Membrana/fisiologia , NF-kappa B/fisiologia , Osteoclastos/fisiologia , Membrana Sinovial/citologia , Artrite Reumatoide/patologia , Antígenos CD40/imunologia , Proteínas de Transporte/análise , Células Cultivadas , Humanos , Quinase I-kappa B/análise , Proteínas I-kappa B/análise , Glicoproteínas de Membrana/análise , Proteína Quinase 1 Ativada por Mitógeno/análise , Inibidor de NF-kappaB alfa , NF-kappa B/análise , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases p38 Ativadas por Mitógeno/análiseRESUMO
Latent transforming growth factor (TGF)-beta-binding protein (LTBP) is required for the assembly, secretion, matrix association, and activation of latent TGF-beta complex. To elucidate the cell specific expression of the genes of LTBP-1 and their splice variants and the factors that regulate the gene expression, we cultured primary human glomerular endothelial cells (HGEC) under different conditions. Basal expression of LTBP-1 mRNA was suppressed in HGEC compared to WI-38 human embryonic lung fibroblasts. High glucose, H(2)O(2), and TGF-beta1 upregulated and vascular endothelial growth factor (VEGF) further downregulated LTBP-1 mRNA in HGEC. RT-PCR with a primer set for LTBP-1S produced many clones but no clone was gained with a primer set for LTBP-1L. Of 12 clones selected randomly, Sca I mapping and DNA sequencing revealed that only one was LTBP-1S and all the others were LTBP-1Sdelta53. TGF-beta1, but not high glucose, H(2)O(2) or VEGF, tended to increase LTBP-1Sdelta53 mRNA. In conclusion, HGEC express LTBP-1 mRNA which is suppressed at basal state but upregulated by high glucose, H(2)O(2), and TGF-beta1 and downregulated by VEGF. Major splice variant of LTBP-1 in HGEC was LTBP-1S 53. Modification of LTBP-1S 53 gene in HGEC may abrogate fibrotic action of TGF-beta1 but this requires confirmation.