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OBJECTIVES: To examine whether or not earlier therapeutic intervention with methotrexate (MTX) prevents the development of rheumatoid arthritis (RA) in patients with recent-onset undifferentiated arthritis (UA) showing high anti-citrullinated peptide antibody (ACPA) titers. METHODS: The patients were divided into two groups, one was treated with MTX (MTX+ group, n = 29), and the other was treated without MTX (MTX- group, n = 19), and other disease-modifying anti-rheumatic drugs were not permitted in the two groups before the primary endpoint was met. The primary endpoint is the occurrence of definite RA, and it was compared in the two groups after 1 year. RESULTS: The percentage of patients who developed definite RA in the MTX+ group (17.2%) was significantly lower than that in the MTX- group (78.9%) (log-rank test, P < 0.001, n = 48); adjusted hazards ratio: 0.028 [95% confidence interval (CI): 0.003-0.250, P = 0.001, n = 39]. Treatment effectiveness was not decreased by major risk factors of RA onset such as smoking habits and human leukocyte antigen-DRB1 shared epitope (SE) (smoking habit, odds ratio [OR]: 0.041 [95% CI: 0.007-0.246] P < 0.001; SE, OR: 0.022 [95% CI: 0.002-0.204] P < 0.001). The safety issues were comparable between the two groups. CONCLUSIONS: This suggests that early therapeutic intervention with MTX could safely prevent the development of RA in patients with recent-onset UA showing high ACPA titers.
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Antirreumáticos/uso terapêutico , Artrite Reumatoide/prevenção & controle , Artrite/tratamento farmacológico , Metotrexato/uso terapêutico , Adulto , Idoso , Artrite Reumatoide/tratamento farmacológico , Autoanticorpos/imunologia , Estudos de Coortes , Epitopos/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Risco , Fumar , Resultado do TratamentoRESUMO
BACKGROUND/AIMS: The early diagnosis of acute cholangitis (AC) is critical for appropriate treatment. METHODOLOGY: Patient records from April 2008 to December 2012 were retrospectively analyzed. Data on white blood cell count and levels of C-reactive protein, total-bilirubin, alkaline phosphatase (ALP), aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transpeptidase (gamma-GTP) were collected from AC patients on the day they underwent endoscopic retrograde cholangiopancreatography (ERCP) for diagnosis and treatment. Data were collected 3 months before ERCP to analyze the rate of change of these variables. Receiver operating characteristics curve analysis was performed. RESULTS: We enrolled 63 patients with AC and 65 patients with non-AC. The threshold values of ALP and gamma-GTP were 1.09 and 1.30, respectively. CONCLUSIONS: 450 (IU/L) and 100 (IU/L), respectively, were thresholds of ALP and gamma-GTP on the day of ERCP. 1.09 and 1.30, respectively, were thresholds of ALP and gamma-GTP rates of change for the diagnosis of AC.
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Fosfatase Alcalina/sangue , Colangite/sangue , Colangite/epidemiologia , gama-Glutamiltransferase/sangue , Doença Aguda , Idoso , Idoso de 80 Anos ou mais , Colangiopancreatografia Retrógrada Endoscópica , Colangite/diagnóstico por imagem , Colangite/cirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Estatísticas não ParamétricasRESUMO
Feeder-free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder-free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA-1-60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA-1-60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder-free culture of hiPS cells requires A or AC to maintain pluripotency.
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Ativinas/farmacologia , Técnicas de Cultura de Células , Células Alimentadoras , Células-Tronco Pluripotentes Induzidas/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Ativinas/antagonistas & inibidores , Fosfatase Alcalina/análise , Animais , Antígenos de Superfície/análise , Benzamidas/farmacologia , Colágeno , Dioxóis/farmacologia , Combinação de Medicamentos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteínas de Homeodomínio/análise , Humanos , Laminina , Fator Inibidor de Leucemia/farmacologia , Camundongos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/análise , Proteoglicanas/análise , Antígenos Embrionários Estágio-Específicos/análiseRESUMO
BACKGROUND/AIMS: To clarify the usefulness of screening ultrasonography (US) to diagnose gastric and colorectal cancer, patient records were analyzed retrospectively. METHODOLOGY: Ultrasonography was performed for patients with abdominal symptoms. They were then subjected to computed tomography (CT) when diagnosed with gastric cancer, colorectal cancer, or bowel obstruction. Patient records were analyzed retrospectively after final diagnosis of gastric cancer or colorectal cancer by endoscopy, surgery or necropsy. RESULTS: Twelve patients were diagnosed with colorectal cancer and six with gastric cancer. The detailed structure of colorectal cancer was visible as wall thickening with US, while cancer was often illustrated as a mass by CT. Loss of stratification was clear with US in 11 patients. US demonstrated wall thickening in 10 patients and a mass in 1 patient, while CT demonstrated wall thickening in 3 patients and a mass in 8 patients. The structure of colorectal cancer was more obvious when using US than when using CT. One patient demonstrated focal wall thickening with US, but this was not detected by CT. CONCLUSIONS: US is useful for diagnosis of gastric cancer and colorectal cancer. US produces more detailed findings in colorectal cancer than CT.
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Neoplasias Colorretais/diagnóstico por imagem , Programas de Rastreamento/métodos , Neoplasias Gástricas/diagnóstico por imagem , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Neoplasias Colorretais/patologia , Endoscopia Gastrointestinal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Valor Preditivo dos Testes , Estudos Retrospectivos , Neoplasias Gástricas/patologia , Tomografia Computadorizada por Raios X , UltrassonografiaRESUMO
Insulin-like growth factor (IGF)-I is up-regulated in pancreatic cancer tissues. Pancreatic cancer cell lines were analyzed in serum-free media as a model of the fibrous tissues that these cells often invade. Pancreatic cancer surgical specimens were immunostained with anti-IGF-I receptor (IGF-IR)ß antibody. The growth of pancreatic cancer cells in serum-free media was also analyzed. Cell lysates were analyzed for protein by western blot analysis. Cells cultured in the presence of picropodophyllin (PPP), LY294002, or PD98059, were subjected to cell proliferation and scratch assays. In addition, BrdU uptake and apoptosis were analyzed in these cells. IGF-IRß was detected in pancreatic cancer cells invading fibrous tissues. NOR-P1 grew most rapidly in serum-free media. The concentrations of IGF-I and IGF-II in the media were higher in NOR-P1 than the other cell lines. Cell proliferation in NOR-P1 cells was enhanced by IGF-I or IGF-II treatment more than in MIA-Paca2 or PK-1 cells. PPP, LY294002, and PD98059 suppressed proliferation and motility of NOR-P1 cells and inhibited BrdU uptake, while PPP induced apoptosis. IGF-IRß may be a potential therapeutic target to inhibit invasion of pancreatic cancer.
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Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro , Desoxiuridina/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like II/farmacologiaRESUMO
BACKGROUND/AIMS: Patients with abdominal symptoms and leukocytosis or elevated C-reactive protein were subjected to abdominal ultrasonography (US) for correct diagnosis. METHODOLOGY: Patients with abdominal symptoms, leukocytosis and elevated C-reactive protein were enrolled. Those with abnormal liver enzymes or radiographs were excluded since either of them might be a clue to proper diagnosis, such as hepatobiliary diseases or bowel obstruction. RESULTS: Total number of patients was 38. Number of acute diverticulitis, colitis, acute appendicitis and enteritis were 8, 7, 7 and 6, respectively. One patient with pelvis tumor and 2 with colon cancer were successfully diagnosed with abdominal US. Colon cancers were confirmed and pelvis tumor was diagnosed as ovarian squamous cell carcinoma with surgical specimens. Sensitivity and specificity were 100% (95% CI: 44-100%) and 97.1% (95% CI: 85-99%), respectively. CONCLUSIONS: Our data clearly recommended that abdominal US be performed carefully for patients with abdominal symptoms and leukocytosis or elevated CRP since potentially they had malignancies.
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Abdome/diagnóstico por imagem , Proteína C-Reativa/análise , Leucocitose/diagnóstico por imagem , Humanos , Sensibilidade e Especificidade , UltrassonografiaRESUMO
We investigated the incidence of symptomatic vertebral fracture in patients who required long-term high-dose glucocorticoid (GC) treatment. The patients with collagen vascular diseases (aged 18 years or older) were registered to Chiba-Shimoshizu Rheumatic Cohort from 1986 to 2006. The study included the patients who were newly treated with the initial dose more than 20 mg prednisolone equivalent per day at least for more than 6 months. Among 700 patients (female/ male: 539/161, mean age: 46.7 years, mean initial GC dose: 39.9 mg/day), 167 patients (23.8%) had at least one symptomatic vertebral fracture. Age and daily GC dose were significantly higher in the symptomatic fracture group than the no symptomatic fracture group. Cox regression model demonstrated that the relative risk for symptomatic vertebral fracture is independently higher in female patients, and in patients with initial higher age, and in those patients with initial higher GC dose and GC dose-increase, but lower with cumulative higher GC dose. High-dose GC treatment causes significantly high prevalence of symptomatic vertebral fracture in patients with collagen vascular disease. Age, female, higher initial GC dose and GC dose-increase are the risk factors for the symptomatic vertebral fracture in those patients.
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Glucocorticoides/efeitos adversos , Fraturas da Coluna Vertebral/epidemiologia , Adulto , Idoso , Estudos de Coortes , Doenças do Colágeno/tratamento farmacológico , Feminino , Glucocorticoides/administração & dosagem , Humanos , Incidência , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Prednisolona/efeitos adversos , Fatores de Risco , Fraturas da Coluna Vertebral/induzido quimicamenteRESUMO
The aim of this study was to evaluate the association between juxtapapillary diverticulum (JD) and acute cholangitis (AC), and to analyze laboratory data to reveal the underlying mechanism. We conducted a retrospective review of 139 patients who underwent endoscopic retrograde cholangiopancreatography (ERCP) between April 2008 and March 2013 for diagnosis or treatment of biliary tract conditions. The Wilcoxon signed-rank test was used for comparison of variables between patients with or without JD. The χ (2) test was used to analyze the association between JD and AC duct dilatation. Logistic regression analysis was performed to identify variables with strong correlation with AC. ERCP was attempted in 139 patients, but in one patient the endoscope did not reach the papilla of Vater because of a partial gastrectomy, and in two patients evaluation for JD was not possible because of duodenal or papilla of Vater cancer. Therefore, 136 patients were included in this study. JD was significantly associated with AC (P<0.0001) and bile-duct dilatation (P=0.0107), and AC was strongly associated with bile duct dilatation (P=0.0013). Alkaline phosphatase levels were significantly elevated in patients with JD (P=0.0237). In AC patients without JD, χ (2) for C-reactive protein was 4.48 (P=0.0342), whereas in AC patients with JD, χ (2) values for the white blood cell count, alkaline phosphatase, and aspartate aminotransferase were 2.62, 3.1, and 3.61, respectively (P=0.025, 0.015, and 0.0336, respectively). JD was strongly associated with AC. Logistic regression analysis suggested that bile flow was disturbed with JD.
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Resistance is one limitation of sorafenib in the treatment of hepatocellular carcinoma (HCC). Insulin-like growth factor-1 receptor (IGF-1R) is involved in cancer cell proliferation. To assess the potential synergistic antitumor effects of picropodophyllin (PPP), an IGF-1R inhibitor, HLF and PLC/PRL/5, HCC cells were treated with PPP alone or PPP in combination with sorafenib, a multikinase inhibitor. Normal human umbilical vein endothelial cells (HUVECs) were also used to analyze the antiangiogenic effects of the drugs. HCC cells and HUVECs were cultured on 96-well plates, and then treated with PPP, with and without the addition of sorafenib. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay and hematoxylin and eosin staining were then performed 48 h later. The HCC cells were also analyzed using scratch assays and hematoxylin and eosin staining after 48 h. The proliferation of HLF, PLC/PRF/5 and HUVEC cells was suppressed by the combination of 0.2 µM PPP and 3 µM sorafenib more effectively than by 10 µM sorafenib alone. The motility of HLF and PLC/PRF/5 cells was also suppressed to a greater extent with the combination of PPP at 0.2 µM and sorafenib at 3 µM than with sorafenib at 10 µM alone. The cells that had been treated with 0.2 µM PPP and 3 µM sorafenib also exhibited pyknotic nuclei, which is characteristic of apoptosis. In conclusion, PPP enhanced sorafenib-mediated suppression of proliferation and motility in HCC cells. Therefore, the combination of PPP and sorafenib may exert antitumor and antiangiogenic effects.
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Transcription factors are essential for the differentiation of human induced pluripotent stem cells (iPS) into specialized cell types. Embryoid body (EB) formation promotes the differentiation of iPS cells. We sought to establish an efficient method of transfection and rotary culture to generate EBs that stably express two genes. The pMetLuc2-Reporter vector was transfected using FuGENE HD (FuGENE), Lipofectamine LTX (LTX), X-tremeGENE, or TransIT-2020 transfection reagents. The media was analyzed using a Metridia luciferase (MetLuc) assay. Transfections were performed on cells adherent to plates/dishes (adherent method) or suspended in the media (suspension method). The 201B7 cells transfected with episomal vectors were selected using G418 (200 µg/mL) or hygromycin B (300 µg/mL). Rotary culture was performed at 2.5 or 9.9 rpm. Efficiency of EB formation was compared among plates and dishes. Cell density was compared at 1.6×10(3),×10(4), and×10(5) cells/mL. The suspended method of transfection using the FuGENE HD reagent was the most efficient. The expression of pEBMulti/Met-Hyg was detected 11 days posttransfection. Double transformants were selected 6 days posttransfection with pEBNK/EGFP-Neo and pEBNK/Cherry-Hyg. Both EGFP and CherryPicker were expressed in all of the surviving cells. EBs were formed most efficiently from cells cultured at a density of 1.6×10(5) cells/mL in six-well plates or 6 cm dishes. The selected cells formed EBs. FuGENE-mediated transfection of plasmids using the suspension method was effective in transforming iPS cells. Furthermore, the episomal vectors enabled us to perform a stable double transfection of EB-forming iPS cells.
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Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Vetores Genéticos/genética , Células-Tronco Pluripotentes Induzidas/citologia , Plasmídeos/genética , Linhagem Celular , HumanosRESUMO
The aim of the present study was to reveal the metabolic disorders most commonly associated with nonalcoholic fatty liver disease (NAFLD). Triglyceride (TG), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), blood glucose (BG) and hemoglobin A1c (HbA1c) were analyzed. NAFLD was diagnosed using abdominal ultrasound (US), and TG, HDL, LDL, BG and HbA1c were immediately collected on the same day and subjected to multivariate regression analysis. Stepwise analysis was performed to select the variables that were closely associated with NAFLD. The patients who were positive for the hepatitis B antigen and hepatitis C antibody were excluded from the study. Additionally, the patients who were prescribed prednisolone or methotrexate were excluded from the study as these agents may cause NAFLD or liver toxicity. The study included 168 and 125 patients with and without NAFLD, respectively. TG, BG and HbA1c were strongly correlated with NAFLD. Among these parameters, TG was the strongest predictor of NAFLD (χ2=9.89, P=0.0017). TG was the parameter that was most strongly associated with NAFLD. In conclusion, elevated TG was a marker of NAFLD.
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In the present study, Frizzled-2 (Fz2), a receptor of the Wnt ligand, was investigated as a potential target of molecular therapy for hepatocellular carcinoma (HCC). Quantitative polymerase chain reaction (PCR) was performed to determine the expression levels of Fz2. A surgical specimen of HCC was immunostained with an Fz2 antibody. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay was performed on HCC cell lines, including HLF and Hep3B, 72 h after the transfection of the short hairpin (sh)RNA of Fz2 (shRNA-Fz2). RNA was isolated from the Hep3B and HLF cells 48 h after transfection and subjected to quantitative PCR. All cell lines had elevated levels of Fz2 compared with those in an adult liver. The highest and lowest expression levels of Fz2 were 246.9±15.7 in the HLF cells and 5.8±1.4 in the Hep3B cells, respectively. Fz2 was expressed in the tumorous HCC tissue, but not in the surrounding non-tumorous tissue. Cell proliferation was suppressed to 28.6±6.4% in the HLF cells and to 29.8±4.3% in the Hep3B cells at 100 ng shRNA-Fz2 per well. Levels of cyclin D1 expression decreased to 65.2±5.9% in the HLF cells and to 60.8±14.6% in the Hep3B cells at 2.5 µg per well. In conclusion, Fz2 was upregulated in the HCC cells. shRNA-Fz2 suppressed the proliferation of the Hep3B and HLF cells, decreasing Fz2 expression. As it was not expressed in the surrounding non-tumorous tissue, Fz2 may be an ideal molecular therapeutic target for HCC.
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In the present study, the threshold values of laboratory data for the diagnosis of non-alcoholic fatty liver disease (NAFLD) were investigated. The study enrolled patients who had undergone abdominal ultrasound (US) between April 2013 and August 2013, and for whom laboratory data were available on the same day. NAFLD was diagnosed following observations of a bright liver or hepatorenal echo contrast on the abdominal US scans. Patients were excluded from the study if they had liver diseases or had been prescribed prednisolone or methotrexate. Receiver operating characteristic curves, the Wilcoxon signed-rank test and Fisher's exact probability test were used for data analysis. In total, 80 NAFLD and 94 non-NAFLD patients were enrolled in the study. The threshold levels of alanine aminotransferase (ALT) and triglyceride (TG) for the diagnosis of NAFLD were 19.0 IU/l and 101 mg/dl, respectively. Patients were divided into two groups according to the levels of ALT and TG. Those with ALT levels of >19 IU/l and TG levels of >101 mg/dl were defined as the positive group, while the remaining patients were classified as the negative group. The specificity and positive predictive value using the combined threshold levels of ALT >19 IU/l and TG >101 mg/dl were 80.9 and 75.0%, respectively. Therefore, the results indicated that ALT levels of >19 IU/l or TG levels of >101 mg/dl were useful markers for the screening of NAFLD. However, NAFLD was more strongly suspected in patients with ALT levels of >19 IU/l and TG levels of >101 mg/dl.
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In the present study, pancreatic cancer cell proliferation was analyzed following the suppression of frizzled (Fz)2 expression. Reverse transcription polymerase chain reaction (PCR) was performed using RNA isolated from pancreatic cancer cell lines, PANC-1, NOR-P1, PK-45H, PK-1, PK-59, MIA-Paca2 and KP4. A surgical specimen of pancreatic cancer was immunostained with antibodies specific to Fz2. Cell proliferation assays were performed with MIA-Paca2 cells transfected with small interfering RNA (siRNA) or short hairpin RNA (shRNA) of Fz2. Fz2 was found to be expressed in all pancreatic cancer cell lines, with the exception of NOR-P1. Immunostaining revealed that Fz2 was not expressed in normal pancreatic tissues, while it was expressed in pancreatic cancer cells. The expression levels of cyclin D1 were analyzed by quantitative PCR. The proliferation and expression of cyclin D1 were suppressed with the siRNA and shRNA of Fz2 in the MIA-Paca2 cells. Therefore, Fz2 is a potential target for the molecular therapy of pancreatic cancer.
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Objective. Symptoms and laboratory data between acute cholangitis (AC) patients treated with and AC patients treated without immunosuppressive drugs (corticosteroids or methotrexate) were compared to identify factors that can be meaningful to the diagnosis of AC. Methods. The Wilcoxon signed-rank test was used for comparison of baseline variables between the patients with AC treated with immunosuppressive drugs and those without it. The chi-squared test was used in the analysis of the symptoms. Results. In total, 69 patients with AC were enrolled. Fifteen patients were treated with immunosuppressants due to rheumatoid arthritis or other collagen diseases. Jaundice was less frequent in the patients treated with immunosuppressive drugs (P = 0.0351). T-Bil level was marginally lower in the patients treated with immunosuppressants (P = 0.086). AST and ALT levels were lower in the patients treated with immunosuppressants (P = 0.0417 and 0.022, respectively). Conclusions. The frequency of jaundice and AST and ALT levels were lower in the patients treated with immunosuppressive drugs. It is recommended that care be taken to evaluate jaundice, AST level, and ALT level in the diagnosis of AC.
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AIM: To investigate the early upper gastrointestinal endoscopy (endoscopy) significantly reduces mortality resulting from upper gastrointestinal (GI) bleeding. METHODS: Upper GI bleeding was defined as 1a, 1b, 2a, and 2b according to the Forrest classification. The hemoglobin (Hb), and C-reactive protein (CRP) were examined at around the day of endoscopy and 3 mo prior to endoscopy. The rate of change was calculated as follows: (the result of blood examination on the day of endoscopy - the results of blood examination 3 mo prior to endoscopy)/(results of blood examination 3 mo prior to endoscopy). Receiver operating characteristic curves were created to determine threshold values. RESULTS: Seventy-nine men and 77 women were enrolled. There were 17 patients with upper GI bleeding: 12 with a gastric ulcer, 3 with a duodenal ulcer, 1 with an acute gastric mucosal lesion, and 1 with gastric cancer. The area under the curve (AUC), threshold, sensitivity, and specificity of Hb around the day of endoscopy were 0.902, 11.7 g/dL, 94.1%, and 77.1%, respectively, while those of CRP were 0.722, 0.5 mg/dL, 70.5%, and 73%, respectively. The AUC, threshold, sensitivity, and specificity of the rate of change of Hb were 0.851, -21.3%, 76.4%, and 82.6%, respectively, while those of CRP were 0.901, 100%, 100%, and 82.5%, respectively. CONCLUSION: Predictors for upper GI bleeding were Hb < 11.7 g/dL, reduction rate in the Hb > 21.3% and an increase in the CRP > 100%, 3 mo before endoscopy.
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Proteína C-Reativa/metabolismo , Hemorragia Gastrointestinal/sangue , Hemoglobinas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Biomarcadores/sangue , Regulação para Baixo , Feminino , Hemorragia Gastrointestinal/diagnóstico , Hemorragia Gastrointestinal/cirurgia , Hemostase Endoscópica , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Curva ROC , Estudos Retrospectivos , Fatores de Risco , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do Tratamento , Regulação para CimaRESUMO
OBJECTIVE: This prospective study aimed to determine whether the comprehensive ultrasonographic assessment of synovial inflammation predicts relapse after discontinuation of treatment with a biologic agent in patients with rheumatoid arthritis (RA) in clinical remission. METHODS: RA patients in clinical remission (Disease Activity Score in 28 joints [DAS28] <2.6) receiving treatment with a biologic agent who agreed to discontinue the treatment were recruited. Patients underwent a comprehensive ultrasound scan on 134 synovial sites in 40 joints and were prospectively followed up for 6 months. Physicians who evaluated the patients during the study period were blinded to the baseline ultrasound findings. RESULTS: Forty-two patients receiving either a tumor necrosis factor antagonist or tocilizumab were enrolled. Using the optimal cutoff values determined by receiver operating characteristic curve analysis, relapse rates were significantly higher in patients whose total ultrasound scores at discontinuation were high than in those whose total ultrasound scores were low (P < 0.001 for both total gray-scale and power Doppler scores), whereas the difference between high and low DAS28 was not statistically significant (P = 0.158 by log rank test). Positive and negative predictive values were 80.0% and 73.3% for the total gray-scale score and 88.9% and 74.2% for the total power Doppler score, respectively. CONCLUSION: In RA patients in clinical remission receiving treatment with a biologic agent, residual synovial inflammation determined by comprehensive ultrasound assessment predicted relapse within a short term after discontinuation of the treatment. Our data provide a rationale and groundwork to conduct a large-scale study for establishment of ultrasound-based strategies to optimize the period of treatment with a biologic agent.
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Antirreumáticos/administração & dosagem , Artrite Reumatoide/diagnóstico por imagem , Artrite Reumatoide/tratamento farmacológico , Produtos Biológicos/administração & dosagem , Sinovite/diagnóstico por imagem , Sinovite/tratamento farmacológico , Ultrassonografia Doppler , Adulto , Idoso , Área Sob a Curva , Intervalo Livre de Doença , Esquema de Medicação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Valor Preditivo dos Testes , Estudos Prospectivos , Curva ROC , Recidiva , Indução de Remissão , Fatores de Risco , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do TratamentoRESUMO
OBJECTIVE: The aim of this prospective multicenter study was to identify biomarkers that can be used to predict therapeutic responses to tocilizumab in patients with rheumatoid arthritis (RA). METHODS: We recruited patients with RA who were treated with tocilizumab for the first time, and determined therapeutic responses at 6 months. In the training cohort (n = 40), gene expression in peripheral blood mononuclear cells (PBMCs) at baseline was analyzed using genome-wide DNA microarray, with 41,000 probes derived from 19,416 genes. In the validation cohort (n = 20), expression levels of the candidate genes in PBMCs at baseline were determined using real-time quantitative polymerase chain reaction (qPCR) analysis. RESULTS: We identified 68 DNA microarray probes that showed significant differences in signal intensity between nonresponders and responders in the training cohort. Nineteen putative genes were selected, and a significant correlation between the DNA microarray signal intensity and the qPCR relative expression was confirmed in 15 genes. In the validation cohort, a significant difference in relative expression between nonresponders and responders was reproduced for 3 type I interferon response genes (IFI6, MX2, and OASL) and MT1G. Receiver operating characteristic curve analysis of models incorporating these genes showed that the maximum area under the curve was 0.947 in predicting a moderate or good response to tocilizumab in the validation cohort. CONCLUSION: Using genome-wide DNA microarray analyses, we identified candidate biomarkers that can be used to predict therapeutic responses to tocilizumab in patients with RA. These findings suggest that type I interferon signaling and metallothioneins are involved in the pathophysiology of RA.
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Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Interferon Tipo I/sangue , Leucócitos Mononucleares/metabolismo , Metalotioneína/sangue , Análise de Sequência com Séries de Oligonucleotídeos , Adulto , Idoso , Artrite Reumatoide/sangue , Artrite Reumatoide/fisiopatologia , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica/genética , Humanos , Interferon Tipo I/genética , Interferon Tipo I/fisiologia , Masculino , Metalotioneína/genética , Metalotioneína/fisiologia , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Reprodutibilidade dos Testes , Resultado do TratamentoRESUMO
BACKGROUND: The Wnt pathway plays an important role in Hepatocarcinogenesis. We analyzed the association of the Wnt pathway with the proliferation of hepatoma cells using Wnt3a and niclosamide, a drug used to treat tapeworm infection. METHODS: We performed an MTS assay to determine whether Wnt3a stimulated proliferation of Huh-6 and Hep3B human hepatoma cell lines after 72 hours of incubation with Wnt3a in serum-free medium. The cells were subjected to hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) after 48 hours of incubation. RNA was isolated 48 hours after addition of Wnt3a or niclosamide, and cyclin D1 expression levels were analyzed by real-time quantitative polymerase chain reaction. The promoter activity of T-cell factor was analyzed by luciferase assay 48 hours after transfection of TOPflash. Western blot analysis was performed with antibodies against ß-catenin, dishevelled 2, and cyclin D1. RESULTS: Cell proliferation increased with Wnt3a. Niclosamide suppressed proliferation with or without Wnt3a. Hematoxylin and eosin and TUNEL staining suggested that apoptosis occurred in cells with niclosamide. Cyclin D1 was upregulated in the presence of Wnt3a and downregulated with addition of niclosamide. The promoter activity of T-cell factor increased with Wnt3a, whereas T-cell factor promoter activity decreased with niclosamide. Western blot analysis showed that Wnt3a upregulated ß-catenin, dishevelled 2, and cyclin D1, while niclosamide downregulated them. CONCLUSION: Niclosamide is a potential candidate for the treatment of hepatoma.
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Genes can be transferred using viral or non-viral vectors. Non-viral methods that use plasmid DNA and short interference RNA (siRNA) have advantages, such as low immunogenicity and low likelihood of genomic integration in the host, when compared to viral methods. Non-viral methods have potential merit, but their gene transfer efficiency is not satisfactory. Therefore, new methods should be developed. Low-frequency ultrasound irradiation causes mechanical perturbation of the cell membrane, allowing the uptake of large molecules in the vicinity of the cavitation bubbles. The collapse of these bubbles generates small transient holes in the cell membrane and induces transient membrane permeabilization. This formation of small pores in the cell membrane using ultrasound allows the transfer of DNA/RNA into the cell. This phenomenon is known as sonoporation and is a gene delivery method that shows great promise as a potential new approach in gene therapy. Microbubbles lower the threshold of cavity formation. Complexes of therapeutic genes and microbubbles improve the transfer efficiency of genes. Diagnostic ultrasound is potentially a suitable sonoporator because it allows the real-time monitoring of irradiated fields.