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The long interspersed nuclear element-1 (LINE-1) retrotransposons are a major family of mobile genetic elements, comprising approximately 17% of the human genome. The methylation state of LINE-1 is often used as an indicator of global DNA methylation levels and it regulates the retrotransposition and somatic insertion of the genetic element. We have previously reported the significant relationship between LINE-1 hypomethylation and poor prognosis in upper gastrointestinal (GI) cancers. However, the causal relationships between LINE-1 hypomethylation, retrotransposition, and tumor-specific insertion in upper GI cancers remain unknown. We used bisulfite-pyrosequencing and quantitative real-time PCR to verify LINE-1 methylation and copy number in tissue samples of 101 patients with esophageal and 103 patients with gastric cancer. Furthermore, we analyzed the LINE-1 retrotransposition profile with an originally developed L1Hs-seq. In tumor samples, LINE-1 methylation levels were significantly lower than non-tumor controls, while LINE-1 copy numbers were markedly increased. As such, there was a significant inverse correlation between the LINE-1 methylation level and copy number in tumor tissues, with lower LINE-1 methylation levels corresponding to higher LINE-1 copy numbers. Of particular importance is that somatic LINE-1 insertions were more numerous in tumor than normal tissues. Furthermore, we observed that LINE-1 was inserted evenly across all chromosomes, and most often within genomic regions associated with tumor-suppressive genes. LINE-1 hypomethylation in upper GI cancers is related to increased LINE-1 retrotransposition and tumor-specific insertion events, which may collectively contribute to the acquisition of aggressive tumor features through the inactivation of tumor-suppressive genes.
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Neoplasias Gastrointestinales , Neoplasias Gástricas , Humanos , Metilación de ADN/genética , Neoplasias Gastrointestinales/genética , Elementos de Nucleótido Esparcido Largo/genética , Neoplasias Gástricas/genética , EsófagoRESUMEN
The in vitro formation of network structures derived from endothelial cells in grafts before transplantation contributes to earlier engraftment. In a previous study, endothelial cells migrated to form a net-shaped structure in co-culture. However, the specific network formation behavior of endothelial cells during migration remains unclear. In this study, we demonstrated the tracing behavior and cell cycle of endothelial cells using Fucci-labeled (Fluorescent Ubiquitination-based Cell Cycle Indicator) endothelial cells. Here, we observed the co-culture of Fucci-labeled human umbilical vein endothelial cells (HUVECs) together with normal human dermal fibroblasts (NHDFs) using time-lapse imaging and analyzed by multicellular concurrent tracking. In the G0/G1 period, HUVECs migrate faster than in the S/G2/M period, because G0/G1 is the mobile phase and S/G2/M is the proliferation phase in the cell cycle. When HUVECs are co-cultured, they tend to move randomly until they find existing tracks that they then follow to form clusters. Extracellular matrix (ECM) staining showed that collagen IV, laminin and thrombospondin deposited in accordance with endothelial cell networks. Therefore the HUVECs may migrate on the secreted ECM and exhibit tracing behavior, where the HUVECs migrate toward each other. These results suggested that ECM and a cell phase contributed to form a network by accelerating cell migration.
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Puntos de Control del Ciclo Celular , Movimiento Celular , Células Endoteliales de la Vena Umbilical Humana/fisiología , Neovascularización Fisiológica , Comunicación Celular , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Matriz Extracelular/metabolismo , Fibroblastos/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Factores de Tiempo , Imagen de Lapso de Tiempo , TransfecciónRESUMEN
OBJECTIVES: We retrospectively examined intraocular pressure variations after visual field examination in primary open angle glaucoma (POAG), together with its influencing factors and its association with 24-hour intraocular pressure variations. SUBJECTS AND METHODS: Subjects were 94 eyes (52 POAG patients) subjected to measurements of 24-hour intraocular pressure and of changes in intraocular pressure after visual field examination using a Humphrey Visual Field Analyzer. Subjects were classified into three groups according to the magnitude of variation (large, intermediate and small), and 24-hour intraocular pressure variations were compared among the three groups. Factors influencing intraocular pressure variations after visual field examination and those associated with the large variation group were investigated. RESULTS: Average intraocular pressure variation after visual field examination was -0.28 ± 1.90 (range - 6.0(-) + 5.0) mmHg. No significant influencing factors were identified. The intraocular pressure at 3 a.m. was significantly higher in the large variation group than other two groups (p < 0.001). Central corneal thickness was correlated with the large variation group (odds ratio = 1.04; 95% confidence interval, 1.01-1.07 ; p = 0.02). CONCLUSION: No particular tendencies in intraocular pressure variations were found after visual field examination. Increases in intraocular pressure during the night might be associated with large intraocular pressure variations after visual field examination.
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Glaucoma de Ángulo Abierto/diagnóstico , Glaucoma de Ángulo Abierto/fisiopatología , Presión Intraocular/fisiología , Pruebas del Campo Visual , Campos Visuales/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de TiempoRESUMEN
Statins are cholesterol-lowering agents that act as inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzymeA (HMG CoA) reductase. Recently, statins have received a lot of attention, especially regarding how statins act on the immune system. Here, the clinical impact of statin intake was examined in patients with resected pancreatic cancer, and the underlying mechanisms were investigated in vitro and in vivo. We found that statin intake was associated with favorable prognostic outcomes in patients with resectable pancreatic cancer. Statins, especially lipophilic statins, exert anti-proliferative effects on pancreatic cancer cells in vitro (simvastatin > fluvastatin > atorvastatin > rosuvastatin > pravastatin). Simvastatin had an anti-proliferative effect on pancreatic cancer cells with decreased the yes-associated protein (YAP)/PDZ-binding motif (TAZ) expression by activating the JNK pathway, and simvastatin treatment with oxaliplatin revealed additive anti-growth effects. Furthermore, lipophilic and hydrophilic statins suppressed programmed cell death ligand 1 (PD-L1) expression by downregulating TAZ. Simvastatin treatment with an anti-PD-1 drug (BP0273) provided immediate anti-growth effects compared to controls, such as anti-PD-1 only and simvastatin only, and suppressed progressive disease during the early period of anti-PD-1 treatment in vivo. In conclusion, Statins display two distinct anti-cancer effects (direct anti-growth effect and elimination of immune suppression by downregulating PD-L1 expression) by targeting YAP/TAZ expression.
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OBJECTIVE: To evaluate the effectiveness of measuring anterior chamber interleukin (IL)-10 and IL-6 concentrations during treatment of vitreoretinal lymphoma with intravitreally injected methotrexate. METHODS: Two patients (3 eyes) underwent repeated weekly intravitreal injections of methotrexate (400 microg of methotrexate/50 microl) for 6 weeks. We monitored the IL-10 and IL-6 concentrations in the anterior chamber and the IL-10/IL-6 ratio at baseline and at the last injections using a standard enzyme-linked immunoassay. RESULTS: Repeated intravitreal injections of methotrexate reduced the vitreous opacity. The IL-10/IL-6 ratio decreased consistently in conjunction with clearance of the vitreous opacity in all 3 eyes, from 39.2 to 0.02, 1.67 to 0.03 and 3.05 to 0.159, respectively. CONCLUSIONS: A decrease in the IL-10/IL-6 ratio seems to be related to a decrease in vitreous opacity. Measuring the IL-10/IL-6 ratio in the anterior chamber may be useful to evaluate the effect of treatment of vitreoretinal lymphoma.
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Antimetabolitos Antineoplásicos/administración & dosificación , Humor Acuoso/metabolismo , Interleucina-10/biosíntesis , Interleucina-6/biosíntesis , Linfoma de Células B/tratamiento farmacológico , Metotrexato/administración & dosificación , Humor Acuoso/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inyecciones , Interleucina-10/análisis , Interleucina-6/análisis , Linfoma de Células B/metabolismo , Masculino , Persona de Mediana Edad , Retina/fisiopatología , Resultado del Tratamiento , Cuerpo Vítreo/fisiopatologíaRESUMEN
OBJECTIVE: We investigated the correlation of age and intraocular pressure (IOP) with visual field damage in patients with normal-tension glaucoma (NTG) and asymmetric visual field defects. METHODS: A total of 88 NTG patients (176 eyes) were investigated. Patients in whom visual field defects were more severe in the eye with higher IOP than in the fellow eye were grouped as IOP-visual field-concordant patients, and the others as IOP-visual field-discordant patients. Mean 24-hour IOP was used as an indicator of IOP, and mean deviation (MD) as an indicator of visual field defects. Patients were also compared by age tertile. RESULTS: The IOP-visual field-concordant group comprised 53 of the 88 patients (60.2%), among whom the >70-years tertile had significantly greater absolute values for MD asymmetry. In contrast, no significant differences in asymmetry by age tertile were seen in the IOP-visual field-discordant group. CONCLUSION: Elevated IOP is a cause of visual field defects in many NTG patients. Among IOP-visual field-concordant patients, particular attention is required to visual field changes in the eyes of patients with higher IOP.
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Glaucoma de Ángulo Abierto/fisiopatología , Presión Intraocular , Campos Visuales , Factores de Edad , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
PURPOSE: To evaluate the relationship between difficulty in daily living and binocular visual field in patients with glaucoma. SUBJECTS AND METHODS: We examined 144 patients with glaucoma who had bilateral visual field disturbances. The mean age was 68 +/- 13 years. Difficulty in daily living was assessed using a questionnaire consisting of 10 questions on daily activities. Each response was rated on a scale of 1 to 4: with a score of 4 for "no difficulty", 3 for "some difficulty", 2 for "great difficulty" and 1 for "cannot manage at all". The binocular visual field was assessed using the Esterman visual field test and the Esterman score was calculated. The relationship between difficulty in daily living and Esterman score was evaluated. RESULTS: The mean total score for difficulty in daily living was 34 +/- 7 (range; 13-40). The mean Esterman score was 79 +/- 24 (range; 0-100). The total score for difficulty in daily living correlates positively with the Esterman score (r2 = 0.48, p < 0.0001). CONCLUSION: The present study indicates that the Esterman score is a good predictor of difficulty daily living.
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Actividades Cotidianas , Glaucoma/fisiopatología , Campos Visuales/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Visión Binocular/fisiologíaRESUMEN
PURPOSE: To study the effect of the concomitant use of latanoprost and brinzolamide on the 24-hour variation in the intraocular pressure (IOP) in patients with normal-tension glaucoma (NTG). METHODS: We studied a total of 44 eyes from 22 NTG patients. Mean 24-hour IOP variation was determined after a washout period of > or =4 weeks. Latanoprost monotherapy was continued in both eyes for 8 weeks. Thereafter, patients were randomized to continue latanoprost monotherapy in 1 eye whereas brinzolamide was added as an adjunct to latanoprost therapy in the other eye. Eight weeks after the initiation of brinzolamide treatment, the 24-hour IOP variation was remeasured. IOP was measured in the sitting position 8 times daily using a Goldmann applanation tonometer before and after treatment. RESULTS: The eyes treated with latanoprost monotherapy and those treated with latanoprost and brinzolamide showed a significant decrease in IOP at all time points. Percent reductions in the diurnal mean IOP (mean IOP at 10 AM, 1 PM, and 4 PM) and in nocturnal mean IOP (mean IOP at 10 PM, 1 AM, and 3 AM) were significantly greater in the eyes treated with the combination of latanoprost and brinzolamide than those with latanoprost alone (diurnal mean IOP: latanoprost and brinzolamide=19.8%, latanoprost=14.1%, P<0.001; nocturnal mean IOP: latanoprost and brinzolamide=13.4%, latanoprost=10.0%, P<0.05). CONCLUSIONS: For the treatment of NTG, the combination of latanoprost and brinzolamide demonstrated additive effects in lowering IOP, not only during the day, but also at night.
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Antihipertensivos/uso terapéutico , Ritmo Circadiano/efectos de los fármacos , Glaucoma de Ángulo Abierto/tratamiento farmacológico , Presión Intraocular/efectos de los fármacos , Prostaglandinas F Sintéticas/uso terapéutico , Sulfonamidas/uso terapéutico , Tiazinas/uso terapéutico , Antihipertensivos/administración & dosificación , Presión Sanguínea , Recuento de Células , Ritmo Circadiano/fisiología , Quimioterapia Combinada , Endotelio Corneal/efectos de los fármacos , Endotelio Corneal/patología , Femenino , Glaucoma de Ángulo Abierto/fisiopatología , Frecuencia Cardíaca , Humanos , Presión Intraocular/fisiología , Latanoprost , Masculino , Persona de Mediana Edad , Prostaglandinas F Sintéticas/administración & dosificación , Sulfonamidas/administración & dosificación , Tiazinas/administración & dosificación , Tonometría OcularRESUMEN
Objectives. To evaluate 24-hour intraocular pressure (IOP) variation in patients with primary open-angle glaucoma (POAG) treated with triple eye drops. Subjects and Methods. The IOP was measured in 74 eyes in 74 POAG patients (seated) on triple therapy (PG analogue, ß-blocker, carbonic anhydrase inhibitor) at about every 3 hours. Results. The peak IOP was 13.5 ± 3.1 at 1:00, and the trough IOP was at 12.6 ± 2.4 mmHg at 7:00. The IOP at 7:00 was significantly lower than that at 10:00, 1:00, and 3:00 (p < 0.05). Based on the time of the peak IOP, we classified the patients into two groups: diurnal (28 eyes) and nocturnal types (37 eyes). There was significant difference at the spherical equivalent between diurnal and nocturnal types (p = 0.014). To assess the influence of reflective error, we conducted subanalysis for two groups: high myopic (26 eyes, ≤-6D) and low/nonmyopic (24 eyes, ≥-2D) groups. In the low/nonmyopia group, the IOP was significantly higher at 1:00 and 3:00 than at 13:00, 16:00, and 7: 00 (p < 0.05). Conclusion. The mean of IOP elevated outside of clinic hour in the POAG patients on triple therapy. The low/nonmyopia patient should be carefully treated because the IOP of the patients at night elevated significantly.
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PURPOSE: To evaluate the noelin 2 gene as a disease-causing factor for open-angle glaucoma (OAG) and the interactions between the noelin 2 (OLFM2), optineurin (OPTN), and myocilin (MYOC) genes. METHODS: OLFM2 was analyzed in 770 Japanese subjects including 215 patients with elevated intraocular pressure (IOP), 277 with normal IOP, 38 with juvenile open-angle glaucoma, and 240 control subjects. Two single-nucleotide polymorphisms (SNPs) in OPTN (c.412G-->A and c.603T-->A) and one SNP in MYOC (c.227G-->A) were examined. Single genes were investigated by univariate analysis and the gene-gene interactions by logistic regression analysis. Associations between genotypes and clinical characteristics at the time of diagnosis were examined. RESULTS: In OLFM2, 12 sequence variants were identified in 770 Japanese subjects. Arg144Gln (exon 4) was identified in two (0.3%) of the patients and in none of the control subjects. Combinations of OLFM2/317A and OPTN/412A or OLFM2/1281T and OPTN/412A were associated with patients with elevated IOP (P = 0.018 or P = 0.012, respectively). The combination of OLFM2/317G and OPTN/603A was significantly associated with elevated IOP (P = 0.018). No significant association was detected between SNPs in OLFM2 and in MYOC. Patients with normal IOP and with OLFM2/678A+OPTN/412G or OLFM2/1281C+OPTN/412G had significantly worse visual field scores (P = 0.022 or 0.030, respectively). CONCLUSIONS: The Arg144Gln mutation in OLFM2 is a possible disease-causing mutation in Japanese patients with OAG. Common polymorphisms in OLFM2 and OPTN may interactively contribute to the development of OAG, indicating a polygenic etiology.
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Proteínas del Citoesqueleto/genética , Proteínas de la Matriz Extracelular/genética , Proteínas del Ojo/genética , Glaucoma de Ángulo Abierto/genética , Glicoproteínas/genética , Polimorfismo de Nucleótido Simple , Factor de Transcripción TFIIIA/genética , Adulto , Anciano , Pueblo Asiatico/genética , Proteínas de Ciclo Celular , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , Genotipo , Humanos , Presión Intraocular , Japón , Proteínas de Transporte de Membrana , Persona de Mediana Edad , Mutación , Hipertensión Ocular/genética , Reacción en Cadena de la Polimerasa , Mapeo de Interacción de ProteínasRESUMEN
PURPOSE: The human trabecular meshwork and ciliary body, which express beta-adrenergic receptors (ADRB1 and ADRB2), control aqueous humor dynamics. We investigated associations of ADRB polymorphisms with open-angle glaucoma (OAG), because ADRB gene polymorphisms alter receptor function. METHODS: We studied 240 Japanese controls and 505 Japanese OAG patients including 211 with primary open-angle glaucoma (POAG), and 294 with normal-tension glaucoma (NTG). Associations of four polymorphisms (Ser49Gly and Arg389Gly in the ADRB1 gene; Arg16Gly and Gln27Glu in the ADRB2 gene) were compared between patients and controls. Age, intraocular pressure (IOP), and visual field defects at diagnosis were examined for associations with polymorphisms. RESULTS: The Arg389Gly polymorphism in the ADRB1 gene showed significantly different allele and genotype frequencies in patients with NTG than in controls (p = 0.004 and 0.006, respectively). Other polymorphisms did not show a significant frequency difference. In POAG patients, carriers of Gly16 in the ADRB2 gene were significantly younger at diagnosis than noncarriers (p<0.001). The IOP at diagnosis was significantly higher in OAG patients carrying 27Glu in the ADRB2 gene than in patients without this allele (p<0.001). Clinical characteristics of OAG patients did not differ significantly in relation to other polymorphisms. CONCLUSIONS: Certain polymorphisms of the ADRB1 and ADRB2 genes influence the pathophysiology of OAG in Japanese patients.
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Pueblo Asiatico/genética , Predisposición Genética a la Enfermedad , Glaucoma de Ángulo Abierto/genética , Polimorfismo Genético , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 2/genética , Anciano , Envejecimiento , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Genes Dominantes , Genes Recesivos , Genotipo , Glaucoma de Ángulo Abierto/diagnóstico , Glaucoma de Ángulo Abierto/fisiopatología , Heterocigoto , Humanos , Presión Intraocular , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
PURPOSE: To evaluate the relationship between central visual field disturbance due to glaucoma and reading performance. SUBJECTS AND METHODS: We evaluated the reading performance of 11 patients (14 eyes) with visual acuities of 1.0 and higher who had absolute scotoma within 3 degrees of the central visual field. The ages of the patients ranged from 37 to 77, with a median of 62 years. Maximum reading speed of texts printed horizontally and vertically was measured using MNREAD-J. Subjective reading difficulties were investigated orally. The relationship between the maximum reading speed of both horizontal and vertical texts, the subjective reading difficulties, the number of quadrants, and the position and continuity of absolute scotoma were evaluated. RESULTS: The mean maximum reading speed for vertical and horizontal texts by the 11 patients was 323 and 335 characters/minute(c/m), respectively, and was not significantly slower than that of normal eyes. In 2 of 14 eyes, a significant (p < 0.05) difference between maximum vertical and horizontal reading speeds was observed. In 3 of 14 eyes, absolute scotoma was distributed continuously in more than 2 adjacent quadrants, and these patients also had subjective reading difficulties. CONCLUSION: Reading difficulty is present in patients having absolute scotoma within 3 degrees of the central visual field when the scotoma involves more than 2 adjacent quadrants.
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Glaucoma/complicaciones , Lectura , Escotoma/etiología , Escotoma/fisiopatología , Campos Visuales , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Agudeza VisualRESUMEN
PURPOSE: Three glaucoma genes have been identified in open-angle glaucoma(OAG). In this study, two of these genes were analyzed in Japanese patients with OAG. SUBJECTS AND METHODS: After informed consent was obtained, the myocilin gene and the optineurin gene were analyzed in 99 Japanese patients with OAG, including 49 cases of primary open-angle glaucoma(POAG) and 50 cases of normal-tension glaucoma(NTG). The patients were outpatients at the Tokyo Metropolitan Police Hospital. Family members were examined for the genes and clinical features if the proband had a mutation. RESULTS: One of the 99 patients had His26Asp mutation in the optineurin gene. None of the 240 subjects serving as controls had this mutation. The proband was a 37-year-old man diagnosed as having NTG. His father had the same mutation, but had normal clinical findings. His mother did not have the mutation, but had ocular hypertension. Polymorphism of Thr34Thr was observed in a wild type for the proband, and heterozygous change was found in his parents. For the myocilin gene, only the Asp208Glu mutation was found in his mother. CONCLUSION: His26Asp mutation in the optineurin gene showed low penetrance in a Japanese family with NTG.
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Glaucoma de Ángulo Abierto/genética , Mutación , Penetrancia , Factor de Transcripción TFIIIA/genética , Adulto , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto/genética , Proteínas del Ojo/genética , Femenino , Glicoproteínas/genética , Humanos , Masculino , Proteínas de Transporte de Membrana , Persona de Mediana EdadRESUMEN
PURPOSE: The local renin-angiotensin system (RAS) is present in the ciliary body and plays a role in regulating aqueous humor dynamics and thus intraocular pressure (IOP). The purpose of this study was to determine whether gene polymorphisms in the RAS increase the risk of development of glaucoma in the Japanese. METHODS: A case-control study was performed in 698 Japanese subjects: 190 patients with primary open-angle glaucoma (POAG), 268 patients with normal-tension glaucoma (NTG), and 240 normal subjects. Ten polymorphisms in seven genes-AGT/Thr174Met and AGT/Met235Thr; REN/I8-83G-->A; ACE/insertion(I)-deletion(D); CMA/-1930A-->G; AGTR1/-731T-->G, AGTR1/-521C-->T, and AGTR1/1166A-->C; AGTR2/3123C-->A; and CYP11B2/-344T-->C were examined. The age, IOP, and visual field defects, all at diagnosis, were examined to determine whether they were associated with the polymorphisms. The effects of oral angiotensin II receptor blocker (ARB) on IOP were examined in association with the AGTR1 and AGTR2 polymorphisms in 20 normal subjects. RESULTS: Of the 10 polymorphisms, the AGTR2/3123C-->A polymorphisms had a significantly different distribution in female patients with NTG; the frequency of the CA+AA genotypes was significantly higher than in female control subjects (P = 0.0095 for CC versus CA+AA). Although no significant difference was seen in the clinical characteristics of female patients with NTG who carried the AGTR2/3123C-->A genotype, patients with CC in the AGTR2 gene had significantly worse visual field scores if they carried ACE/ID+DD (i.e., D carriers; P = 0.012). ARB significantly lowered IOP in normal subjects, but the male subjects with the AGTR2/3123A genotype had significantly less lowering of IOP than those with the C genotype (P = 0.014). CONCLUSIONS: Angiotensin II receptor gene polymorphisms may be associated with the risk of glaucoma in the Japanese population.
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Glaucoma de Ángulo Abierto/genética , Polimorfismo Genético , Receptores de Angiotensina/genética , Administración Oral , Adulto , Anciano , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Antagonistas de Receptores de Angiotensina , Bencimidazoles/farmacología , Compuestos de Bifenilo , Estudios de Casos y Controles , Estudios Cruzados , Método Doble Ciego , Femenino , Genotipo , Glaucoma de Ángulo Abierto/epidemiología , Humanos , Presión Intraocular , Japón/epidemiología , Masculino , Factores de Riesgo , Tetrazoles/farmacologíaRESUMEN
PURPOSE: Endothelin 1 (ET-1), a potent vasoconstrictor, may affect regulation of intraocular pressure and ocular vessel tone. Thus, ET-1 and its receptors may contribute to development of glaucoma. We investigated whether gene polymorphisms of ET-1 (EDN1) and its receptors ETA (EDNRA) and ETB (EDNRB) were associated with glaucoma phenotypes and clinical features. METHODS: We studied 224 normal Japanese controls and 426 open angle glaucoma (OAG) patients including 176 with primary open angle glaucoma (POAG) and 250 with normal tension glaucoma (NTG). Nine single nucleotide polymorphisms were detected among the participants using the Invader assay; four for EDN1 (T-1370G, +138/ex1 del/ins, G8002A, K198N), four for EDNRA (G-231A, H323H, C+70G, C+1222T), and one for EDNRB (L277L). Genotype distributions were compared between normal controls and OAG. Age at diagnosis, untreated maximum intraocular pressure (IOP), and visual field defects at diagnosis were examined for association with polymorphisms. RESULTS: Of the 9 polymorphisms, genotype distributions showed no significant differences between OAG patients and controls adjusted by age. The GG genotype of EDNRA/C+70G was associated with worse visual field defects in NTG patients (p=0.014; Mann-Whitney U test, and p=0.027; logistic regression analysis). CONCLUSIONS: The polymorphism of EDNRA/C+70G may be related to NTG risk factors.
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Endotelina-1/genética , Glaucoma de Ángulo Abierto/genética , Receptor de Endotelina A/genética , Anciano , Femenino , Genotipo , Glaucoma de Ángulo Abierto/fisiopatología , Humanos , Presión Intraocular , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Receptor de Endotelina B/genética , Factores de Riesgo , Trastornos de la Visión/fisiopatología , Campos VisualesRESUMEN
The purpose of this study was to evaluate the apoptosis and necrosis induced by five kinds of statins in IM-9 human lymphoblasts with fluorescence-enhanced flow cytometry using avidin-biotin complex. IM-9 human lymphoblasts (2 x 10(4) cells/cm2) were seeded into tissue culture plates and incubated with five kinds of statins. Statin-treated cells were first incubated with biotin-annexin V, followed by addition of avidin-FITC and propidium iodide, and then subjected to flow cytometry. The fluorescence intensity was enhanced using an avidin-biotin complex system, resulting in successful separate determination of the statin-induced apoptosis and necrosis by flow cytometry, which enabled us to quantitatively evaluate the statin-induced cell damage. Flow cytometric analysis results in the intensity of statin-induced apoptosis in IM-9 cells as follows: atorvastatin cerivastatin>fluvastatin simvastatin>pravastatin. The intensity of statin-induced necrosis in IM-9 cells was expressed as follows: atorvastatin cerivastatin>fluvastatin simvastatin>pravastatin. The total damage of IM-9 cells induced by five kinds of statins were expressed as the sum of both percentages of apoptosis and necrosis as follows: atorvastatin cerivastatin>fluvastatin simvastatin>pravastatin. Our studies show that fluorescence enhancement with avidin-biotin complex is useful for the identification and quantitation of annexin-positive apoptosis cells and thus, the fluorescence-enhanced flow cytometry was shown to be applicable for screening of statins as new anti-leukemia agents.
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Apoptosis , Citometría de Flujo/métodos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/análisis , Microscopía Fluorescente/métodos , Necrosis , Atorvastatina , Avidina/química , Biotina/química , Línea Celular , Separación Celular/métodos , Células Cultivadas , Industria Farmacéutica , Ensayos de Selección de Medicamentos Antitumorales , Ácidos Grasos Monoinsaturados/farmacología , Fluoresceína-5-Isotiocianato/farmacología , Fluvastatina , Ácidos Heptanoicos/farmacología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Indoles/farmacología , Leucemia/tratamiento farmacológico , Preparaciones Farmacéuticas/análisis , Pravastatina/farmacología , Propidio/farmacología , Piridinas/farmacología , Pirroles/farmacología , Simvastatina/farmacología , Factores de TiempoRESUMEN
For the production of tumor-specific vaccines, including dendritic cell (DC) vaccines, the tumor cells themselves are an ideal source. Floating tumor cells in the ascites fluid from patients with malignant ascites are a good candidate source, but it is not easy to obtain pure tumor cells from ascites because of various types of cell contamination as well as protein aggregates. We here report an effective method to recover pure tumor cells from malignant ascites. We used lavage fluid from 13 patients with malignant ascites who were treated with modified cell-free and concentrated ascites reinfusion therapy (KM-CART). Cellular components were separated from the lavage fluid by centrifugation, enzymatic digestion and hemolysis. Tumor cells were purified by depleting CD45(+) leukocytes with antibody-conjugated magnetic beads. The tumor cell lysate was extracted by freeze-and-thaw cycles. The mean obtained total cell number was 7.50 × 10(7) cells (range 4.40 × 10(6)-2.48 × 10(8) cells). From this fraction, 6.39 × 10(6) (range 3.23 × 10(5)-2.53 × 10(7)) CD45(-) cells were collected, and the tumor cell purity was over 80 % defined as CD45(-)CD326(+). A sufficient amount of tumor lysate, average = 2416 µg (range 25-8743 µg), was extracted from CD45(-)CD326(+) tumor cells. We here established an effective method to produce highly purified tumor cells from KM-CART lavage fluid. The clinical feasibility of this simple preparation method for generating tumor lysate should be examined in clinical studies of DC vaccines.
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Congenital syphilis (CS) is a public health burden in both developing and developed countries. We report two cases of CS in premature neonates with severe clinical manifestations; Patient 1 (gestational age 31 weeks, birth weight 1423 g) had disseminated idiopathic coagulation (DIC) while Patient 2 (gestational age 34 weeks and 6 days, birth weight 2299 g) had refractory syphilitic meningitis. Their mothers were single and had neither received antenatal care nor undergone syphilis screening. Both neonates were delivered via an emergency cesarean section and had birth asphyxia and transient tachypnea of newborn. Physical examination revealed massive hepatosplenomegaly. Laboratory testing of maternal and neonatal blood showed increased rapid plasma reagin (RPR) titer and positive Treponema pallidum hemagglutination assay. Diagnosis of CS was further supported by a positive IgM fluorescent treponemal antibody absorption test and large amounts of T. pallidum spirochetes detected in the placenta. Each neonate was initially treated with ampicillin and cefotaxime for early bacterial sepsis/meningitis that coexisted with CS. Patient 1 received fresh frozen plasma and antithrombin III to treat DIC. Patient 2 experienced a relapse of CS during initial antibiotic treatment, necessitating parenteral penicillin G. Treatment was effective in both neonates, as shown by reductions in RPR. Monitoring of growth and neurological development through to age 4 showed no evidence of apparent delay or complications. Without adequate antenatal care and maternal screening tests for infection, CS is difficult for non-specialists to diagnose at birth, because the clinical manifestations are similar to those of neonatal sepsis and meningitis. Ampicillin was insufficient for treating CS and penicillin G was necessary.
RESUMEN
PURPOSE: To investigate sequence variations in the optineurin (OPTN) gene and their association with TNF-alpha polymorphisms in Japanese patients with glaucoma. METHODS: The OPTN gene was analyzed in blood samples from 629 Japanese subjects. There were 194 patients with primary open-angle glaucoma (POAG), 217 with normal-tension glaucoma (NTG), and 218 with no eye disease (control subjects). The gene was screened for mutations by denaturing high-performance liquid chromatography. Genotyping of three polymorphisms of -308G-->A, -857C-->T, and -863C-->A in the TNF-alpha promoter region was performed. The associations between the genotypes and age, intraocular pressure (IOP), and visual field defects at the time of diagnosis were examined. RESULTS: A possible glaucoma-causing mutation, His26Asp, was identified in 1 of the 411 Japanese patients with glaucoma. A c.412G-->A (Thr34Thr) polymorphism in the OPTN gene was significantly associated with POAG (genotype frequency, P = 0.011; allele frequency, P = 0.003). The frequency of TNF-alpha/-857T and optineurin/412A carriers was significantly higher (P = 0.006) in patients with POAG than in control subjects. Among the patients with POAG who were carriers of TNF-alpha/-857T, the optineurin/412A carriers had significantly worse (P = 0.020) visual field scores than the non-optineurin/412A ones. The frequency of TNF-alpha/-863A and optineurin/603A (or Lys98) carriers was significantly higher in patients with POAG (P = 0.008) or NTG (P = 0.027) than in control subjects. Among the patients with POAG who were carriers of TNF-alpha/-863A, the ones with optineurin/603A (or Lys98) had significantly worse (P = 0.026) visual field scores than did those with non-optineurin/603A (or Lys98). CONCLUSIONS: These findings demonstrated that the OPTN gene is associated with POAG rather than NTG in the Japanese. Statistical analysis showed a possible interaction between polymorphisms in the OPTN and the TNF-alpha genes that would increase the risk for glaucoma.
Asunto(s)
Pueblo Asiatico/genética , Variación Genética , Glaucoma de Ángulo Abierto/genética , Presión Intraocular , Polimorfismo Genético , Factor de Transcripción TFIIIA/genética , Factor de Necrosis Tumoral alfa/genética , Adulto , Envejecimiento , Alelos , Ácido Aspártico , Proteínas de Ciclo Celular , Cromatografía Líquida de Alta Presión , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Glaucoma de Ángulo Abierto/fisiopatología , Heterocigoto , Histidina , Humanos , Masculino , Proteínas de Transporte de Membrana , Persona de Mediana Edad , Mutación , Treonina , Campos VisualesRESUMEN
PURPOSE: To screen for mutations in the MYOC gene in Japanese patients with primary open-angle glaucoma (POAG) using denaturing high-performance liquid chromatography (DHPLC). PATIENTS AND METHODS: Blood samples were collected from 171 patients with POAG and 100 controls from seven institutions in Japan. For high-throughput analysis, seven exonic regions were amplified by polymerase chain reaction using DNA pooled from three patients; each DNA pool was then analyzed chromatographically. For analysis of a small number of samples, 7 exonic regions were amplified separately but simultaneously with annealing at 58 degrees C in each patient and then chromatographed, using 7 wells of the same 96-well plate per sample. When chromatographic patterns were abnormal by either method, the PCR products of the individual samples were sequenced. RESULTS: Four glaucoma-causing mutations were identified in five POAG patients (2.9%). One missense mutation, Phe369Leu, is new; and three others, Ile360Asn, Ala363Thr, and Thr448Pro, have been reported in Japanese patients. Phe369Leu was associated with adult onset POAG. CONCLUSIONS: Mutations in the MYOC gene were demonstrated chromatographically in 2.9% of our Japanese POAG patients. The use of pooled DNAs with DHPLC analysis is a time- and labor-saving technique. All mutations detected appear to be specific to Japanese patients.