RESUMEN
BACKGROUND: In the phase III KEYNOTE-189 study (NCT02578680), pembrolizumab plus pemetrexed and platinum-based chemotherapy (pemetrexed-platinum) significantly improved overall survival (OS) and progression-free survival (PFS) in patients with previously untreated metastatic nonsquamous non-small-cell lung cancer (NSCLC) versus placebo plus pemetrexed-platinum. We report updated efficacy outcomes from the protocol-specified final analysis, including outcomes in patients who crossed over to pembrolizumab from pemetrexed-platinum and in patients who completed 35 cycles (â¼2 years) of pembrolizumab. PATIENTS AND METHODS: Eligible patients were randomized 2 : 1 to receive pembrolizumab 200 mg (n = 410) or placebo (n = 206) every 3 weeks (for up to 35 cycles, â¼2 years) plus four cycles of pemetrexed (500 mg/m2) and investigators' choice of cisplatin (75 mg/m2) or carboplatin (area under the curve 5 mg·min/ml) every 3 weeks, followed by pemetrexed until progression. Patients assigned to placebo plus pemetrexed-platinum could cross over to pembrolizumab upon progression if eligibility criteria were met. The primary endpoints were OS and PFS. RESULTS: After a median follow-up of 31.0 months, pembrolizumab plus pemetrexed-platinum continued to improve OS [hazard ratio (HR), 0.56; 95% confidence interval (CI), 0.46-0.69] and PFS (HR, 0.49; 95% CI, 0.41-0.59) over placebo plus pemetrexed-platinum regardless of programmed death-ligand 1 expression. Objective response rate (ORR) (48.3% versus 19.9%) and time to second/subsequent tumor progression on next-line treatment (PFS2; HR, 0.50; 95% CI, 0.41-0.61) were improved in patients who received pembrolizumab plus pemetrexed-platinum. Eighty-four patients (40.8%) from the placebo plus pemetrexed-platinum group crossed over to pembrolizumab on-study. Grade 3-5 adverse events occurred in 72.1% of patients receiving pembrolizumab plus pemetrexed-platinum and 66.8% of patients receiving placebo plus pemetrexed-platinum. Fifty-six patients completed 35 cycles (â¼2 years) of pembrolizumab; ORR was 85.7% and 53 (94.6%) were alive at data cut-off. CONCLUSIONS: Pembrolizumab plus pemetrexed-platinum continued to show improved efficacy outcomes compared with placebo plus pemetrexed-platinum, with manageable toxicity. These findings support first-line pembrolizumab plus pemetrexed-platinum in patients with previously untreated metastatic nonsquamous NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Pemetrexed/uso terapéutico , Platino (Metal)/uso terapéutico , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Background: Analysis of circulating cell-free DNA (cfDNA) is under intensive investigation for its potential to identify tumor somatic mutations. We have now explored the usefulness of such liquid biopsy testing with both the digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) during treatment of patients with the epidermal growth factor receptor (EGFR) inhibitor afatinib. Patients and methods: Eligible patients had advanced lung adenocarcinoma with EGFR activating mutations and were treated with afatinib. Plasma samples were collected before and during (4 and 24 weeks) afatinib treatment as well as at disease progression. Tumor and plasma DNA were analyzed by dPCR and NGS. Results: Thirty-five patients were enrolled. The objective response rate and median progression-free survival (PFS) were 77.1% and 13.8 months, respectively. Tumor and plasma DNA were available for 32 patients. dPCR and NGS detected EGFR activating mutations in 81.3% and 71.9% of baseline cfDNA samples, respectively. In 19 patients treated with afatinib for ≥24 weeks, the number of EGFR mutant alleles detected in cfDNA by dPCR declined rapidly and markedly after treatment onset, becoming undetectable or detectable at only a low copy number (<10 copies per milliliter) at 4 weeks. Median PFS was slightly longer for patients with undetectable EGFR mutant alleles in cfDNA at 4 weeks than for those in whom such alleles were detectable (14.3 versus 10.0 months). A total of 45 somatic mutations was identified in baseline tumor DNA, and 30 (66.7%) of these mutations were identified in cfDNA by NGS. Allele frequency for somatic mutations in cfDNA determined by NGS changed concordantly during afatinib treatment with the number of EGFR mutant alleles determined by dPCR. Conclusions: Monitoring of cfDNA by dPCR is informative for prediction of afatinib efficacy, whereas that by NGS is reliable and has the potential to identify mechanisms of treatment resistance.
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Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , ADN Tumoral Circulante/genética , Receptores ErbB/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Quinazolinas/uso terapéutico , Adenocarcinoma/sangre , Adenocarcinoma/enzimología , Adenocarcinoma del Pulmón , Afatinib , ADN Tumoral Circulante/sangre , Receptores ErbB/metabolismo , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Biopsia Líquida , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/enzimología , Masculino , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa/métodos , Estudios Prospectivos , Quinazolinas/efectos adversosRESUMEN
We describe successful treatment of 3 cases of human herpesvirus 6 (HHV-6) encephalitis/myelitis following cord blood transplantation (CBT). Ganciclovir (GCV) (10 mg/kg/day) reduced HHV-6 load to undetectable levels in cerebrospinal fluid (CSF). Early dose reduction in the presence of HHV-6 detectable in CSF resulted in an increased HHV-6 load. GCV was capably shifted to valganciclovir (VGCV) with an almost equivalent concentration. GCV/VGCV may be effective for HHV-6 encephalitis/myelitis after CBT, although HHV-6 load in CSF should be monitored.
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Antivirales/uso terapéutico , Encefalitis Viral/tratamiento farmacológico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Herpesvirus Humano 6/aislamiento & purificación , Mielitis/tratamiento farmacológico , Infecciones por Roseolovirus/tratamiento farmacológico , Acondicionamiento Pretrasplante/efectos adversos , Carga Viral/efectos de los fármacos , Adulto , Antivirales/administración & dosificación , Preescolar , ADN Viral , Encefalitis Viral/líquido cefalorraquídeo , Encefalitis Viral/virología , Femenino , Sangre Fetal , Ganciclovir/administración & dosificación , Ganciclovir/análogos & derivados , Ganciclovir/uso terapéutico , Humanos , Masculino , Mielitis/líquido cefalorraquídeo , Mielitis/virología , Agonistas Mieloablativos/efectos adversos , Infecciones por Roseolovirus/líquido cefalorraquídeo , Infecciones por Roseolovirus/virología , Resultado del Tratamiento , Valganciclovir , Adulto JovenRESUMEN
BACKGROUND: We evaluated the safety, maximum-tolerated dose (MTD), pharmacokinetics, recommended dose for phase II (P2RD), and preliminary anticancer activity of a combination eribulin and S-1 therapeutic in metastatic breast cancer patients pretreated with anthracycline and taxane. METHOD: Patients aged 20-74 years were recruited. In level 1, patients received S-1 (65 mg m(-2)) from day 1 to 14, and eribulin (1.1 mg m(-2)) on day 1 and 8 in a 21-day cycle. In level 2, eribulin was increased to 1.4 mg m(-2). In level 3, S-1 was increased to 80 mg m(-2). RESULTS: Twelve patients were enrolled into three cohorts. Planned dose escalation was completed, with one case exhibiting dose-limiting toxicity (grade 3 hypokalaemia) at level 3, without reaching the MTD. The P2RD was determined to be level 2 (eribulin 1.4 mg m(-2) and S-1 65 mg m(-2)). The most common grade 3 or 4 toxicity was neutropenia (83.3%), followed by febrile neutropenia (25.0%). Five of eleven patients (41.7%) with measurable disease had a partial response. Pharmacokinetics were characterised by dose-dependent elimination and nonlinear exposure. CONCLUSION: Dose level 3 was not tolerated owing to febrile neutropenia development. Thus, intermediate dose level 2 was recommended for further evaluation. Preliminary antitumour activity warrants further investigation in this setting.
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Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Neoplasias de la Mama/tratamiento farmacológico , Furanos/administración & dosificación , Furanos/farmacocinética , Cetonas/administración & dosificación , Cetonas/farmacocinética , Ácido Oxónico/administración & dosificación , Ácido Oxónico/farmacocinética , Tegafur/administración & dosificación , Tegafur/farmacocinética , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Combinación de Medicamentos , Femenino , Furanos/uso terapéutico , Humanos , Cetonas/uso terapéutico , Dosis Máxima Tolerada , Persona de Mediana Edad , Metástasis de la Neoplasia/tratamiento farmacológico , Ácido Oxónico/uso terapéutico , Tegafur/uso terapéutico , Adulto JovenRESUMEN
Using serum-containing culture, we examined whether AGM-S3 stromal cells, alone or in combination with hematopoietic growth factor(s), stimulated the proliferation of CD34(+) cells from patients with juvenile myelomonocytic leukemia (JMML). AGM-S3 cells in concert with stem cell factor plus thrombopoietin increased the numbers of peripheral blood CD34(+) cells to approximately 20-fold of the input value after 2 weeks in nine JMML patients with either PTPN11 mutations or RAS mutations, who received allogeneic hematopoietic transplantation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) also augmented the proliferation of JMML CD34(+) cells on AGM-S3 cells. The expansion potential of CD34(+) cells was markedly low in four patients who achieved spontaneous hematological improvement. A large proportion of day-14-cultured CD34(+) cells were negative for CD38 and cryopreservable. Cultured JMML CD34(+)CD38(-) cells expressed CD117, CD116, c-mpl, CD123, CD90, but not CXCR4, and formed GM and erythroid colonies. Day-7-cultured CD34(+) cells from two of three JMML patients injected intrafemorally into immunodeficient mice stimulated with human GM-CSF after transplantation displayed significant hematopoietic reconstitution. The abilities of OP9 cells and MS-5 cells were one-third and one-tenth, respectively, of the value obtained with AGM-S3 cells. Our culture system may provide a useful tool for elucidating leukemogenesis and for therapeutic approaches in JMML.
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Células Madre Embrionarias/efectos de los fármacos , Regulación Leucémica de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Leucemia Mielomonocítica Juvenil/genética , Células del Estroma/efectos de los fármacos , ADP-Ribosil Ciclasa 1/genética , ADP-Ribosil Ciclasa 1/metabolismo , Adolescente , Animales , Antígenos CD34/genética , Antígenos CD34/metabolismo , Proliferación Celular/efectos de los fármacos , Células Clonales , Técnicas de Cocultivo , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/patología , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Leucemia Mielomonocítica Juvenil/metabolismo , Leucemia Mielomonocítica Juvenil/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mutación , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/trasplante , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas p21(ras) , Transducción de Señal , Células del Estroma/metabolismo , Células del Estroma/patología , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Rubella virus is the causative agent of rubella. The symptoms are usually mild, and characterized by a maculopapular rash and fever. However, rubella infection in pregnant women sometimes can result in the birth of infants with congenital rubella syndrome (CRS). Global efforts have been made to reduce and eliminate CRS. Although a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detection of rubella virus has been reported, the primers contained several mismatched nucleotides with the genomes of currently circulating rubella virus strains. In the present study, a new RT-LAMP assay was established. The detection limit of this assay was 100-1000PFU/reaction of viruses for all rubella genotypes, except for genotype 2C, which is not commonly found in the current era. Therefore, the new RT-LAMP assay can successfully detect all current rubella virus genotypes, and does not require sophisticated devices like TaqMan real-time PCR systems. This assay should be a useful assay for laboratory diagnosis of rubella and CRS.
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Técnicas de Diagnóstico Molecular/métodos , Virus de la Rubéola/aislamiento & purificación , Rubéola (Sarampión Alemán)/diagnóstico , Rubéola (Sarampión Alemán)/virología , Virología/métodos , Femenino , Humanos , Recién Nacido , Embarazo , Virus de la Rubéola/genética , Sensibilidad y EspecificidadAsunto(s)
Leucemia Mielomonocítica Juvenil/tratamiento farmacológico , Leucemia Mielomonocítica Juvenil/genética , Mercaptopurina/farmacología , Mercaptopurina/uso terapéutico , Mutación , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Preescolar , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Lactante , Leucemia Mielomonocítica Juvenil/metabolismo , Masculino , Transducción de Señal , Resultado del TratamientoRESUMEN
PURPOSE: To determine ombrabulin's maximum tolerated dose and dose recommended for Japanese patients with advanced solid tumors and to assess its antitumor activity and overall safety and pharmacokinetic profiles. METHODS: This was a multi-center, open-label, sequential-cohort, dose-escalation phase I study of ombrabulin, a vascular disrupting agent, administered once every 3 weeks. Patients were treated with 15.5, 25, 35, or 50 mg/m(2) ombrabulin over a 30-min intravenous infusion. The recommended dose was the highest dose at which <33 % of all evaluable patients experienced dose-limiting toxicities (DLTs) during the first treatment cycle or 50 mg/m(2) (recommended in Caucasian patients) if the previous definition was not met. RESULTS: Fifteen patients were treated. No DLT occurred with 15.5, 25, or 35 mg/m(2) ombrabulin. In the 50 mg/m(2) group, one patient had Grade 3 lymphopenia, and another experienced Grade 2 hypertension and Grade 3 diarrhea judged as DLTs. The most frequent related adverse events in this group were diarrhea, nausea, and hypertension. Two patients had Grade 3 anemia, one at the 15.5 mg/m(2) and the other at the 50 mg/m(2). No AEs necessitating dose reduction or Grade 4 AEs were observed. Overall, five patients had stable disease. Pharmacokinetic parameters were comparable to those in non-Japanese patients. CONCLUSIONS: Ombrabulin treatment once every 3 weeks was well tolerated in Japanese patients with advanced solid tumors. The dose recommended is 50 mg/m(2), as in Caucasian patients. The safety and pharmacokinetic profiles were comparable between Japanese and Caucasian patients (funded by Sanofi; ClinicalTrials.gov number, NCT00968916).
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Neoplasias/tratamiento farmacológico , Serina/análogos & derivados , Pueblo Asiatico , Estudios de Cohortes , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Infusiones Intravenosas , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Neoplasias/irrigación sanguínea , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica/tratamiento farmacológico , Serina/administración & dosificación , Serina/efectos adversos , Serina/farmacocinéticaAsunto(s)
Enfermedades del Sistema Nervioso Autónomo/genética , Trastornos del Conocimiento/genética , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades por Prión/genética , Adulto , Enfermedades del Sistema Nervioso Autónomo/complicaciones , Trastornos del Conocimiento/complicaciones , Femenino , Eliminación de Gen , Humanos , Enfermedades del Sistema Nervioso Periférico/complicaciones , Enfermedades por Prión/complicacionesRESUMEN
BACKGROUND: A variety of hereditary spinocerebellar ataxia (SCA) develops a broad spectrum of both ataxia and non-ataxia symptoms. Cognitive and affective changes are one such non-ataxia symptoms, but have been described only in hereditary SCAs with exonic CAG gene expansion. METHODS: We newly found intronic hexanucleotide GGCCTG gene expansion in NOP56 gene as the causative mutation (=SCA36) in nine unrelated Japanese familial SCA originating from Asida river area in the western part of Japan, thus nicknamed Asidan for this mutation. These patients show unique clinical balance of cerebellar ataxia and motor neuron disease (MND), locating on the crossroad of these two diseases. In the nine families, 14 patients were clinically examined and genetically confirmed to Asidan. In the present study, we examined cognitive and affective analyses on 12 patients (seven men and five women) who agreed to join the examination with average age at onset of 53.1 ± 3.2 years, average duration of 12.1 ± 5.2 years, and current average age at 65.1 ± 6.2 years. RESULTS: The 12 Asidan patients demonstrated a significant decrease in their frontal executive functions measured by frontal assessment battery (FAB) and Montreal cognitive assessment (MoCA) compared with age- and gender-matched controls, whilst mini-mental state examination (MMSE) and Hasegawa dementia score-revised (HDS-R) were within normal range. The decline of frontal executive function was related to their disease duration and scale for the assessment and rating of ataxias (SARA). They also demonstrated mild depression and apathy. Single-photon emission tomography (SPECT) analysis showed that these Asidan patients showed decline of regional cerebral blood flow (rCBF) in a particular areas of cerebral cortices such as Brodmann areas 24 and 44-46. CONCLUSION: These data suggest that the patients with Asidan mutation show unique cognitive and affective characteristics different from other hereditary SCAs with exonal CAG expansion or MND.
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Trastornos del Conocimiento/genética , Enfermedad de la Neurona Motora/complicaciones , Enfermedad de la Neurona Motora/genética , Mutación , Proteínas Nucleares/genética , Ataxias Espinocerebelosas/complicaciones , Ataxias Espinocerebelosas/genética , Edad de Inicio , Anciano , Femenino , Humanos , Japón , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
Structural and functional abnormalities in the neurovascular unit (NVU) have been recently observed in Alzheimer's disease (AD). Statins, which are used clinically for reducing cholesterol levels, can also exert beneficial vascular actions. Thus, we examined the protective effects of statins on NVU disturbances in a mouse AD model. Amyloid precursor protein (APP) transgenic (Tg) mice were used as a model of AD. Atorvastatin (30 mg/kg/day, p.o.) or pitavastatin (3 mg/kg/day, p.o.) were administered from 5 to 20 months of age. Changes in the NVU, including the endothelium and basement membrane, as well as astrogliosis and matrix metalloproteinase-9 (MMP-9) activation, were assessed. There was a reduction in immunopositive staining for N-acetyl glucosamine oligomer (NAGO) in the endothelium and in collagen IV in the APP vehicle (APP/Ve) group, with collagen IV staining most weakest near senile plaques (SPs). There was also an increase in intensity and number of glial fibrillary acidic protein (GFAP)-positive astrocytes, particularly around the SP, where MMP-9 was more strongly labeled. Double immunofluorescent analysis showed that astrocytic endfeet had detached from the capillary endothelium in the APP/Ve group. Treatment with atorvastatin or pitavastatin ameliorated the activation of MMP-9. Overall, these data suggest that statins may have therapeutic potential for AD by protecting NVU.
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Enfermedad de Alzheimer/fisiopatología , Corteza Cerebral/fisiopatología , Circulación Cerebrovascular/efectos de los fármacos , Ácidos Heptanoicos/farmacología , Fármacos Neuroprotectores/farmacología , Pirroles/farmacología , Quinolinas/farmacología , Precursor de Proteína beta-Amiloide/genética , Animales , Atorvastatina , Western Blotting , Corteza Cerebral/irrigación sanguínea , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones TransgénicosRESUMEN
BACKGROUND: It remains unclear whether response rate (RR) is related to survival benefit in phase III trials of advanced cancer treated with molecular targeted agents (MTA) in combination with standard therapies. MATERIALS AND METHODS: We carried out a systematic search of PubMed for randomized phase III trials of four solid tumors examining the efficacy of MTA when added to a standard therapy. We examined whether there were any associations between RR increment obtained by the addition of targeted agents (DeltaRR) and survival benefit in phase III trials. RESULTS: We identified 26 phase III trials of MTA with a total of 21 156 patients and 29 experimental arms of MTA. Studies which showed significant survival benefit had higher DeltaRR compared with those which did not show significant benefit. In the receiver operating characteristic curve analysis, using a 7% gain as threshold value for DeltaRR allowed assessment of survival benefit with high sensitivity and specificity. There were also significant relationships between DeltaRR and hazard ratios for overall survival and progression-free survival in the linear regression analysis. CONCLUSION: RR increment obtained by the addition of MTA to a standard therapy may be useful to predict survival benefit in clinical phase III trials of advanced cancer.
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Antineoplásicos/uso terapéutico , Ensayos Clínicos Fase III como Asunto , Neoplasias/tratamiento farmacológico , Ensayos Clínicos Controlados Aleatorios como Asunto , Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Humanos , Análisis de SupervivenciaRESUMEN
AIM: Minor recurrent aphthous stomatitis (MRAS) is a common, painful and inflammatory ailment of the oral cavity with juvenile onset and unknown aetiology. The purpose of this study was to evaluate the potential of ascorbate (vitamin C) to reduce the frequency of MRAS and severity of pain. PATIENTS AND METHODS: Sixteen MRAS patients (9 boys and 7 girls: mean age, 12.0 +/- 2.4 years old) were assigned to take an oral dosage of 2000 mg/m(2)/day ascorbate. SUBJECTS: Their baseline frequency of outbreaks and the level of pains were compared during the treatment; in addition, a crossover clinical trial was performed. Polymorphonuclear leucocytes play a role in the pathogenesis, and then superoxide anion production was evaluated in prior to ascorbate treatment. RESULTS: The data indicated a statistically significant 50% reduction in oral ulcer outbreaks and a decline of pain level. Neutrophils were primed for superoxide anion production in the patients with MRAS. CONCLUSION: Ascorbate may modulate the generation of reactive oxygen species and augment neutrophil apoptosis, which could prevent neutrophil-mediated inflammation. Ascorbate seems to be effective, but the findings of our study were preliminary and it should be re-evaluated with a larger randomized controlled clinical trials.
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Antioxidantes/uso terapéutico , Ácido Ascórbico/uso terapéutico , Estomatitis Aftosa/prevención & control , Administración Oral , Adolescente , Niño , Estudios Cruzados , Esquema de Medicación , Femenino , Humanos , Masculino , Neutrófilos/metabolismo , Dolor/prevención & control , Prevención Secundaria , Índice de Severidad de la Enfermedad , Superóxidos/metabolismo , Resultado del TratamientoRESUMEN
Fusarium oxysporum produces three kinds of asexual spores, microconidia, macroconidia, and chlamydospores. We previously found that the transcript level of the nitrite reductase gene of F. oxysporum, named FoNIIA, was markedly upregulated during conidiation compared with during vegetative growth. FoNIIA was also found to be positively regulated by Ren1 that is a transcription regulator controlling development of microconidia and macroconidia. In this study, we analyzed the function of FoNIIA in conidiation of F. oxysporum. Conidiation cultures showed markedly higher level of accumulation of FoNiiA protein as well as FoNIIA mRNA than vegetative growth cultures. FoNIIA protein was significantly decreased in cultures of the REN1 disruption mutant compared with that of the wild type. These results confirmed that FoNIIA expression is upregulated during conidiation and is positively regulated by REN1. The FoNIIA disruption mutants produced microconidia, macroconidia, and chlamydospores, which were morphologically indistinguishable from those of the wild type. The mutants, however, produced significantly fewer macroconidia than the wild type, although the wild type and mutant strains produced similar numbers of microconidia and chlamydospores. These results demonstrate that nitrite reductase is involved in quantitative control of macroconidium formation as well as nitrate utilization in F. oxysporum.
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Fusarium/genética , Nitrito Reductasas/genética , Secuencia de Aminoácidos , Clonación Molecular , Cartilla de ADN , Oscuridad , Proteínas Fúngicas/genética , Fusarium/enzimología , Fusarium/crecimiento & desarrollo , Luz , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Microbiología del Suelo , Solanum tuberosum/microbiología , Esporas Fúngicas/enzimología , Esporas Fúngicas/genética , Transcripción Genética , Regulación hacia ArribaRESUMEN
Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for "immediate-early 1") protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.
Asunto(s)
Antígenos Virales/metabolismo , Infecciones por Citomegalovirus/metabolismo , Citomegalovirus/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Anticoagulantes/farmacología , Línea Celular , Técnicas de Cocultivo , Citomegalovirus/genética , Citomegalovirus/aislamiento & purificación , Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/virología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/virología , Heparina/farmacología , Humanos , Concentración 50 Inhibidora , Pulmón/citología , Pulmón/embriología , Microscopía Fluorescente , Pruebas de Neutralización , Proteína de la Leucemia Promielocítica , Factores de TiempoRESUMEN
In order to establish a reliable method for the detection of human herpesvirus-6 (HHV-6) B antigens in peripheral blood mononuclear cells (PBMCs) collected from HHV-6 infected patients, we created a polyclonal antibody against the HHV-6 B U90 protein (IEA/ex3) and used it to examine the expression of this protein in virus-infected cells and patients' PBMCs. This antibody reacted with 170 and 195 kDa proteins in HHV-6 B-infected cord blood mononuclear cells. The IEA/ex3 antigen was detected in cord blood mononuclear cells at 6 hr post-infection, and the number of infected cells reached its maximum at 48 hr post-infection. The antigen stained in a punctate pattern and partially localized to the promyelocytic leukemia (PML) protein body. We also examined 60 PBMC samples from 60 febrile children (3-19 months old) and detected IEA/ex3 antigen in the PBMCs by laser-scanning microscopy. HHV-6 was isolated from 31 of the 60 samples. The sensitivity and specificity of the antigen detection were 84% (26/31) and 97% (28/29), respectively, in the samples with virus detected. The mean number of antigen-positive PBMCs was 409/10(6) cells in 20 samples with viral isolation. A significant correlation (r = 0.566; P = 0.008) was observed between the viral load and number of antigen-positive cells. Although IEA/ex3 antigen was detected by laser-scanning microscopy in PBMCs (without cultivation) collected from six patients with isolated virus, it was detected in only one sample by conventional fluorescence microscopy. Increasing the intensity by cultivation (24 hr) resulted in a higher detection rate (5/6) even by conventional fluorescence microscopy, which is available in most hospital laboratories.
Asunto(s)
Antígenos Virales/análisis , Herpesvirus Humano 6/inmunología , Infecciones por Roseolovirus/diagnóstico , Proteínas Virales/análisis , Anticuerpos Antivirales , Antígenos Virales/biosíntesis , Western Blotting , Células Cultivadas , Regulación Viral de la Expresión Génica , Herpesvirus Humano 6/metabolismo , Humanos , Lactante , Leucocitos Mononucleares/química , Leucocitos Mononucleares/virología , Microscopía Confocal , Microscopía Fluorescente , Infecciones por Roseolovirus/virología , Sensibilidad y Especificidad , Carga Viral , Proteínas Virales/biosíntesisAsunto(s)
Interferón-alfa/uso terapéutico , Panencefalitis Esclerosante Subaguda/tratamiento farmacológico , Administración Oral , Adolescente , Anticuerpos Antivirales/sangre , Quimioterapia Combinada , Femenino , Humanos , Inyecciones Espinales , Inosina Pranobex/administración & dosificación , Inosina Pranobex/uso terapéutico , Interferón-alfa/administración & dosificación , Virus del Sarampión/inmunología , Panencefalitis Esclerosante Subaguda/inmunologíaRESUMEN
Oita virus 296/1972 was isolated from the blood of a wild horseshoe bat, Rhinolophus cornutus (Temminck) in 1972. We investigated the pathogenicity of this virus in mice in relation to its histological, immunohistochemical and ultrastructural characteristics and the entire sequence of nucleoprotein gene. This virus caused lethal encephalitis in mice through intracerebral route. This susceptibility of mice was until 3 weeks of age. Immunohistochemical analysis using the convalescent sera obtained from survived adult mice after intracerebral inoculation revealed that many neurons were positive in the cytoplasm, besides no cross reactivity with normal and rabies virus-infected mouse brain tissues to this anti-sera. Ultrastructural analysis disclosed many bullet-shaped and enveloped virions in neurons. These morphological characteristics of the virions are consistent of that of viruses in the family Rhabdoviridae. Budding from endoplasmic membrane suggests that this virus has a similarity with lyssaviruses. Molecular analysis of cDNA coding a tentative nucleoprotein sequence revealed homology with those of viruses in the family Rhabdoviridae. Distance matrix analysis of this gene sequence with those of other rhabdoviruses isolated from mammals disclosed the discrete position of this virus in the phylogenic tree of rhabdoviridae infecting mammals and we renamed this virus as Oita rhabdovirus.
Asunto(s)
Quirópteros/virología , Infecciones por Rhabdoviridae/patología , Rhabdoviridae/patogenicidad , Factores de Edad , Secuencia de Aminoácidos , Animales , Reacciones Cruzadas , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Datos de Secuencia Molecular , Neuronas/patología , Neuronas/virología , Nucleoproteínas/química , Nucleoproteínas/genética , Filogenia , Rhabdoviridae/clasificación , Rhabdoviridae/genética , Rhabdoviridae/aislamiento & purificación , Infecciones por Rhabdoviridae/virología , Alineación de Secuencia , Telencéfalo/ultraestructura , Telencéfalo/virología , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
To establish the toxicities and maximum tolerated dose (MTD) of nedaplatin with gemcitabine, and to observe their antitumour activity, we conducted a combination phase I study in advanced non-small-cell lung cancer (NSCLC). Patients received nedaplatin (60-100 mg m(-2) given intravenously over 90 min) on day 1, and gemcitabine (800-1000 mg m(-2) given intravenously over 30 min) on days 1, 8, every 3 weeks. In total, 20 patients with locally advanced or metastatic NSCLC who received no prior chemotherapy or one previous chemotherapy regimen were enrolled. The most frequent toxicities were neutropenia and thrombocytopenia; nonhaematological toxicities were generally mild. Three out of six patients experienced dose-limiting toxicities (neutropenia, thrombocytopenia and delayed anaemia) at dose level 4, 100 mg m(-2) nedaplatin with 1000 mg m(-2) gemcitabine, which was regarded as the MTD. There were three partial responses, for an overall response rate of 16.7%. The median survival time and 1-year survival rate were 9.1 months and 34.1%, respectively. This combination is well tolerated and active for advanced NSCLC. The recommended dose is 80 mg m(-2) nedaplatin with 1000 mg m(-2) gemcitabine. This combination chemotherapy warrants a phase II study and further evaluation in prospective randomised trials with cisplatin- or carboplatin-based combinations as first-line chemotherapy for advanced NSCLC.