RESUMEN
UNLABELLED: What is already known about this subject African Americans are disproportionately affected by obesity and other metabolic risk factors in comparison to White Americans. Increasing prevalence of obesity has been associated with concomitant increases in childhood hypertension, dyslipidaemia and type 2 diabetes. Oxidative stress is associated with obesity in both adults and children. What this study adds Oxidative stress is positively associated with total body fat and truncal fat, but not with body mass index (BMI) or BMI z-score in healthy youth. Oxidative stress is associated with diastolic blood pressure in African American but not in White American healthy youth. BACKGROUND: Oxidative stress is elevated in obese youth, but less is known regarding racial disparities in the relationship of oxidative stress with metabolic risk factors. OBJECTIVES: To determine the relationship between oxidative stress and metabolic risk factors, adiposity, leptin, adiponectin and cardiovascular fitness (VO2PEAK ) in healthy African American and White American youth. METHODS: A marker of oxidative stress (F2 -isoprostane), validated markers of metabolic risk factors, fitness and body composition were measured in African American (n = 82) and White American (n = 76) youth (8-17 years old) recruited over a range of BMI percentiles (4th to 99th). RESULTS: F2 -isoprostane concentration was positively correlated with percentage body fat (r = 0.198) and percentage truncal fat (r = 0.173), but was not different between African American and White American males and females (P = 0.208). African American youth had significantly higher mean systolic and diastolic blood pressure (P = 0.023 and P = 0.011, respectively), body weight, BMI percentile and Tanner stage. After adjusting for gender, age, BMI and Tanner stage, African American youth varied from White Americans in the association of F2 -isoprostane with diastolic blood pressure (P = 0.047), but not with systolic blood pressure, triglycerides, VO2PEAK or homeostatic model assessment for insulin resistance (all P > 0.05). CONCLUSIONS: Oxidative stress, as measured by urinary F2 -isoprostane concentrations, was positively associated with percent body fat and truncal fat in youth. Oxidative stress levels were similar among African American and White American youth. Among markers of the metabolic syndrome, a significant difference between African American and White American youth was demonstrated only in the association of oxidative stress with diastolic blood pressure.
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Adiposidad/etnología , Negro o Afroamericano/estadística & datos numéricos , F2-Isoprostanos/sangre , Síndrome Metabólico/etnología , Síndrome Metabólico/prevención & control , Estrés Oxidativo , Triglicéridos/sangre , Población Blanca/estadística & datos numéricos , Adiponectina/sangre , Adolescente , Glucemia/metabolismo , Presión Sanguínea , Niño , Femenino , Humanos , Leptina/sangre , Masculino , Datos de Secuencia Molecular , Factores de Riesgo , Estados Unidos/epidemiologíaRESUMEN
BACKGROUND: This study was designed to determine the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of brivanib in patients with advanced/metastatic solid tumors. PATIENTS AND METHODS: Ninety patients enrolled in this two-part, phase I open-label study of oral brivanib alaninate. The primary objectives of this study were (in part A) dose-limiting toxicity, maximum tolerated dose (MTD) and the lowest biologically active dose level and (in part B) the optimal dose/dose range. The secondary objectives of this study were preliminary evidence of antitumor activity, PK and PD. RESULTS: Across part A (open-label dose escalation and MTD) and part B (open-label dose optimization), 68 patients received brivanib alaninate. Brivanib demonstrated a manageable toxicity profile at doses of 180-800 mg. Most toxic effects were mild. Systemic exposure of the active moiety brivanib increased linearly ≤1000 mg/day. The MTD was 800 mg/day. Forty-four patients were treated at the MTD: 20 with 800 mg continuously, 11 with 800 mg intermittently and 13 with 400 mg b.i.d. doses. Partial responses were confirmed in two patients receiving brivanib ≥600 mg. Dynamic contrast-enhanced magnetic resonance imaging demonstrated statistically significant decreases in parameters reflecting tumor vascularity and permeability after multiple doses in the 800-mg continuous q.d. and 400-mg b.i.d. dose cohorts. CONCLUSION: In patients with advanced/metastatic cancer, brivanib demonstrates promising antiangiogenic and antitumor activity and manageable toxicity at doses ≤800 mg orally q.d., the recommended phase II study dose.
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Alanina/análogos & derivados , Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Triazinas/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Alanina/farmacología , Alanina/uso terapéutico , Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Metástasis de la Neoplasia , Neoplasias/irrigación sanguínea , Neoplasias/mortalidad , Neovascularización Patológica , Triazinas/uso terapéuticoRESUMEN
OBJECTIVE: To review and summarize the efficacy, mechanisms of action, and cost of the options available when choosing antiplatelet agents for secondary stroke prevention. DATA SOURCES: This article is based on a review of the literature found with MEDLINE, CINAHL, and Cochrane Reviews (1980-June 2000) and abstracts from relevant international scientific meetings. We searched for the terms aspirin, ticlopidine, dipyridamole, antiplatelet, and clopidogrel. STUDY SELECTION: English-language articles, both reviews and original studies, were evaluated, and all information considered relevant was included in this review. In addition, guidelines from the American Heart Association are included. DATA SYNTHESIS: Aspirin is a relatively inexpensive and effective agent for secondary stroke prevention, and lower doses of aspirin appear as effective as higher doses. Ticlopidine has been used alone or in combination with aspirin, but serious adverse effects have limited its use. Clopidogrel has emerged as a safe and effective alternative to ticlopidine and lacks some of the serious adverse effects associated with ticlopicine, but is not superior to aspirin in secondary stroke prevention. Unlike previous studies, one recent trial showed that dipyridamole in combination with aspirin is superior to aspirin alone. CONCLUSIONS: Antiplatelet therapy is a key component of secondary prevention strategies in ischemic stroke. While aspirin has been the cornerstone in the management of stroke, other classes of antiplatelet drugs present new opportunities to optimize antiplatelet therapy.
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Isquemia Miocárdica/prevención & control , Inhibidores de Agregación Plaquetaria/uso terapéutico , Accidente Cerebrovascular/prevención & control , Ticlopidina/análogos & derivados , Aspirina/administración & dosificación , Aspirina/efectos adversos , Aspirina/uso terapéutico , Ensayos Clínicos como Asunto , Clopidogrel , Análisis Costo-Beneficio , Dipiridamol/administración & dosificación , Dipiridamol/efectos adversos , Dipiridamol/uso terapéutico , Humanos , Ticlopidina/efectos adversos , Ticlopidina/uso terapéuticoRESUMEN
The use of reverse transcription-PCR (RT-PCR) to analyze cells in the blood of cancer patients for the detection of mRNA expressed in tumor cells has implications for both the prognosis and the monitoring of cancer patients for the efficacy of established or experimental therapies. Carcinoembryonic antigen (CEA) is expressed on approximately 95% of colorectal, gastric, and pancreatic tumors, and on the majority of breast, non-small cell lung, and head and neck carcinomas. CEA shed in serum is useful as a marker in only approximately 50% of colorectal cancer patients and rarely is shed by some other carcinoma types. RT-PCR has been used previously to detect CEA mRNA in cells in the blood and lymph nodes of cancer patients. Under the assay conditions validated in the studies reported here, 34 of 51 (67%) patients with different stages of colorectal cancer had blood cells that were positive by RT-PCR for CEA mRNA, whereas none of 18 patients with colonic polyps were positive; 2 of 60 apparently healthy individuals (who were age and sex matched with the carcinoma patients and were part of a colon cancer screening program as controls) were marginally positive. The results of CEA PCR in the blood of the carcinoma patients and the other groups showed strong statistical correlation with the disease (P2 < 0.0001). Analyses were carried out to detect both serum CEA protein levels and CEA mRNA in blood cells of colorectal carcinoma patients by RT-PCR. For all stages of disease, 18 of 51 patients (35%) were positive for serum CEA, whereas 35 of 51 (69%) were positive by RT-PCR. More importantly, only 5 of 23 (20%) of stage B and C colorectal cancer patients were positive for serum CEA, whereas 16 of 23 (70%) were positive by RT-PCR. The use of two other serum markers (CA19.9 and CA72-4) for colorectal cancer in combination with serum CEA scored two additional patients as positive; both were positive by RT-PCR for CEA mRNA. Pilot long-term longitudinal studies conducted before and after surgery identified some patients with CEA mRNA in blood cells that were negative for all serum markers, who eventually developed clinical metastatic disease. The studies reported here are the first to correlate RT-PCR results for CEA mRNA in blood cells with one or more serum markers for patients with different stages of colorectal cancer, and are the first long-term longitudinal studies to use RT-PCR to detect CEA mRNA in blood cells of cancer patients. Larger cohorts will be required in future studies to define the impact, if any, of this technology on prognosis and/or disease monitoring.
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Adenocarcinoma/sangre , Biomarcadores de Tumor/sangre , Antígeno Carcinoembrionario/genética , Neoplasias Colorrectales/sangre , Células Neoplásicas Circulantes/inmunología , ARN Mensajero/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Carbohidratos Asociados a Tumores/sangre , Antígenos de Carbohidratos Asociados a Tumores/genética , Biomarcadores de Tumor/genética , Antígeno CA-19-9/sangre , Antígeno CA-19-9/genética , Antígeno Carcinoembrionario/biosíntesis , Antígeno Carcinoembrionario/sangre , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Femenino , Humanos , Inmunohistoquímica , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Células Neoplásicas Circulantes/metabolismo , ARN Mensajero/genética , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: The goal of this study was to specifically estimate the effectiveness of platelet releasate, a widely available treatment administered by a proprietary group of wound care centers (WCCs) for the treatment of diabetic neuropathic foot ulceration. RESEARCH DESIGN AND METHODS: Treatment effectiveness was estimated in a retrospective cohort study controlling for treatment selection bias using logistic regression-derived propensity scores. RESULTS: Platelet releasate was more effective than standard care. The relative risk for a wound to heal after treatment with platelet releasate compared with standard care at a WCC varied from 1.14 (95% CI 1.03-1.27) to 1.59 (1.49-1.70). The effect was greatest in those with the most severe wounds, i.e., large wounds that affect deeper anatomical structures. CONCLUSIONS: Within the limitations of the ability of propensity score analysis to control for selection bias, platelet releasate is more effective than standard therapy. This effect is more pronounced in more severe wounds. Unfortunately, severe wounds have not been evaluated in randomized clinical trials of new interventions. We encourage the inclusion of these patients in future trials.
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Plaquetas/fisiología , Pie Diabético/tratamiento farmacológico , Neuropatías Diabéticas/tratamiento farmacológico , Sustancias de Crecimiento/uso terapéutico , Factor de Crecimiento Derivado de Plaquetas/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Anciano , Plaquetas/metabolismo , Estudios de Cohortes , Bases de Datos Factuales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
BACKGROUND: Diabetic foot ulcers are a common problem and result in more than 85,000 lower extremity amputations each year in the United States. Studies have suggested that between 25% and 50% of costs related to inpatient diabetes care may be directly attributable to the diabetic foot. Novel treatments for these wounds, while expensive, have been reported to improve healing rates, although no formal cost effectiveness analyses have been performed in order to address the cost effectiveness of a given therapy. OBJECTIVE: To estimate the cost effectiveness of common treatment strategies for diabetic neuropathic foot ulcers. METHODS: Four main options are available for the treatment of diabetic foot ulcers: (1) standard care (SC), (2) standard treatment in a specialized wound care center (WCC), (3) treatment with becaplermin, (4) or treatment with platelet releasate (PR). We utilized effectiveness data from published clinical trials, meta-analyses, and a database that includes data on 26,599 patients with these wounds. Effectiveness was assessed as a percentage of ulcers healed at 20 and 32 weeks. RESULTS: Baseline effectiveness (with 95% confidence intervals) for SC, becaplermin, PR, and WCC care were 30.9% (26.6, 35.1), 43.0% (37.3, 48.7), 36.8% (35.4, 38.2), and 35.6% (34.8, 36.4), respectively. Cost:effectiveness ratios for PR versus SC and becaplermin versus SC were 414.40 and 36.59, respectively. Therefore the incremental cost of increasing the odds of healing by 1% over standard therapy was $414.40 for PR and $36.59 for becaplermin. CONCLUSIONS: PR, becaplermin, and WCC care all provided improved healing rates over standard care, and becaplermin was less expensive and more effective than PR after 20 weeks of care.
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Pie Diabético/economía , Pie Diabético/terapia , Instituciones de Atención Ambulatoria , Anticoagulantes/uso terapéutico , Becaplermina , Análisis Costo-Beneficio , Humanos , Factor de Crecimiento Derivado de Plaquetas/uso terapéutico , Proteínas Proto-Oncogénicas c-sisRESUMEN
Activation of T cells usually requires two signals. Signal 1 is mediated via a peptide-MHC on the APC; signal 2 is mediated via a costimulatory molecule on the APC surface. We demonstrate here that naive CD4(+) T cells actually acquire the costimulatory molecule CD80 (B7-1) from syngeneic APCs after activation. This phenomenon was demonstrated showing acquisition of CD80 by T cells from CD80/CD86 (B7-2) knockout mice, and by treating T cells with cyclohexamide to further rule out endogenous expression of CD80 by T cells. Moreover, no CD80 mRNA could be detected in T cells that had acquired CD80. The amount of acquisition of CD80 by T cells was shown to be directly related to both the strength of signal 1 and the amount of CD80 on the APC. Specificity of this acquisition was also shown by the lack of acquisition by T cells from CD28 knockout mice (implicating CD28 in this process), the lack of acquisition of CD40 (another molecule on the APC surface) by T cells, and confocal microscopy studies. We demonstrate for the first time that 1) naive T cells, following acquisition of CD80 from APCs, were themselves shown to be capable of acting as APCs; and 2) memory T cells that have acquired CD80 from APCs undergo apoptosis in the presence of increased levels of signal 1. Thus we demonstrate both immunostimulatory and immunoregulatory functions as a result of CD80 acquisition by different T cell populations.
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Antígeno B7-1/biosíntesis , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Presentación de Antígeno/genética , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Apoptosis/genética , Apoptosis/inmunología , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Antígenos CD28/genética , Antígenos CD28/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células COS , Línea Celular , Células Cultivadas , Cicloheximida/farmacología , Relación Dosis-Respuesta Inmunológica , Memoria Inmunológica/genética , Interfase/genética , Interfase/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Unión Proteica/genética , Unión Proteica/inmunología , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/análisis , Transducción de Señal/genética , Transducción de Señal/inmunología , Especificidad de la Especie , Subgrupos de Linfocitos T/efectos de los fármacos , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
OBJECTIVE: To estimate the effect of various risk factors on the probability that neuropathic diabetic foot ulcers will heal within 20 weeks of care. DESIGN AND SETTING: A pooled or meta-analysis of individual patient data from the standard care arms of 5 randomized clinical trials was conducted. We analyzed 586 subjects with diabetes mellitus who had a neuropathic diabetic foot ulcer. All patients received good wound care, debridement, and "off-loading" of the wound. MAIN OUTCOME MEASURE: Multivariable logistic regression was used to calculate the magnitude of the association of each risk factor with patients having healed wounds. RESULTS: Logistic regression odds ratios (ORs; 95% confidence intervals [95% CIs]) revealed that those patients with a diabetic neuropathic foot ulcer that healed within 20 weeks using standard care were more likely to have a smaller wound (OR = 0.67; 95% CI, 0.55-0.81), a wound that existed for a shorter period (OR = 0.73; 95% CI, 0.61-0.87), and be nonwhite (OR = 0.64; 95% CI, 0.43-0.96) compared with patients whose wounds did not heal within 20 weeks. The patient's age (OR = 0.99; 95% CI, 0.89-1.01), serum level of glycosylated hemoglobin at the start of the study (OR = 1.03; 95% CI, 0.97-1.10), and sex (OR = 1. 02; 95% CI, 0.69-1.50) were unassociated with the probability of wound healing. Substantial heterogeneity was not found among the studies. CONCLUSIONS: A standard care regimen for diabetic neuropathic foot ulcers is most likely to be effective for patients who have wounds that are small and of brief duration. This information should help dermatologists decide initially whether to use standard care, to try a new treatment, or to refer the patient to a specialty center.
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Pie Diabético/patología , Pie Diabético/terapia , Cicatrización de Heridas , Adulto , Anciano , Anciano de 80 o más Años , Ensayos Clínicos como Asunto , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Factores de Riesgo , Factores de TiempoRESUMEN
OBJECTIVES: To determine the effects of ipsilateral hip and knee position on intraneural sciatic nerve pressures. DESIGN: Intraneural sciatic nerve pressures measured in intact, fresh cadaveric specimens relative to ipsilateral hip and knee positions. LOCATION: Medical school anatomy laboratory. SUBJECTS: Randomly acquired adult cadavers. INTERVENTION: Pressure transducer placed within the sciatic nerve distal to the femoral attachment of the gluteus maximus. Intraneural pressures measured with the hip placed in 0, 45, and 90 degrees of flexion while the knee was positioned in 90, 45, and 0 degrees of flexion. MAIN OUTCOME MEASUREMENTS: Tissue fluid pressures within the sciatic nerve relative to the position of the ipsilateral hip and knee. Tissue fluid pressure within the sciatic nerve exceeded previously defined critical thresholds for alteration of neural microcirculation and function. Although increased intraneural pressures were realized as the hip was positioned in greater flexion and the knee was extended, clinically relevant pressures were realized only when the hip was flexed to 90 degrees and the knee was fully extended. Pressures with the limbs in these positions were fifty-five millimeters of mercury (range 38 to 74 millimeters of mercury). RESULTS: With the hip held flexed to 90 degrees, statistically significantly increased intraneural pressures were measured as the knee was extended from 90 to 45 degrees of flexion (p = 0.048) and again from 45 to 0 degrees of flexion (p < or = 0.01). With the knee positioned in 45 degrees of flexion, statistically significantly increased intraneural pressures were measured as the hip was flexed from 45 to 90 degrees (p < or = 0.0062). When the knee was held fully extended, statistically significantly increased intraneural pressures were measured as the hip was flexed from 0 to 45 degrees of flexion (p = 0.0006) and again when the hip was brought from 45 to 90 degrees of flexion (p < or = 0.01). CONCLUSIONS: Intraneural sciatic nerve pressures are influenced by the position of the ipsilateral hip and knee. The magnitude of the pressure elevation appears to be related to the excursion of the nerve as the linear distance between the greater sciatic notch and the distal aspect of the leg increases. Intraneural tissue fluid pressures measured within a localized section of the sciatic nerve appeared to exceed published critical thresholds for alterations of blood flow and neural function only when the hip was flexed to 90 degrees and the knee was fully extended.
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Articulación de la Cadera/fisiología , Articulación de la Rodilla/fisiología , Nervio Ciático/fisiología , Adulto , Cadáver , Humanos , PresiónRESUMEN
Few epidemiologic studies have examined the effect of clinical risk factors on the probability that a patient with a chronic wound will heal or develop another wound. Curative Health Services maintains one of the few databases that contain detailed patient record information on patients with chronic wounds. The purpose of this study was to evaluate the reliability and validity of using this database to study individuals with diabetic neuropathic foot ulcers. 154 patient medical records were randomly selected from the database and abstracted using a standardized questionnaire and protocol. We assessed three key variables: diagnosis of diabetic neuropathic foot ulcer, whether the patient healed, and if the patient received an autologous product called platelet releasate. These variables in the database very accurately agreed with the information in the patient medical records, with positive predictive values of 98% (95% confidence interval [0.89, 0.99]), 93% (95% confidence interval [0. 68, 0.99]), and 100%, respectively. We have shown that, with respect to these three variables, the database is very accurate when compared to the medical record. It therefore represents a valuable tool with which to study patients with diabetic insensate foot ulcers.
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Pie Diabético/epidemiología , Bases de Datos Factuales , Métodos Epidemiológicos , Humanos , Sistemas de Registros Médicos Computarizados , Reproducibilidad de los ResultadosRESUMEN
Venous leg ulcers, which may take months to heal, account for 40-70% of all lower extremity chronic wounds. New treatment options for venous leg ulcers have recently been proposed, and therefore deciding which patients are candidates for these novel-and often expensive-treatments is an important task. Moreover, researchers conducting clinical trials often wish to enroll patients who are unlikely to heal in order to minimize sample sizes needed and research costs. Our purpose was to assess the use of percentage change in venous leg ulcer area over the first few weeks of treatment as a prognostic indicator of healing or non-healing at 24 weeks. We conducted a cohort study based on an existing data set from a multicentre randomized clinical trial that enrolled 104 patients. Wounds were measured using digital planimetry for 4 consecutive weeks following the inception of good wound care. Utilizing the Wilcoxon rank sum (Mann-Whitney) test, we found that percentage change in area over time distinguished between those who healed and those who failed to heal after 24 weeks of good wound care (P < 0.05). The rate of healing, or area healed per week, did not differentiate between those who healed at 24 weeks and those who did not, as all patients had similar rates of healing over the first 4 weeks of treatment. Percentage change in area from baseline to week 4 provided the best combination of positive and negative predictive values (68.2%, 74.7%) and the largest area under the receiver operating characteristic curve (0.75). Thus, percentage change in area over the first 4 weeks of treatment represents a practical and predictive measure of complete wound healing by 24 weeks.
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Úlcera Varicosa/patología , Cicatrización de Heridas/fisiología , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Curva ROC , Estadísticas no ParamétricasRESUMEN
B7-1 is a co-stimulatory molecule that provides a second signal for T-cell activation. Several studies have demonstrated that vaccination with a vector containing genes encoding B7-1 and an antigen appears to be efficacious at promoting immune responsiveness to the antigen. To evaluate the safety of such a protocol and determine the effect of the B7-1 vector on immune responsiveness, female C57BL/6 mice were administered Wyeth wild-type vaccinia virus (V-WT) or V-WT containing the gene for B7-1 (rV-B7-1) as a single s. c. injection or 3 monthly s.c. injections. Immunologic parameters were evaluated in half of the mice and general toxicity in the other half. Immunologic end points included determination of splenic lymphocyte phenotypes, mitogen-induced T- and B-cell proliferation, T-cell proliferation in response to alloantigens, cell-mediated cytotoxicity (CMC), natural killer cell activity and serum anti-nuclear antibody (ANA) titers. No significant signs of general toxicity were noted. The primary immunologic effect was an increase in the ability of spleen cells to lyse allogeneic targets and to proliferate in response to allogeneic stimulation. Numbers of splenic CD8(+) cells were also increased. These effects were more pronounced after 3 vaccinations than after a single vaccination. Minimal differences in ANA were observed between mice immunized with V-WT and rV-B7-1. In addition, no serum antibodies against B7-1 were detected in any mice. The data suggest that vaccination with rV-B7-1 augments CMC with minimal toxicity.
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Antígeno B7-1/inmunología , Citotoxicidad Inmunológica , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/toxicidad , Alanina Transaminasa/sangre , Fosfatasa Alcalina/sangre , Animales , Anticuerpos Antinucleares/sangre , Antígeno B7-1/genética , Recuento de Células Sanguíneas , Nitrógeno de la Urea Sanguínea , Femenino , Inmunidad Celular , Ratones , Ratones Endogámicos C57BL , Virus VacciniaRESUMEN
Mammalian cells express several isoforms of beta-thymosin, a major actin monomer sequestering factor, including thymosins beta4, beta10, and beta15. Differences in actin-binding properties of different beta-thymosin family members have not been investigated. We find that thymosin beta15 binds actin with a 2.4-fold higher affinity than does thymosin beta4. Mutational analysis was performed to determine the amino acid differences in thymosin beta15 that specify its increased actin-affinity. Previous work with thymosin beta4 identified an alpha-helical domain, as well as a conserved central motif, as crucial for actin binding. Mutational analysis confirms that these domains are also vital for actin binding in thymosin beta15, but that differences in these domains are not responsible for the variation in actin-binding properties between thymosins beta4 and beta15. Truncation of the unique C-terminal residues in thymosin beta15 inhibits actin binding, suggesting that this domain also has an important role in mediating actin-binding affinity. Replacement of the 10 C-terminal amino acids of thymosin beta15 with those of thymosin beta4 did, however, reduce the actin-binding affinity of the hybrid relative to thymosin beta15. Similarly, replacement of the thymosin beta4 C-terminal amino acids with those of thymosin beta15 led to increased actin binding. We conclude that functional differences between closely related beta-thymosin family members are, in part, specified by the C-terminal variability between these isoforms. Such differences may have consequences for situations where beta-thymosins are differentially expressed as in embryonic development and in cancer.
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Actinas/metabolismo , Variación Genética , Timosina/genética , Timosina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Movimiento Celular/fisiología , Masculino , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Gle1p is an essential, nuclear pore complex (NPC)-associated RNA export factor. In a screen for high copy suppressors of a GLE1 mutant strain, we identified the FG-nucleoporin Rip1p and the DEAD-box protein Rat8p/Dbp5p, both of which have roles in RNA export; we also found Ymr255p/Gfd1p, a novel inessential protein. All three high copy suppressors interact with the C-terminal domain of Gle1p; immunoelectron microscopy localizations indicate that Gle1p, Rip1p and Rat8p/Dbp5p are present on the NPC cytoplasmic fibrils; Rip1p was also found within the nucleoplasm and on the nuclear baskets. In vivo localizations support the hypothesis that Rip1p contributes to the association of Gle1p with the pore and that Gle1p, in turn, provides a binding site for Rat8p/Dbp5p at the NPC. These data are consistent with the view that Gle1p, Rip1p, Rat8p/Dbp5p and Ymr255p/Gfd1p associate on the cytoplasmic side of the NPC to act in a terminal step of RNA export. We also describe a human functional homologue of Rip1p, called hCG1, which rescues Rip1p function in yeast, consistent with the evolutionary conservation of this NPC-associated protein.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Fúngicas/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , ARN Helicasas , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citoplasma/metabolismo , ARN Helicasas DEAD-box , Cartilla de ADN/genética , Humanos , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Mutación , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Supresión Genética , TemperaturaRESUMEN
Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP.Rad was also reconverted to GTP.Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Nucleósido-Difosfato Quinasa/metabolismo , Factores de Transcripción/metabolismo , Proteínas ras/metabolismo , Animales , ADN/biosíntesis , Diabetes Mellitus/metabolismo , Activación Enzimática , Genes Supresores de Tumor , Glucosa/metabolismo , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Modelos Biológicos , Proteínas de Unión al GTP Monoméricas/genética , Nucleósido Difosfato Quinasas NM23 , Metástasis de la Neoplasia/genética , Nucleósido-Difosfato Quinasa/genética , Unión Proteica , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/genéticaRESUMEN
A new era involving the evaluation of recombinant vaccines for colon cancer has begun with the concurrent emergence of insights and technologies in the fields of molecular biology and immunology. These advances include (I) the identification and cloning of an array of genes associated with the neoplastic process, such as oncogenes, suppressor genes, genes encoding oncofetal antigens, and tissue lineage determinants; (2) the development of a variety of viral and bacterial vectors to deliver and present gene products; (3) the identification of numerous T-cell costimulatory molecules and the knowledge of their mode of action; (4) the cloning and analysis of the modes of action of an array of cytokines and other immunomodulatory molecules; and (5) a more sophisticated knowledge of the mode(s) of antigen presentation and T-cell activation.
Asunto(s)
Vacunas contra el Cáncer , Neoplasias Colorrectales/prevención & control , Adyuvantes Inmunológicos , Animales , Presentación de Antígeno , Antígenos de Neoplasias , Antígeno Carcinoembrionario , Ensayos Clínicos como Asunto , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Citocinas , Evaluación Preclínica de Medicamentos , Epítopos , Regulación Neoplásica de la Expresión Génica , Genes ras , Vectores Genéticos , Humanos , Activación de Linfocitos , Mutación Puntual , Linfocitos TRESUMEN
Many human cancers have been shown to contain activated forms of the Ras proto-oncogene. Mutations comprising amino acid changes at codons 12, 13 and 61 therefore represent unique targets for cancer immunotherapy. Recombinant Vaccinia viruses encoding point mutated Ras oncogenes have raised issues concerning the safety and transforming ability of these recombinant vaccines. Vaccinia virus, a representative of the orthopox virus genus, is a large DNA virus that is cytopathogenic and that replicates in the cytoplasm of the infected cell. However, it remains unclear whether orthopox viruses are capable of genetic interactions with infected cells. Our studies show that DNA isolated from cells infected with a recombinant Vaccinia virus expressing mutated Ras constituted a poor reagent for transfection into NIH3T3 cells for transformation analysis. Stable integration of a recombinant Vaccinia virus expressing mutant Ras DNA was not detected in recipient cells. This study also demonstrates that the crossover plasmids used to generate the recombinant virus where the activated Ras gene is under the control of a Vaccinia virus early promoter had low but detectable transforming efficiency in the NIH3T3 transformation assay. Analysis of the transfected cells indicated that Ras transcription was initiated upstream of the Vaccinia virus promoter. The introduction of wobble mutations as well as the truncation of the Ras protein removed the transforming capabilities of the crossover vector. This study demonstrates the potential problems and solutions in the use of point mutated oncogenes in live vectors for cancer vaccine development.