RESUMO
How recycling receptors are segregated from down-regulated receptors in the endosome is unknown. In previous studies, we demonstrated that substitutions in the transferrin receptor (TR) transmembrane domain (TM) convert the protein from an efficiently recycling receptor to one that is rapidly down regulated. In this study, we demonstrate that the "signal" within the TM necessary and sufficient for down-regulation is Thr(11)Gln(17)Thr(19) (numbering in TM). Transplantation of these polar residues into the wild-type TR promotes receptor down-regulation that can be demonstrated by changes in protein half-life and in receptor recycling. Surprisingly, this modification dramatically increases the TR internalization rate as well ( approximately 79% increase). Sucrose gradient centrifugation and cross-linking studies reveal that propensity of the receptors to self-associate correlates with down-regulation. Interestingly, a number of cell surface proteins that contain TM polar residues are known to be efficiently down-regulated, whereas recycling receptors for low-density lipoprotein and transferrin conspicuously lack these residues. Our data, therefore, suggest a simple model in which specific residues within the TM sequences dramatically influence the fate of membrane proteins after endocytosis, providing an alternative signal for down-regulation of receptor complexes to the well-characterized cytoplasmic tail targeting signals.
Assuntos
Antígenos de Diferenciação de Linfócitos B/química , Endocitose/fisiologia , Antígenos de Histocompatibilidade Classe II/química , Receptores de Superfície Celular/metabolismo , Receptores da Transferrina/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação de Linfócitos B/efeitos dos fármacos , Antígenos de Diferenciação de Linfócitos B/fisiologia , Embrião de Galinha , Reagentes de Ligações Cruzadas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Fibroblastos , Meia-Vida , Antígenos de Histocompatibilidade Classe II/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/fisiologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/efeitos dos fármacos , Receptores da Transferrina/química , Receptores da Transferrina/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
OBJECTIVES: We evaluated a novel protocol of dual-isotope, gated single-photon emission computed tomographic (SPECT) imaging combined with low and high dose dobutamine as a single test for the characterization of various types of altered myocardial dysfunction. BACKGROUND: Myocardial perfusion tomography and echocardiography have been used separately for the assessment of myocardial viability. However, it is possible to assess perfusion, function and contractile reserve using gated SPECT imaging. METHODS: We studied 54 patients with ischemic cardiomyopathy using rest and 4 h redistribution thallium-201 imaging and dobutamine technetium-99m sestamibi SPECT imaging. The sestamibi images were acquired 1 h after infusion of the maximal tolerated dose of dobutamine and again during infusion of dobutamine at a low dose to estimate contractile reserve. Myocardial segments were defined as hibernating, stunned, remodeled or scarred. RESULTS: Severe regional dysfunction was present in 584 (54%) of 1,080 segments. Based on the combination of function and perfusion characteristics in these 584 segments, 24% (n = 140) were labeled as hibernating; 23% (n = 136) as stunned; 30% (n = 177) as remodeled; and 22% (n = 131) as scarred. Contractile reserve, represented by improvement in wall motion/thickening by low dose dobutamine, was observed in 83% of stunned, 59% of hibernating, 35% of remodeled and 13% of scarred myocardial segments (p<0.05). CONCLUSIONS: It is possible with this new imaging technique to characterize dysfunctional myocardium as stunned, hibernating, remodeled and nonviable. These subtypes often coexist in the same patient.
Assuntos
Isquemia Miocárdica/diagnóstico por imagem , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Idoso , Cardiotônicos/administração & dosagem , Dobutamina/administração & dosagem , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miocárdio Atordoado/diagnóstico por imagemRESUMO
The purpose of this Phase I study was to determine the feasibility of using an anti-erbB-2-encoding adenovirus (Ad21) to treat erbB-2-overexpressing ovarian cancer. Recurrent ovarian cancer patients were treated i.p. with Ad21 in dosages ranging from 1 x 10(9) to 1 x 10(11) pfu. Patients were monitored after treatment for evidence of clinical toxicity and efficacy. Peritoneal aspirates and serum samples were obtained to assess for evidence of gene transfer/expression, for generation of wild-type vector, and antiadenoviral humoral response. Fifteen patients were treated per study specifications. Treatment-specific grade 1/2 fever was experienced by 9 of 15 (60%) patients. Other transient grade 1/2 constitutional, pain, and gastrointestinal symptoms were also experienced. No dose-limiting vector-related toxicity was experienced. Of 13 patients evaluable for response, 5 (38%) had stable disease and 8 (61%) had evidence of progressive disease. One patient with nonmeasurable disease normalized her CA125 at the 8-week evaluation, and one patient with nonmeasurable disease remained without clinical evidence of disease for 6 months after treatment. PCR analysis of peritoneal aspirates demonstrated the presence of Ad21 in 84.6%, 84.6%, and 61.6% of evaluable specimens at days 2, 14, and 56 after treatment, respectively. No wild-type virus was detected. Reverse transcription-PCR analysis demonstrated expression of the anti-erbB-2 sFv-encoding gene in 10 of 14 evaluable patients at day 2. Five of six evaluable patients had an increase in antiadenovirus antibody titer. This study suggests that adenoviral-mediated gene therapy using an anti-erbB-2-directed intrabody is feasible in the context of human ovarian cancer.
Assuntos
Fragmentos de Imunoglobulinas/genética , Neoplasias Ovarianas/terapia , Receptor ErbB-2/imunologia , Adenovírus Humanos/genética , Idoso , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Genes erbB-2/imunologia , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Humanos , Fragmentos de Imunoglobulinas/imunologia , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Malignant gliomas remain incurable with current interventions. Encouraging investigational approaches include the use of genetically modified herpes simplex-1 (HSV-1) viruses as direct cytotoxic agents. Combining attenuated HSV-1 with standard therapy, human U-87 malignant glioma xenografts grown in the hind limb or intracranially in athymic nude mice were exposed to ionizing radiation, inoculated with genetically modified HSV R3616, or received both virus and radiation. The combination of virus with fractionated ionizing radiation suggests a synergistic action and results in reduced tumor volumes and longer survivals when compared with treatment with either modality alone.
Assuntos
Neoplasias Encefálicas/terapia , Vacinas Anticâncer/virologia , Glioma/terapia , Herpesvirus Humano 1 , Animais , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/virologia , Vacinas Anticâncer/uso terapêutico , Terapia Combinada , Feminino , Glioma/mortalidade , Glioma/radioterapia , Glioma/virologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Transplante de Neoplasias , Distribuição Aleatória , Neoplasias de Tecidos Moles/mortalidade , Neoplasias de Tecidos Moles/radioterapia , Neoplasias de Tecidos Moles/terapia , Neoplasias de Tecidos Moles/virologia , Taxa de Sobrevida , Células Tumorais Cultivadas , Raios XRESUMO
The principal objectives of this trial were twofold: (a) to examine the safety and relative efficacy of intradermal needle injection versus s.c. jet administration of a carcinoembryonic antigen (CEA)-encoding recombinant vaccinia virus (rV-CEA) over a limited dose range and (b) to evaluate CEA-specific immune responses or antitumor effects induced by rV-CEA vaccination. Patients were randomly assigned to one of two groups, depending upon the technique of vaccine administration. All 20 patients received two doses of 10(7) or 10(8) pfu of rV-CEA at a 4-week interval. Toxicity was limited to modest local inflammation at the inoculation site as well as low-grade fever and increased fatigue, each affecting fewer than 20% of the patients. No evidence of CEA-specific lymphoproliferation, interleukin 2 release, delayed-type hypersensitivity, or antibody response was observed. Thus, the efficacy comparison between the two administration techniques was based upon the induction of immune responses to the vaccinia virus vector. Both techniques induced vaccinia-specific lymphoproliferation, interleukin 2 release, and antibody responses of comparable magnitude and frequency as well as protected 80% of patients against pustule formation following vaccinia scarification. Thus, there is no compelling reason to recommend one administration technique over the other based upon toxicity or efficacy. We have selected s.c. jet injection for subsequent trials of rV-CEA based on the ability to accommodate larger injection volumes, enhanced standardization between clinicians, and avoidance of needles that could transmit the vaccine or blood-borne pathogens to health care workers. We recommend use of 10(8) pfu doses for subsequent trials of recombinant vaccinia virus vaccines based upon the favorable toxicity profile and more consistent local pustule formation indicative of an adequate inoculation of live virus. No objective clinical responses to the rV-CEA vaccine were observed among this population of patients with widely metastatic adenocarcinoma.
Assuntos
Adenocarcinoma/secundário , Adenocarcinoma/terapia , Vacinas Anticâncer/administração & dosagem , Antígeno Carcinoembrionário/genética , Vaccinia virus/genética , Adenocarcinoma/imunologia , Adulto , Idoso , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Antígeno Carcinoembrionário/administração & dosagem , Antígeno Carcinoembrionário/biossíntese , Antígeno Carcinoembrionário/imunologia , Feminino , Humanos , Injeções Intradérmicas , Injeções Subcutâneas , Interleucina-2/imunologia , Interleucina-2/metabolismo , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Vaccinia virus/imunologiaRESUMO
BACKGROUND: Longitudinal studies in infants and children suggest that low total energy expenditure (EE) (TEE) and parental body composition are important predisposing factors to obesity. OBJECTIVE: The aim of this study was to examine potential predictors of changes in total or percentage body fat over 2.7 y in premenarcheal girls. DESIGN: We studied 47 normal-weight prepubertal girls aged 4.8-8.9 y in 3 visits. The girls' age, total and percentage body fat at baseline, sleep EE (SEE) and activity-related EE (AEE) adjusted for fat-free mass (FFM) and total body fat, mothers' and fathers' total and percentage body fat and FFM at baseline, and time to follow-up visits were measured; 24-h EE and SEE were measured by whole-room indirect calorimetry. AEE was calculated as TEE minus (SEE + 0.1 TEE), with the assumption that the thermic effect of food was 10% of TEE. The girls' body composition was measured at each visit and that of the parents was measured at the time of the girls' enrollment by using dual-energy X-ray absorptiometry. RESULTS: From baseline to the first (mean: 1.6 y) and the second (mean: 2.7 y) follow-up visits, the girls' mean (+/-SD) change in total fat adjusted for FFM was 1.2 +/- 2.7 and 3.3 +/- 4.0 kg, respectively, and the mean change in percentage body fat was -2.0 +/- 5.0% and -0. 8 +/- 5.9%, respectively. Fathers' total and percentage body fat were the main predictors of changes in the girls' total and percentage body fat. For the first follow-up visit, SEE, girls' age at baseline, and AEE were significant predictors of percentage body fat. CONCLUSION: Fathers' total and percentage body fat were predictors of changes in body fat of premenarcheal girls during a 2. 7-y period.
Assuntos
Tecido Adiposo , Composição Corporal , Pai , Absorciometria de Fóton , Calorimetria Indireta , Criança , Pré-Escolar , Estudos de Coortes , Metabolismo Energético , Feminino , Humanos , Estudos Longitudinais , MenarcaRESUMO
Lifetime data classified according to categorical variables under the proportionality of the hazard functions of response variables for various treatment combinations is assumed. The proposed model is a combination of Cox's proportional hazards model and ANOVA model. The existence of a solution to the marginal likelihood function is examined for the case of 2 x 2 two-way classification. We provide an easily verifiable condition for the existence of a unique estimate.
Assuntos
Análise de Sobrevida , Humanos , Tábuas de Vida , Matemática , Probabilidade , Modelos de Riscos ProporcionaisRESUMO
In the course of clinical (or preclinical) trial studies, it is a common practice to conduct a relatively large number of tests to extract the maximum level of information from the study. It has been known that as the number of tests (or endpoints) increases, the probability of falsely rejecting at least one hypothesis also increases. Single-step methods such as the Bonferroni, Sidák, or James approximation procedure have been used to adjust the p-values for each hypothesis. To reduce the conservatism (i.e., underestimating type I error) possessed by the aforementioned methods, Holm proposed a so-called "free-step-down" procedure. This adjustment can be made even less conservative by incorporating the dependence structure of endpoints at each adjustment step of the procedure. That is done by sequentially applying James's approximation procedure for correlated endpoints at each step, referred to as the Free-James method. This article primarily compares the power of the Free-James method to the power of the Bonferroni and James single-step-down and the Holm free-step-down methods. Two definitions of power are considered: (a) the probability of correctly rejecting at least one hypothesis when it is true, and (b) the probability of correctly rejecting all hypotheses that are true. Monte Carlo simulations show that the Free-James method is as good as other methods under definition (a) and the most powerful under definition (b) for various sample sizes, numbers of endpoints, and correlations.
Assuntos
Ensaios Clínicos como Assunto/métodos , Estatística como Assunto/métodos , Humanos , Método de Monte Carlo , Análise MultivariadaRESUMO
A typical animal carcinogenicity experiment routinely analyzes approximately 10-30 tumor sites. Comparisons of tumor responses between dosed and control groups and dose-related trend tests are often evaluated for each individual tumor site/type separately. p-Value adjustment approaches have been proposed for controlling the overall Type I error rate or familywise error rate (FWE). However, these adjustments often result in reducing the power to detect a dose effect. This paper proposes using weighted adjustments by assuming that each tumor can be classified as either class A or class B based on prior considerations. The tumors in class A, which are considered as more critical endpoints, are given less adjustment. Two weighted methods of adjustments are presented, the weighted p adjustment and weighted alpha adjustment. A Monte Carlo simulation shows that both weighted adjustments control the FWE well. Furthermore, the power increases if a treatment-dependent tumor is analyzed as in class A tumors and the power decreases if it is analyzed as in class B tumors. A data set from a National Toxicology Program (NTP) 2-year animal carcinogenicity experiment with 13 tumor types/sites observed in male mice was analyzed using the proposed methods. The modified poly-3 test was used to test for increased carcinogenicity since it has been adopted by the NTP as a standard test for a dose-related trend. The unweighted adjustment analysis concluded that there was no statistically significant dose-related trend. Using the Food and Drug Administration classification scheme for the weighted adjustment analyses, two rare tumors (with background rates of 1% or less) were analyzed as class A tumors and 11 common tumors (with background rates higher than 1%) as class B. Both weighted analyses showed a significant dose-related trend for one rare tumor.
Assuntos
Testes de Carcinogenicidade/estatística & dados numéricos , Interpretação Estatística de Dados , Neoplasias Experimentais/induzido quimicamente , Animais , Carcinógenos/toxicidade , Masculino , Camundongos , Modelos Estatísticos , Método de Monte Carlo , Neoplasias Experimentais/classificaçãoRESUMO
Herpes zoster or shingles is a frequent occurrence in both elderly individuals and immunocompromised hosts. The pain associated with herpes zoster is the most debilitating complication of the disease. It can be described as acute pain and post-herpetic neuralgia or zoster associated pain (ZAP). The latter definition encompasses pain from the onset of disease through its resolution and provides a convenient analytic tool for evaluation of antiviral therapy. A heuristic examination of ZAP historical data suggests the existence of three phases of pain resolution: the acute, subacute and chronic phases. The subacute and chronic phases comprise the post-herpetic neuralgia (PHN) stage. Common analytic methods, such as a Kaplan-Meier survival function or a Cox's model, have been used to assess the pain. However, such approaches do not adequately allow for phase comparison. Notably, in the clinical trial setting the comparison of specific treatment effects on the latter stages of pain are of the greatest medical relevance since this is the most debilitating phase of the illness. In order to incorporate the phase-specific information in the modelling of time to cessation of ZAP, we assumed the hazard function was a stepwise constant. Utilizing the full likelihood function, we obtained the maximum likelihood estimate for the transition times (that is, change-points), and other parameters of medical importance. The standard error of the change-point estimates were obtained through a bootstrapping method. The asymptotic properties of the parameter estimates are also discussed. Hence, the rates of pain resolution across all phases can be examined in order to precisely define the existence of multiple phases. In addition, the covariates effect can be examined across phases and populations, thereby allowing us to translate potential efficacy of a standard therapy to different populations. These results can be utilized in the design of clinical trials or in targeting the outcome for a specific phase while controlling for the effect of other variables.
Assuntos
Ensaios Clínicos como Assunto , Interpretação Estatística de Dados , Herpes Zoster/complicações , Herpes Zoster/tratamento farmacológico , Funções Verossimilhança , Neuralgia/diagnóstico , Neuralgia/virologia , Medição da Dor/métodos , Modelos de Riscos Proporcionais , Doença Aguda , Idoso , Análise de Variância , Antivirais/uso terapêutico , Viés , Doença Crônica , Fatores de Confusão Epidemiológicos , Convalescença , Progressão da Doença , Modificador do Efeito Epidemiológico , Humanos , Medição da Dor/normas , Análise de Sobrevida , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: We have previously reported that intramuscular, intradermal or epidermal gene gun administration of a plasmid encoding carcinoembryonic antigen (CEA) under transcriptional regulatory control of the cytomegalovirus (CMV) early promoter/enhancer elicits CEA-specific humoral and cellular immune responses in mice with resultant immunoprotection against challenge with syngeneic, CEA-expressing colon adenocarcinoma cells. METHODS: In the present work, we examine the ability of this DNA vaccine construct (pCEA) to elicit CEA-specific immunity following intrasplenic administration. Groups of mice were immunized with pCEA by intrasplenic or intramuscular injection. Six weeks later, mice were evaluated for the presence of anti-CEA humoral responses and were challenged with syngeneic, CEA-expressing colon carcinoma cells. RESULTS: Intrasplenic administration of pCEA produced a frequency of CEA-specific antibody responses comparable to that elicited by intramuscular pCEA inoculation. Both intrasplenic and intramuscular administration of pCEA generated IgG2a antibody responses to CEA, consistent with the induction of T helper-1-biased immune responses. In addition, partial immunoprotection against tumor challenge was observed after a single plasmid DNA dose by either route of administration. Subsequent studies revealed that antibody responses to intrasplenic DNA vaccination are dose and schedule dependent. CONCLUSION: These findings support future investigations of DNA vaccination strategies that specifically promote the uptake of plasmid by splenocytes.
Assuntos
Antígeno Carcinoembrionário/imunologia , Neoplasias do Colo/prevenção & controle , Vacinas de DNA/farmacologia , Animais , Formação de Anticorpos , Antígeno Carcinoembrionário/genética , Neoplasias do Colo/imunologia , Citomegalovirus/genética , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Injeções , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Baço , Células Tumorais Cultivadas , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
The purpose of this phase I study was to determine the potential efficacy of adenoviral-mediated suicide gene therapy in women with recurrent ovarian cancer. Fourteen patients were treated intraperitoneally with herpes simplex virus-thymidine kinase (HSV-TK)-encoding adenovirus (AdHSV-TK) in dosages ranging between 1x10(9) and 1x10(11) pfu. Beginning 2 days later, ganciclovir (GCV) was administered intravenously at a dose of 5 mg/kg bid for 14 days. Transient vector-associated fever was experienced by 4 of 14 (29%) treated patients. Other possible vector-associated constitutional symptoms, abdominal pain, and gastrointestinal symptoms were experienced by 6 of 14 (43%) treated patients. No other dose-limiting vector-specific side effects were noted. Of the 13 patients evaluable for response, 5 (38%) had stable disease and 8 (62%) had evidence of progressive disease. Molecular analysis of evaluable ascites samples demonstrated the presence of transgene DNA and RNA in most patients 2 days following Ad HSV-TK administration. Ten of 11 evaluable patients had an increase in anti-adenovirus antibody titer. These results suggest that treatment with AdHSV-TK in combination with GCV is feasible in the context of human ovarian cancer and tolerated at the dosages studied.