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BACKGROUND: Prostate-specific membrane antigen (PSMA) is a well-characterized target that is overexpressed selectively on prostate cancer cells. PSMA antibody-drug conjugate (ADC) is a fully human IgG1 monoclonal antibody conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE), which is designed to specifically bind PSMA-positive cells, internalize, and then release its cytotoxic payload into the cells. PSMA ADC has demonstrated potent and selective antitumor activity in preclinical models of advanced prostate cancer. A Phase 1 study was conducted to assess the safety, pharmacokinetics, and preliminary antitumor effects of PSMA ADC in subjects with treatment-refractory prostate cancer. METHODS: In this first-in-man dose-escalation study, PSMA ADC was administered by intravenous infusion every three weeks to subjects with progressive metastatic castration-resistant prostate cancer (mCRPC) who were previously treated with docetaxel chemotherapy. The primary endpoint was to establish a maximum tolerated dose (MTD). The study also examined the pharmacokinetics of the study drug, total antibody, and free MMAE. Antitumor effects were assessed by measuring changes in serum prostate-specific antigen (PSA), circulating tumor cells (CTCs), and radiologic imaging. RESULTS: Fifty-two subjects were administered doses ranging from 0.4 to 2.8 mg/kg. Subjects had a median of two prior chemotherapy regimens and prior treatment with abiraterone and/or enzalutamide. Neutropenia and peripheral neuropathy were identified as important first-cycle and late dose-limiting toxicities, respectively. The dose of 2.5 mg/kg was determined to be the MTD. Pharmacokinetics were approximately dose-proportional with minimal drug accumulation. Reductions in PSA and CTCs in subjects treated with doses of ≥1.8 mg/kg were durable and often concurrent. CONCLUSIONS: In an extensively pretreated mCRPC population, PSMA ADC demonstrated acceptable toxicity. Antitumor activity was observed over dose ranges up to and including 2.5 mg/kg. The observed anti-tumor activity supported further evaluation of this novel agent for the treatment of advanced metastatic prostate cancer.
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Anticorpos Monoclonais , Neoplasias da Próstata , Idoso , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Relação Dose-Resposta a Droga , Monitoramento de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos , Humanos , Imunoglobulinas Intravenosas/administração & dosagem , Imunoglobulinas Intravenosas/farmacocinética , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Células Neoplásicas Circulantes/patologia , Oligopeptídeos/metabolismo , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Plate fixation is a recognized treatment for pelvic ring injuries involving disruption of the pubic symphysis. Although fixation failure is well known, it is unclear whether early or late fixation failure is clinically important. QUESTIONS/PURPOSES: We therefore determined (1) the incidence and mode of failure of anterior plate fixation for traumatic pubic symphysis disruption; (2) whether failure of fixation was associated with the types of pelvic ring injury or pelvic fixation used; (3) the complications, including the requirement for reoperation or hardware removal; and (4) whether radiographic followup of greater than 1 year alters subsequent management. METHODS: We retrospectively reviewed 148 of 178 (83%) patients with traumatic symphysis pubis diastasis treated by plate fixation between 1994 and 2008. Routine radiographic review, pelvic fracture classification, method of fixation, incidence of fixation failure, timing and mode of failure, and the complications were recorded after a minimum followup of 12 months (mean, 45 months; range, 1-14 years). RESULTS: Hardware breakage occurred in 63 patients (43%), of which 61 were asymptomatic. Breakage was not related to type of plate, fracture classification, or posterior pelvic fixation. Five patients (3%) required revision surgery for failure of fixation or symptomatic instability of the symphysis pubis, and seven patients (5%) had removal of hardware for other reasons, including late deep infection in three (2%). Routine radiographic screening as part of annual followup after 1 year did not alter management. CONCLUSIONS: Our observations suggest the high rate of late fixation failure after plate fixation of the symphysis pubis is not clinically important.
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Placas Ósseas , Fixação Interna de Fraturas/métodos , Complicações Pós-Operatórias/epidemiologia , Diástase da Sínfise Pubiana/cirurgia , Sínfise Pubiana/cirurgia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Fixação Interna de Fraturas/instrumentação , Consolidação da Fratura , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/etiologia , Sínfise Pubiana/diagnóstico por imagem , Sínfise Pubiana/lesões , Diástase da Sínfise Pubiana/epidemiologia , Radiografia , Estudos Retrospectivos , Reino Unido/epidemiologia , Adulto JovemRESUMO
BACKGROUND: PRO 140 is a humanized CCR5 monoclonal antibody that has demonstrated potent antiviral activity when it is administered intravenously to adults infected with CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1). This study is the first to evaluate subcutaneous administration. METHODS: A randomized, double-blind, placebo-controlled study was conducted among 44 subjects with HIV-1 RNA levels of >5000 copies/mL, CD4(+) cell counts of >300 cells/microL, no receipt of antiretroviral therapy for >or=12 weeks, and only R5 HIV-1 detectable. Subjects received placebo, 162 mg of PRO 140, or 324 mg of PRO 140 weekly for 3 weeks or 324 mg of PRO 140 every other week for 2 doses by means of subcutaneous infusion. Subjects were monitored for 58 days for safety, antiviral effects, and PRO 140 serum concentrations. RESULTS: Subcutaneous PRO 140 demonstrated potent and prolonged antiretroviral activity. Mean log(10) reductions in HIV-1 RNA level were 0.23, 0.99 (P=.009), 1.37 (P<.001), and 1.65 (P<.001) for the placebo, 162 mg weekly, 324 mg biweekly, and 324 mg weekly dose groups, respectively. Viral loads remained suppressed between successive doses. Treatment was generally well tolerated. CONCLUSIONS: This trial demonstrates proof of concept for a monoclonal antibody administered subcutaneously in HIV-1 infected individuals. Subcutaneous PRO 140 offers the potential for significant dose-dependent HIV-1 RNA suppression and infrequent patient self-administration. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00642707 .
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Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Anticorpos Anti-HIV/administração & dosagem , Anticorpos Anti-HIV/farmacologia , Inibidores da Fusão de HIV/administração & dosagem , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Adulto , Anticorpos Monoclonais Humanizados , Esquema de Medicação , Feminino , Humanos , Injeções Subcutâneas , Masculino , Pessoa de Meia-IdadeRESUMO
A two-step developability assessment workflow is described to screen variants of recombinant protein antigens under various formulation conditions to rapidly identify stable, aluminum-adjuvanted, multi-dose vaccine candidates. For proof-of-concept, a series of sequence variants of the recombinant non-replicating rotavirus (NRRV) P[8] protein antigen (produced in Komagataella phaffii) were compared in terms of primary structure, post-translational modifications, antibody binding, conformational stability, relative solubility and preservative compatibility. Based on these results, promising P[8] variants were down-selected and the impact of key formulation conditions on storage stability was examined (e.g., presence or absence of the aluminum-adjuvant Alhydrogel and the preservative thimerosal) as measured by differential scanning calorimetry (DSC) and antibody binding assays. Good correlations between rapidly-generated developability screening data and storage stability profiles (12 weeks at various temperatures) were observed for aluminum-adsorbed P[8] antigens. These findings were extended and confirmed using variants of a second NRRV antigen, P[4]. These case-study results with P[8] and P[4] NRRV variants are discussed in terms of using this vaccine formulation developability workflow to better inform and optimize formulation design with a wide variety of recombinant protein antigens, with the long-term goal of rapidly and cost-efficiently identifying low-cost vaccine formulations for use in low and middle income countries.
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Vacinas contra Rotavirus , Rotavirus , Antígenos , Proteínas Recombinantes , SaccharomycetalesRESUMO
The anti-CCR5 antibody PRO 140 has shown potent and prolonged antiretroviral activity in subjects infected with CCR5-tropic (R5) HIV-1. Prior studies have examined single intravenous doses ranging up to 5 mg/kg of body weight or up to three subcutaneous doses ranging up to 324 mg. Here we report the results of a randomized, double-blind, placebo-controlled trial that examined the antiviral activity, tolerability, and pharmacokinetics of single 5-mg/kg and 10-mg/kg intravenous infusions of PRO 140 in 31 treated subjects. Eligibility criteria included HIV-1 RNA levels of >5,000 copies/ml, CD4(+) cell counts of >300/µl, no antiretroviral therapy for ≥12 weeks, and detection of only R5 HIV-1 in the original Trofile assay. Following poststudy testing with an enhanced-sensitivity Trofile assay, one subject treated with 10 mg/kg was reclassified as having dual/mixed-tropic virus at screening, and the data for that subject were censored from efficacy analyses. The mean maximum reduction of the HIV-1 RNA level from the baseline level was 1.8 log(10) units for both the 5-mg/kg and 10-mg/kg doses (P < 0.0001 relative to placebo). Viral loads reached their nadir at day 12 posttreatment and remained significantly (P < 0.01) reduced through day 29 for both PRO 140 dose groups. Treatment was generally well tolerated, with no dose-limiting toxicity being observed. Peak serum concentrations and overall exposures increased proportionally with dose. In summary, single 5-mg/kg and 10-mg/kg doses of PRO 140 exhibited potent, long-lived antiviral activity and were generally well tolerated. The findings further delineate the safety and antiviral properties of this novel, long-acting antiretroviral agent.
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Anticorpos Monoclonais/uso terapêutico , Antagonistas dos Receptores CCR5 , Anticorpos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Adulto , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Feminino , Anticorpos Anti-HIV/administração & dosagem , Infecções por HIV/sangue , Humanos , Injeções Intraventriculares , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Receptores CCR5/metabolismo , Adulto JovemRESUMO
To meet the challenges of global health, vaccine design and development must be reconsidered to achieve cost of goods as low as 15¢ per dose. A new recombinant protein-based rotavirus vaccine candidate derived from non-replicative viral subunits fused to a P2 tetanus toxoid CD4(+) T cell epitope is currently under clinical development. We have sought to simplify the existing manufacturing process to meet these aims. To this end, we have taken a holistic process development approach to reduce process complexity and costs while producing a product with the required characteristics. We have changed expression system from Escherichia coli to Pichia pastoris, to produce a secreted product, thereby reducing the number of purification steps. However, the presence of proteases poses challenges to product quality. To understand the effect of fermentation parameters on product quality small-scale fermentations were carried out. Media pH and fermentation duration had the greatest impact on the proportion of full-length product. A novel acidic pH pulse strategy was used to minimize proteolysis, and this combined with an early harvest time significantly increased the proportion of full-length material (60-75%). An improved downstream process using a combination of CIEX and AIEX to further reduce proteases, resulted in maintaining product quality (95% yield).
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Técnicas de Cultura Celular por Lotes , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus/biossíntese , Saccharomycetales/genética , Fermentação/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Proteólise , Rotavirus/patogenicidade , Infecções por Rotavirus/virologia , Vacinas contra Rotavirus/química , Vacinas contra Rotavirus/genética , Saccharomycetales/químicaRESUMO
BACKGROUND: Recombinant human parathyroid hormone (1-84) (PTH) increases bone mass and strength and improves bone quality by stimulating new bone formation. OBJECTIVE: To determine the safety of PTH and its effect on the incidence of vertebral fractures in postmenopausal women with osteoporosis. DESIGN: 18-month, randomized, double-blind, placebo-controlled, parallel-group study. SETTING: 168 centers in 9 countries. PATIENTS: 2532 postmenopausal women with low bone mineral density at the hip or lumbar spine. INTERVENTIONS: Women received 100 mug of recombinant human PTH or placebo daily by subcutaneous injection. All received calcium, 700 mg/d, and vitamin D3, 400 U/d. MEASUREMENTS: New or worsened vertebral fractures (primary outcome) and changes in bone mineral density and safety (secondary outcomes). RESULTS: 67.2% of patients who received at least 1 dose of the study drug completed the study. Parathyroid hormone reduced the risk for new or worsened vertebral fractures, but in sensitivity analyses, the magnitude of the reduction was changed with assumptions about fracture incidence in patients who did not complete the study (relative risk assuming no fractures, 0.42 [95% CI, 0.24 to 0.72] [P = 0.001]; relative risk assuming fracture incidence observed in all patients who completed the trial, 0.60 [CI, 0.36 to 1.00] [P = 0.05]; relative risk assuming fracture incidence observed in the placebo group, 0.62 [CI, 0.37 to 1.04] [P = 0.07]). Compared with placebo, mean bone mineral density increased at the spine by 6.9% (CI, 6.4% to 7.4%) and at the hip by 2.1% (CI, 1.7% to 2.5%) but decreased at the forearm in the PTH-treated group. Parathyroid hormone treatment increased the percentage of participants with hypercalciuria, hypercalcemia, and nausea by 24% (CI, 20% to 27%), 23% (CI, 21% to 26%), and 14% (CI, 11% to 16%), respectively, compared with placebo. LIMITATIONS: Baseline serum PTH and vitamin D levels were not measured. Many patients discontinued the trial prematurely. CONCLUSIONS: Parathyroid hormone (1-84) reduced the overall risk for new or worsened vertebral fracture in postmenopausal women with osteoporosis. Hypercalciuria, hypercalcemia, and nausea were more common in women who took the drug. Although the magnitude of the reduction was sensitive to assumptions about fracture incidence in patients who did not complete the study, the findings suggest that PTH provides an alternative therapeutic option for fracture prevention.
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Densidade Óssea/efeitos dos fármacos , Osteoporose Pós-Menopausa/tratamento farmacológico , Hormônio Paratireóideo/uso terapêutico , Fraturas da Coluna Vertebral/prevenção & controle , Fosfatase Alcalina/sangue , Biomarcadores/sangue , Método Duplo-Cego , Feminino , Humanos , Hipercalcemia/induzido quimicamente , Hipercalciúria/induzido quimicamente , Incidência , Vértebras Lombares/lesões , Pessoa de Meia-Idade , Náusea/induzido quimicamente , Osteoporose Pós-Menopausa/fisiopatologia , Hormônio Paratireóideo/efeitos adversos , Estudos Prospectivos , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Risco , Sensibilidade e Especificidade , Fraturas da Coluna Vertebral/epidemiologiaRESUMO
The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130-140, 2018.
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Ensaios de Triagem em Larga Escala , Pichia/genética , Proteínas Recombinantes/genética , Fermentação , Proteínas Recombinantes/química , Saccharomyces cerevisiae/genéticaRESUMO
To overcome yearly efforts and costs for the production of seasonal influenza vaccines, new approaches for the induction of broadly protective and long-lasting immune responses have been developed in the past decade. To warrant safety and efficacy of the emerging crossreactive vaccine candidates, it is critical to understand the evolution of influenza viruses in response to these new immune pressures. Here we applied unique molecular identifiers in next generation sequencing to analyze the evolution of influenza quasispecies under in vivo antibody pressure targeting the hemagglutinin (HA) long alpha helix (LAH). Our vaccine targeting LAH of hemagglutinin elicited significant seroconversion and protection against homologous and heterologous influenza virus strains in mice. The vaccine not only significantly reduced lung viral titers, but also induced a well-known bottleneck effect by decreasing virus diversity. In contrast to the classical bottleneck effect, here we showed a significant increase in the frequency of viruses with amino acid sequences identical to that of vaccine targeting LAH domain. No escape mutant emerged after vaccination. These results not only support the potential of a universal influenza vaccine targeting the conserved LAH domains, but also clearly demonstrate that the well-established bottleneck effect on viral quasispecies evolution does not necessarily generate escape mutants.
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Reações Cruzadas/imunologia , Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Domínios Proteicos/imunologia , Quase-Espécies , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Reações Cruzadas/genética , Epitopos/imunologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Sequenciamento de Nucleotídeos em Larga Escala , Imunização , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Camundongos , Mutação , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Conformação Proteica em alfa-Hélice , Quase-Espécies/genética , Quase-Espécies/imunologia , Carga ViralRESUMO
Pulmonary function tests such as flow-volume loops and reconstructive radiological imaging may aid the detection of large airway obstruction prior to corrective surgery for severe scoliosis. Intraoperative use of halo-gravity traction may help to reduce the severity of the scoliosis, and thus the extrinsic compression or torsion of the airways.
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STUDY DESIGN: Radiologic imaging measurement study. PURPOSE: To assess the accuracy of detecting lateral mass and facet joint injuries of the subaxial cervical spine on plain radiographs using computed tomography (CT) scan images as a reference standard; and the integrity of morphological landmarks of the lateral mass and facet joints of the subaxial cervical spine. OVERVIEW OF LITERATURE: Injuries of lateral mass and facet joints potentially lead to an unstable subaxial cervical spine and concomitant neurological sequelae. However, no study has evaluated the accuracy of detecting specific facet joint injuries. METHODS: Eight spinal surgeons scored four sets of the same, randomly re-ordered, 30 cases with and without facet joint injuries of the subaxial cervical spine. Two surveys included conventional plain radiographs series (test) and another two surveys included CT scan images (reference). Facet joint injury characteristics were assessed for accuracy and reliability. Raw agreement, Fleiss kappa, Cohen's kappa and intraclass correlation coefficient statistics were used for reliability analysis. Majority rules were used for accuracy analysis. RESULTS: Of the 21 facet joint injuries discerned on CT scan images, 10 were detected in both plain radiograph surveys (sensitivity, 0.48; 95% confidence interval [CI], 0.26-0.70). There were no false positive facet joint injuries in either of the first two X-ray surveys (specificity, 1.0; 95% CI, 0.63-1.0). Five of the 11 cases with missed injuries had an injury below the lowest visible articulating level on radiographs. CT scan images resulted in superior inter- and intra-rater agreement values for assessing morphologic injury characteristics of facet joint injuries. CONCLUSIONS: Plain radiographs are not accurate, nor reliable for the assessment of facet joint injuries of the subaxial cervical spine. CT scans offer reliable diagnostic information required for the detection and treatment planning of facet joint injuries.
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Verapamil has been shown to attenuate the extent of myocardial injury in murine models of chronic Trypanosoma cruzi infection. Infected mice treated with verapamil have significantly lower myocardial expression of inducible nitric oxide synthase and cytokines and substantially less inflammatory infiltrate and myocyte necrosis at necropsy. In the present study, we examined the cardiac structural and functional correlates of verapamil treatment in CD1 mice infected with the Brazil strain of T. cruzi using serial transthoracic echocardiography. There were four groups: uninfected- untreated control, uninfected-verapamil-treated, infected-untreated control, and infected-verapamil-treated. Verapamil was given in drinking water (1 gm/l) continuously from the day of infection for a total of 120 days. Mice were evaluated at baseline, 40 and 150 days p.i. Mice in the untreated-infected group compared with the mice in the infected-verapamil-treated group showed thinning of the left ventricular wall (0.84 +/- 0.02-vs-0.92 +/- 0.04, P<0.05 mm), increase in the left ventricular end-diastolic diameter (3.27 +/- 0.15-vs-2.74 +/- 0.05 mm, P<0.05) and reduction in percent fractional shortening (37 +/- 2-vs-53 +/- 4%, P<0.05). No differences in these parameters were noted among mice in the uninfected-untreated and uninfected-verapamil-treated groups. Furthermore, right ventricular dilation was more severe in mice from the infected-untreated group as compared with those in the infected- verapamil-treated group (visual grade 1.9 +/- 0.4-vs-1.0 +/- 0.2, P<0.05). At necropsy, the extent of myocardial injury, as determined histologically, was significantly greater in the infected-untreated mice. These data provide cardiac structural and functional correlates for the previously observed cardioprotective effects of verapamil in chronic chagasic cardiomyopathy.
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Bloqueadores dos Canais de Cálcio/farmacologia , Cardiotônicos/farmacologia , Doença de Chagas/tratamento farmacológico , Miocárdio/patologia , Trypanosoma cruzi/metabolismo , Função Ventricular/efeitos dos fármacos , Verapamil/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/uso terapêutico , Cardiotônicos/uso terapêutico , Doença de Chagas/diagnóstico por imagem , Doença de Chagas/patologia , Doença de Chagas/fisiopatologia , Colágeno/análise , Colágeno/biossíntese , Modelos Animais de Doenças , Ecocardiografia , Histocitoquímica , Masculino , Camundongos , Trypanosoma cruzi/efeitos dos fármacos , Verapamil/uso terapêuticoRESUMO
BACKGROUND CONTEXT: In 2007, the Subaxial Cervical Spine Injury Classification (SLIC) system was introduced demonstrating moderate reliability in an internal validation study. PURPOSE: To assess the agreement on the SLIC system using clinical data from a spinal trauma population and whether the SLIC treatment algorithm outcome improved agreement on treatment decisions among surgeons. STUDY DESIGN: An external classification validation study. PATIENT SAMPLE: Twelve spinal surgeons (five consultants and seven fellows) assessed 51 randomly selected cases. OUTCOME MEASURES: Raw agreement, Fleiss kappa, and intraclass correlation coefficient statistics were used for reliability analysis. Majority rules and latent class modeling were used for accuracy analysis. METHODS: Fifty-one randomly selected cases with significant injuries of the cervical spine from a prospective consecutive series of trauma patients were assessed using the SLIC system. Neurologic details, plain radiographs, and computed tomography scans were available for all cases as well as magnetic resonance imaging in 21 cases (41%). No funds were received in support of this study. The authors have no conflict of interest in the subject of this article. RESULTS: The inter-rater agreement on the most severely affected level of injury was strong (κ=0.76). The agreement on the morphologic injury characteristics was poor (κ=0.29) and agreement on the integrity of the discoligamentous complex was average (κ=0.46). The inter-rater agreement on the treatment verdict after the total SLIC injury severity score was slightly lower than the surgeons' agreement on personal treatment preference (κ=0.55 vs. κ=0.63). Latent class analysis was not converging and did not present accurate estimations of the true classification categories. Based on these findings, no second survey for testing intrarater agreement was performed. CONCLUSIONS: We found poor agreement on the morphologic injury characteristics of the SLIC system, and its treatment algorithm showed no improved agreement on treatment decisions among surgeons. The authors discuss that the reproducibility of the SLIC system is likely to improve when unambiguous true morphologic injury characteristics are being implemented.
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Algoritmos , Vértebras Cervicais/lesões , Traumatismos da Coluna Vertebral/classificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Traumatismos da Medula Espinal/etiologia , Traumatismos da Coluna Vertebral/complicações , Adulto JovemRESUMO
BACKGROUND: The current goal of human immunodeficiency virus type 1 (HIV-1) therapy is to maximally suppress viral replication. Securing this goal requires new drugs and treatment classes. The chemokine receptor CCR5 provides an entry portal for HIV-1, and PRO 140 is a humanized monoclonal antibody that binds to CCR5 and potently inhibits CCR5-tropic (R5) HIV-1 in vitro. METHODS: A randomized, double-blind, placebo-controlled, dose-escalating study was conducted in 39 individuals with HIV-1 RNA levels or =5000 copies/mL, CD4(+) cell counts > or =250 cells/microL, no antiretroviral therapy for 3 months, and only R5 HIV-1 detectable. Cohorts were randomized 3:10 to receive placebo or doses of PRO 140 of 0.5, 2, or 5 mg/kg. Subjects were monitored for 58 days for safety, antiviral effects, and serum concentrations of PRO 140. RESULTS: PRO 140 was generally well tolerated and demonstrated potent, rapid, prolonged, and dose-dependent antiviral activity. Mean reductions in HIV-1 RNA level of 0.58 log(10), 1.20 log(10) (P= .0002) and 1.83 log(10) (P= .0001) were observed for the 0.5-, 2-, and 5-mg/kg dose groups, respectively. Reductions in mean viral load of > or =10-fold were observed within 4 days and persisted for 2-3 weeks after treatment. CONCLUSIONS: This trial established clear proof of concept for PRO 140 as a potent antiretroviral agent with extended activity after a single dose. TRIAL REGISTRATION: ISRCTN Register: ISRCTN45537485 .
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Fármacos Anti-HIV/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , Fármacos Anti-HIV/sangue , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais Humanizados , Área Sob a Curva , Relação Dose-Resposta a Droga , Método Duplo-Cego , Farmacorresistência Viral , Feminino , Anticorpos Anti-HIV/sangue , HIV-1/efeitos dos fármacos , Humanos , Contagem de Linfócitos , Linfócitos/imunologia , Masculino , RNA Viral/sangue , Receptores CCR5 , Fatores de TempoRESUMO
Sequence motifs (L domains) have been described in viral structural proteins. Mutations in these lead to a defect at a late stage in virus assembly and budding. For several viruses, recruitment of an endosomal sorting complexes required for transport 1 subunit (Tsg101), a component of the class E vacuolar protein sorting (EVPS) machinery, is a prerequisite for virion budding. To effect this, Tsg101 interacts with the PT/SAP L domain. We have identified candidate L-domain motifs, PSAP, PPPI, and YEIL, in the prototypic foamy virus (PFV) Gag protein, based on their homology to known viral L domains. Mutation of the PSAP and PPPI motifs individually reduced PFV egress, and their combined mutation had an additive effect. When PSAP was mutated, residual infectious PFV release was unaffected by dominant negative Vps4 (an ATPase involved in the final stages of budding), and sensitivity to dominant negative Tsg101 was dramatically reduced, suggesting that the PSAP motif functions as a conventional class E VPS-dependent L domain. Consistent with this notion, yeast two-hybrid analysis showed a PSAP motif-dependent interaction between PFV Gag and Tsg101. Surprisingly, PFV release which is dependent on the PPPI motif was Vps4-independent and was partially inhibited by dominant negative Tsg101, suggesting that PPPI functions by an unconventional mechanism to facilitate PFV egress. Mutation of the YEIL sequence completely abolished particle formation and also reduced the rate of Gag processing by the viral protease, suggesting that the integrity of YEIL is required at an assembly step prior to budding and YEIL is not acting as an L domain.
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Produtos do Gene gag/química , Estrutura Terciária de Proteína/fisiologia , Spumavirus/fisiologia , Replicação Viral , Sequência de Aminoácidos , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Transporte Proteico/fisiologia , Alinhamento de Sequência , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Risedronate, a bisphosphonate, was used to treat CD-1 mice infected with the Brazil strain of Trypanosoma cruzi. When given by subcutaneous injection 3 times/week, there was a significant reduction in mortality, however, the myocardial pathology and right ventricular dilation was unchanged in these mice compared to control animals. In C57BL/6 mice infected with the Tulahuen strain, there was no change in mortality in response to risedronate treatment. These data suggest that this class of compounds has activity against T. cruzi in vivo and illustrate the utility of imaging and pathologic studies as adjuncts in the evaluation of therapeutic compounds as treatments for experimental Chagas' disease. In addition, it underscores the need to use different strains of T. cruzi.
Assuntos
Cardiomiopatia Chagásica/tratamento farmacológico , Doença de Chagas/tratamento farmacológico , Ácido Etidrônico/análogos & derivados , Trypanosoma cruzi/efeitos dos fármacos , Animais , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/patologia , Doença de Chagas/parasitologia , Doença de Chagas/patologia , Modelos Animais de Doenças , Ácido Etidrônico/administração & dosagem , Ácido Etidrônico/farmacologia , Ácido Etidrônico/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Ácido Risedrônico , Análise de Sobrevida , Tripanossomicidas/administração & dosagem , Tripanossomicidas/farmacologia , Tripanossomicidas/uso terapêuticoRESUMO
Inositol 1,4,5-trisphosphate (IP(3)) receptors from cerebellum and recombinant type 1 IP(3) receptors expressed in Sf9 cells had indistinguishable affinities for IP(3) ( K (d)=6.40+/-0.48 nM) and adenophostin A ( K (d)=0.89+/-0.05 nM). In cytosol-like medium, each of the three mammalian IP(3) receptor subtypes when expressed in Sf9 cells bound adenophostin A with greater affinity than IP(3). It has been suggested that adenophostin A binds with high affinity only in the presence of ATP, but we found that adenophostin A similarly displaced [(3)H]IP(3) from type 1 IP(3) receptors whatever the ATP concentration. N-terminal fragments of the type 1 receptor were expressed with and without the S1 splice site; its removal had no effect on [(3)H]IP(3) binding to the 1-604 protein, but abolished binding to the 224-604 protein. The 1-604 fragment and full-length receptor bound adenophostin A with the same affinity, but the fragment had 3-fold greater affinity for IP(3), suggesting that C-terminal residues selectively inhibit IP(3) binding. The 224-604S1(+) fragment bound IP(3) and adenophostin A with increased affinity, but as with the 1-604 fragment it bound adenophostin A with only 2-fold greater affinity than IP(3). High-affinity binding of adenophostin A may be partially determined by its 2'-phosphate interacting more effectively than the 1-phosphate of IP(3) with residues within the IP(3)-binding core. This may account for the 2-fold greater affinity of adenophostin A relative to IP(3) for the minimal IP(3)-binding domain. In addition we suggest that C-terminal residues, which impede access of IP(3), may selectively interact with adenophostin A to allow it unhindered access to the IP(3)-binding domain.
Assuntos
Adenosina/análogos & derivados , Adenosina/química , Canais de Cálcio/química , Receptores Citoplasmáticos e Nucleares/química , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Cerebelo/metabolismo , Relação Dose-Resposta a Droga , Receptores de Inositol 1,4,5-Trifosfato , Insetos , Cinética , Ligantes , Modelos Químicos , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
The inositol 1,4,5-trisphosphate receptor (IP(3)R) is a tetrameric intracellular Ca(2+) channel, which mediates the release of Ca(2+) from the endoplasmic reticulum in response to many different extracellular stimuli. We present a 3D structure of the type 1 IP(3)R obtained by electron microscopy and single-particle analysis that reveals its domain organization. The IP(3)R has a flower-like appearance with fourfold symmetry and is made up of three distinct domains connected by slender links. By relating the organization of the structural domains to secondary-structure predictions and biochemical data we develop a model in which structural domains are mapped onto the amino acid sequence to deduce the location of the channel region and the cytoplasmic inositol 1,4,5-trisphosphate-binding and modulatory subdomains. The structure of the IP(3)R is compared with that of other tetrameric cation channels. The channel domain is similar in size and shape to its counterparts in the ryanodine receptor and the Shaker voltage-gated K(+) channel.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/ultraestrutura , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/ultraestrutura , Animais , Canais de Cálcio/isolamento & purificação , Membrana Celular/química , Cerebelo/química , Citoplasma/ultraestrutura , Processamento de Imagem Assistida por Computador , Receptores de Inositol 1,4,5-Trifosfato , Canais Iônicos/química , Canais Iônicos/isolamento & purificação , Microscopia Eletrônica , Conformação Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Receptores Citoplasmáticos e Nucleares/isolamento & purificaçãoRESUMO
Parathyroid hormone (PTH) sensitive adenylyl cyclase activity (ACA) in SaOS-2 cells varies as a function of cell passage. In early passage (EP) cells (< 6), ACA in response to PTH and forskolin (FOR) was relatively low and equivalent, whereas in late passage (LP) cells (> 22), PTH exceeded FOR dependent ACA. Potential biochemical mechanisms for this passage dependent change in ACA were considered. In EP, prolonged exposure to pertussis toxin (PT) markedly enhanced ACA activity in response to PTH, Isoproterenol and Gpp(NH)p, whereas ACA in response to FOR was decreased. In contrast, the identical treatment of LP with PT diminished all ACA in response to PTH, Gpp(NH)p, and FOR. The dose dependent effects of PT on subsequent [(32)P]ADP-ribosylation of its substrates, GTPase activity, as well as FOR-dependent ACA, were equivalent in EP and LP. The relative amounts of G(alpha)i and G(alpha)s proteins, as determined both by Western blot, PT and cholera toxin (CT) dependent [(32)P]ADP-ribosylation, were quantitatively similar in EP and LP. Western blot levels of G(alpha)s and G(alpha)i proteins were not influenced by prior exposure to PT. Both PT and CT dependent [(32)P]ADP-ribosylation were dose-dependently decreased following exposure to PT. However, the PT-dependent decline in CT-dependent [(32)P]ADP-ribosylation occurred with enhanced sensitivity in LP. The protein synthesis inhibitor cycloheximide partially reversed the PT associated decrease in FOR dependent ACA in EP. In contrast, cycloheximide completely reversed the PT associated decrease in FOR and as well as PTH dependent ACA in LP. G(alpha)s activity, revealed by cyc(-) reconstitution, was not altered either by cell passage or exposure to PT. The results suggest that the coupling between the components of the complex may be pivotally important in the differential responsiveness of early and late passage SaOS-2 cells to PTH.
Assuntos
Adenilil Ciclases/metabolismo , Neoplasias Ósseas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Osteoblastos/metabolismo , Osteossarcoma/metabolismo , Hormônio Paratireóideo/farmacologia , Toxina Adenilato Ciclase , Diferenciação Celular/efeitos dos fármacos , Toxina da Cólera/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , GTP Fosfo-Hidrolases/metabolismo , Guanilil Imidodifosfato/farmacologia , Humanos , Osteoblastos/efeitos dos fármacos , Toxina Pertussis , Inibidores da Síntese de Proteínas/farmacologia , Fatores de Tempo , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The distances between the inositol 1,4,5-trisphosphate (IP(3))-binding sites of tetrameric IP(3) receptors were probed using dimers of IP(3) linked by poly(ethylene glycol) (PEG) molecules of differing lengths (1-8 nm). Each of the dimers potently stimulated (45)Ca(2+) release from permeabilized cells expressing predominantly type 1 (SH-SY5Y cells) or type 2 (hepatocytes) IP(3) receptors. The shortest dimers, with PEG linkers of an effective length of 1.5 nm or less, were the most potent, being 3-4-fold more potent than IP(3). In radioligand binding experiments using cerebellar membranes, the shortest dimers bound with highest affinity, although the longest dimer (8 nm) also bound with almost 4-fold greater affinity than IP(3). The affinity of monomeric IP(3) with only the PEG attached was 2-fold weaker than IP(3), confirming that the increased affinity of the dimers requires the presence of both IP(3) motifs. The increased affinity of the long dimer probably results from the linked IP(3) molecules binding to sites on different receptors, because the dimer bound with greater affinity than IP(3) to cerebellar membranes, where receptors are densely packed, but with the same affinity as IP(3) to purified receptors. IP(3) and the IP(3) dimers, irrespective of their length, bound with similar affinity to a monomeric IP(3)-binding domain of the type 1 IP(3) receptor expressed in bacteria. Short dimers therefore bind with increased affinity only when the receptor is tetrameric. We conclude that the four IP(3)-binding sites of an IP(3) receptor may be separated by as little as 1.5 nm and are therefore likely to be placed centrally in this large (25 x 25 nm) structure, consistent with previous work indicating a close association between the central pore and the IP(3)-binding sites of the IP(3) receptor.