RESUMO
INTRODUCTION: The aim of this study was to examine the role of palliative percutaneous secondary lesions bone treatment by comparing the visual analogue scale (VAS) scores of cryoablation plus vertebroplasty versus radiofrequency (RF) plus vertebroplasty so as to determine their feasibility, reliability and efficacy in a short-term series. METHODS: Combined RF thermal ablation plus osteoplasty or cryoablation plus osteoplasty was performed in osteolytic secondary bone localisations in 30 consecutive patients who were suffering from pain refractory to conservative therapies. We evaluated pain with the VAS during the preoperative period and at four hours, 24 hours, one week, one month, three months and six months post procedure. RESULTS: There were no statistically significant differences in the VAS score between patients treated with cryoablation plus osteoplasty and those treated with RF ablation plus osteoplasty at one week (p-value is 0.34), one month (p-value is 1), three months (p-value is 0.68) and six months (p-value is 0.65) post procedure. Patients treated with cryoablation plus vertebroplasty have less pain at four hours (p-value less than 0.001) and 24 hours (p-value less than 0.001) than patients treated with RF ablation plus vertebroplasty. CONCLUSION: Both RF ablation and cryoablation are optimal techniques in the treatment of painful bone metastatic cancer. Cryoablation achieves less treatment-related pain during the early period of follow-up and better volume control by real-time depiction of ablation margins.
Assuntos
Neoplasias Ósseas/patologia , Administração Cutânea , Idoso de 80 Anos ou mais , Algoritmos , Criocirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Metástase Neoplásica , Osteólise/terapia , Medição da Dor , Ondas de Rádio , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Tempo , Resultado do Tratamento , VertebroplastiaRESUMO
PURPOSE: This study aimed at exploring the feasibility of high-field diffusion-weighted magnetic resonance imaging (DW-MRI) (3 T) and to correlate apparent diffusion coefficient (ADC) values with tumour cellularity in renal malignancies. MATERIALS AND METHODS: Thirty-seven patients (ten healthy volunteers and 27 patients with suspected renal malignancy) underwent T1-, T2-weighted and T1-weighted contrast-enhanced magnetic resonance imaging (MRI). Diffusion-weighted images were obtained with a single-shot spin-echo echo-planar imaging (SE-EPI) sequence with a b value of 500 s/mm(2). All lesions were surgically resected, and mean tumour cellularity was calculated. Comparison between tumour cellularity and mean ADC value was performed using simple linear regression analysis. RESULTS: The mean ADC value in normal renal parenchyma was 2.35+/-0.31 x 10(-3) mm(2)/s, whereas mean ADC value in renal malignancies was 1.72+/-0.21 x 10(-3) mm(2)/s. In our population, there were no statistically significant differences between ADC values of different histological types. The analysis of mean ADC values showed an inverse linear correlation with cellularity in renal malignancies (r=-0.73, p<0.01). CONCLUSIONS: DW-MRI is able to differentiate between normal and neoplastic renal parenchyma on the basis of tissue cellularity.
Assuntos
Carcinoma de Células Renais/diagnóstico , Imagem de Difusão por Ressonância Magnética/métodos , Neoplasias Renais/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/cirurgia , Estudos de Casos e Controles , Meios de Contraste , Diagnóstico Diferencial , Difusão , Imagem de Difusão por Ressonância Magnética/normas , Imagem Ecoplanar/métodos , Estudos de Viabilidade , Feminino , Humanos , Neoplasias Renais/patologia , Neoplasias Renais/cirurgia , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
During the development of a new drug product, it is a common strategy to develop a first-generation process with the aim to rapidly produce material for pre-clinical and early stage clinical trials. At a later stage of the development, a second-generation process is then introduced with the aim to supply late-stage clinical trials as well as market needs. This work was aimed at comparing the performance of two different CHO cell culture processes (perfusion and fed-batch) used for the production of a therapeutically active recombinant glycoprotein at industrial pilot-scale. The first-generation process was based on the Fibra-Cel packed-bed perfusion technology. It appeared during the development of the candidate drug that high therapeutic doses were required (>100mg per dose), and that future market demand would exceed 100 kg per year. This exceeded by far the production capacity of the first-generation process, and triggered a change of technology from a packed-bed perfusion process with limited scale-up capabilities to a fed-batch process with scale-up potential to typical bioreactor sizes of 15m(3) or more. The productivity per bioreactor unit volume (in product m(-3)year(-1)) of the fed-batch process was about 70% of the level reached with the first-generation perfusion process. However, since the packed-bed perfusion system was limited in scale (0.6m(3) maximum) compared to the volumes reached in suspension cultures (15m(3)), the fed-batch was selected as second-generation process. In fact, the overall process performance (in product year(-1)) was about 18-fold higher for the fed-batch compared to the perfusion mode. Data from perfusion and fed-batch harvests samples indicated that comparable product quality (relative abundance of monomers dimers and aggregates; N-glycan sialylation level; isoforms distribution) was obtained in both processes. To further confirm this observation, purification to homogeneity of the harvest material from both processes, followed by a complementary set of studies (e.g. full physico-chemical characterization, assessment of in vitro and in vivo bioactivity, comparative pharmacokinetics and pharmacodynamics studies in relevant species, etc.) would be required. Finally, this illustrates the need to fix the production process early during the development of a new drug product in order to minimize process conversion efforts and to shorten product development time lines.
Assuntos
Reatores Biológicos , Células CHO/fisiologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Modelos Biológicos , Proteínas Recombinantes/biossíntese , Animais , Biotecnologia/instrumentação , Biotecnologia/métodos , Proliferação de Células , Simulação por Computador , Desenho Assistido por Computador , Cricetinae , Cricetulus , Desenho de Equipamento , Análise de Falha de Equipamento , Perfusão , Controle de QualidadeRESUMO
BACKGROUND: A commercial preparation of recombinant human chorionic gonadotrophin (r-hCG, Ovitrelle) was launched in 2001. Generally, hCG is available in two formats: human chorionic gonadotrophin (u-hCG), derived from the urine of pregnant females, and r-hCG produced by DNA based biotechnology. METHOD: The analytical characteristics of a highly purified u-hCG (Gonasi HP) were assessed and compared, for the first time, with the recombinant derived r-hCG (Ovitrelle). Gonasi HP is produced by extracting and purifying hCG from urine to obtain a specific bioactivity of 5000 IU/mg protein. Ovitrelle is produced via a recombinant derived mammalian cell line and purified to obtain a specific activity of 26 000 IU/mg. RESULTS AND CONCLUSION: It has been documented that commercially available u-hCG preparations can contain a number of urine derived protein contaminants as well as hCG related metabolites. This is also the case for Gonasi HP, where hCG related molecules and other proteins were found to be present, including epidermal growth factor (EGF) and eosinophil derived neurotoxin (EDN). It was also demonstrated that this preparation contained high levels of oxidised hCG. r-hCG was confirmed to be essentially intact hCG, free from contaminant proteins and with very low levels of oxidised hCG.
Assuntos
Gonadotropina Coriônica/análise , Gonadotropina Coriônica/química , Gonadotropina Coriônica/urina , Cromatografia Líquida de Alta Pressão , Densitometria , Eletroforese em Gel de Poliacrilamida , Neurotoxina Derivada de Eosinófilo/análise , Fator de Crescimento Epidérmico/análise , Humanos , Immunoblotting , Peso Molecular , Proteínas Recombinantes/análiseRESUMO
The lymphocyte activation gene-3 (LAG-3), selectively transcribed in human activated T and NK cells, encodes a ligand for major histocompatibility complex (MHC) class II molecules. Like CD4, LAG-3 ectodomain is composed of four Ig-like domains (D1-D4). Nothing is known about the LAG-3 regions or residues required to form a stable MHC class II binding site. In contrast to CD4, soluble LAG-3 molecules stably interact with MHC class II molecules expressed on the cell surface. In addition, the first two N-terminal domains of soluble LAG-3 (D1 and D2) molecules, alone, are capable of binding MHC class II. From a LAG-3 model structure, we designed mutants and tested their ability to bind MHC class II molecules in an intercellular adhesion assay. We found residues on the membrane-distal, CDR1-2-containing top face of D1 that are essential for either binding or repulsing MHC class II proteins. Most of these residues are clustered at the base of a large extra-loop structure that is a hallmark of the LAG-3 D1 Ig-like domain. In addition, as for CD4, oligomerization of LAG-3 on the cell surface may be required to form a stable MHC binding site because mutation of three residues in the ABED beta-strands containing side of D1 results in a dominant negative effect (i.e., binding inhibition of coexpressed wild-type LAG-3).
Assuntos
Antígenos CD4/química , Antígenos HLA-D/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Antígenos CD/química , Sítios de Ligação , Células COS , Antígenos HLA-D/química , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Cinética , Ligantes , Ativação Linfocitária , Proteínas de Membrana/biossíntese , Modelos Moleculares , Modelos Estruturais , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Linfócitos T/imunologia , Transfecção , Células Tumorais Cultivadas , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
In recent years PCR-based gene cloning strategies have found wide application in molecular biology, due to the power, speed, and relative simplicity of the PCR methodology. We have set up a novel PCR cloning strategy to isolate homologous genes, which is based on the capture of the cDNA sequence(s) of interest with a biotinylated probe and streptavidin-coupled magnetic beads followed by PCR amplification of the selected molecules. This method does not require sequence information on 5' and 3' regions of the cDNA of interest and permits gene isolation to be sensitive, fast, simple, and specific even when the conventional screening procedures give rise to high backgrounds. By using this technique, which we propose to call gene-capture PCR (GC-PCR) cloning, we were able to isolate the full-length murine lymphocyte activation gene 3 (LAG-3) cDNA from total RNA of activated thymocytes. The GC-PCR technique represents a powerful tool for easy isolation not only of homologous genes from related species, but also of genes sharing conserved regions of suitable length, gene variants, and gene encoding proteins where only limited knowledge of the amino acid sequence exists.
Assuntos
Antígenos CD , Clonagem Molecular/métodos , Proteínas de Membrana/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Aminoácidos , Animais , Sequência de Bases , Separação Imunomagnética/métodos , Camundongos , Técnicas de Sonda Molecular , Dados de Sequência Molecular , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Interleukin-6 (IL-6) is known to be an important modulator of acute phase (AP) protein expression in hepatocytes both in vivo and in vitro. In the present study the inducing activity of IL-6 on the expression of the AP protein haptoglobin (HP) by the human hepatoma cell line HepG2, has been evaluated. HP mRNA inducibility was analysed by Northern and slot-blot hybridization, while HP protein was detected by means of an ELISA procedure. A dose-response relationship from 0.3 to 4.8 ng/ml of a human recombinant IL-6 preparation derived from a Chinese hamster ovary (CHO) cell line was observed after 48 h of treatment. Comparable results were obtained by analysing both HP mRNA expression and HP protein secretion. Detectable induction of HP protein secretion was observed with as little as 25 pg/ml of IL-6. The effect of IL-6 was potentiated by dexamethasone, while an inhibition on HP mRNA inducibility could be prevented by lowering the foetal calf serum (FCS) concentration to 1%. Preliminary data indicate that neither IL-1 beta nor TNF-alpha were able to induce significantly HP mRNA expression and protein secretion. The activity ratio between two IL-6 preparations (from CHO and E. coli cells) obtained with a conventional IL-6 bioassay (i.e., T1165 cell growth assay) was comparable to that obtained in the induction of HP expression. The nominal specific activity of the CHO-derived IL-6 was two to three times higher with both responses.