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1.
N Engl J Med ; 381(10): 923-932, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31483963

RESUMO

BACKGROUND: Pulmonary alveolar proteinosis is a disease characterized by abnormal accumulation of surfactant in the alveoli. Most cases are autoimmune and are associated with an autoantibody against granulocyte-macrophage colony-stimulating factor (GM-CSF) that prevents clearing of pulmonary surfactant by alveolar macrophages. An open-label, phase 2 study showed some therapeutic efficacy of inhaled recombinant human GM-CSF in patients with severe pulmonary alveolar proteinosis; however, the efficacy in patients with mild-to-moderate disease remains unclear. METHODS: We conducted a double-blind, placebo-controlled trial of daily inhaled recombinant human GM-CSF (sargramostim), at a dose of 125 µg twice daily for 7 days, every other week for 24 weeks, or placebo in 64 patients with autoimmune pulmonary alveolar proteinosis who had a partial pressure of arterial oxygen (Pao2) while breathing ambient air of less than 70 mm Hg (or <75 mm Hg in symptomatic patients). Patients with severe pulmonary alveolar proteinosis (Pao2 <50 mm Hg) were excluded to avoid possible exacerbation of the disease in patients who were assigned to receive placebo. The primary end point was the change in the alveolar-arterial oxygen gradient between baseline and week 25. RESULTS: The change in the mean (±SD) alveolar-arterial oxygen gradient was significantly better in the GM-CSF group (33 patients) than in the placebo group (30 patients) (mean change from baseline, -4.50±9.03 mm Hg vs. 0.17±10.50 mm Hg; P = 0.02). The change between baseline and week 25 in the density of the lung field on computed tomography was also better in the GM-CSF group (between-group difference, -36.08 Hounsfield units; 95% confidence interval, -61.58 to -6.99, calculated with the use of the Mann-Whitney U test and the Hodges-Lehmann estimate of confidence intervals for pseudo-medians). Serious adverse events developed in 6 patients in the GM-CSF group and in 3 patients in the placebo group. CONCLUSIONS: In this randomized, controlled trial, inhaled recombinant human GM-CSF was associated with a modest salutary effect on the laboratory outcome of arterial oxygen tension, and no clinical benefits were noted. (Funded by the Japan Agency for Medical Research and Development and the Ministry of Health, Labor, and Welfare of Japan; PAGE ClinicalTrials.gov number, NCT02835742; Japan Medical Association Center for Clinical Trials number, JMA-IIA00205.).


Assuntos
Doenças Autoimunes/tratamento farmacológico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Fatores Imunológicos/uso terapêutico , Proteinose Alveolar Pulmonar/tratamento farmacológico , Administração por Inalação , Adulto , Idoso , Autoanticorpos/sangue , Doenças Autoimunes/diagnóstico por imagem , Método Duplo-Cego , Esquema de Medicação , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/efeitos adversos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/efeitos adversos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Oxigênio/sangue , Proteinose Alveolar Pulmonar/diagnóstico por imagem , Proteinose Alveolar Pulmonar/imunologia , Capacidade de Difusão Pulmonar , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Fumar/efeitos adversos , Tomografia Computadorizada por Raios X , Teste de Caminhada
2.
Gan To Kagaku Ryoho ; 46(9): 1421-1425, 2019 Sep.
Artigo em Japonês | MEDLINE | ID: mdl-31530782

RESUMO

Docetaxel(DTX)plus ramucirumab(RAM)therapy is recommended as second-line or later treatment by the Japanese lung cancer guideline. However, febrile neutropenia(FN)is a frequent complication with this therapy. Efforts for reducing FN risk are essential. We administered pegfilgrastim, a durable granulocyte colony-stimulating factor, as primary prophylaxis for FN to all patients. We also reduced the dose of DTX according to its toxicity. Moreover, we used RAM monotherapy. Herein, we report the results of these efforts regarding DTX plus RAM therapy. We retrospectively reviewed the therapeutic results and occurrence of various adverse effects in 11 patients who started receiving DTX plus RAM therapy in our department between August 2016 and December 2017. Median number of DTX plus RAM cycles was 8(1-25). The following best effects were noted: 2(18%)patients, complete response: 5(45%), partial response: 2(18%), stable disease: and 2(18%), nonevaluable. No patient showed progressive disease. The overall response rate was 63.6%, and the disease control rate was 81.8%. Median progression-free survival was 127 days, and the 1-year progression-free survival rate was 27.3%. The median overall survival duration was not reached, and the 1-year overall survival rate was 53.0%. Adverse effects higher than Grade 3 occurred in 2 cases. FN was not observed. By using pegfilgrastim as primary prophylaxis, we could suppress FN onset in patients; furthermore, we observed better overall response and disease control rates than those observed in clinical trials.


Assuntos
Anticorpos Monoclonais/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas , Docetaxel/efeitos adversos , Neutropenia Febril , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neutropenia Febril/prevenção & controle , Filgrastim , Fator Estimulador de Colônias de Granulócitos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Recombinantes , Estudos Retrospectivos
3.
Zhonghua Xue Ye Xue Za Zhi ; 40(8): 673-677, 2019 Aug 14.
Artigo em Chinês | MEDLINE | ID: mdl-31495135

RESUMO

Objective: To compare the differences in population pharmacokinetic (PK) parameters between two recombinant coagulation factor Ⅷ (FⅧ) preparations, Kogenate FS and Advate, in patients with hemophilia A, and to provide the theoretical basis of precise individualized treatment for those patients. Methods: Patients with moderate or severe hemophilia A who had at least one injection of Kogenate FS or Advate at 41 international hemophilia centers were enrolled as subjects from the WAPPS-Hemo project since January 2015 to December 2017. The half-lives of the two drugs and the time of FⅧ activity reaching 2% (TAT 2%) were calculated, and the differences of PK between the two drugs among different age and dose subgroups were further analyzed. Results: ①The mean age of patients in the Kogenate FS (n=117) and Advate groups (n=120) were (27.6±17.7) and (23.4±16.2) years old, respectively. All patients in the two groups were males. ②The administration doses in the Kogenate FS and Advate groups were (31.5±13.1) IU/kg and (38.17±14.83) IU/kg, respectively; the half-lives of the two drugs were (12.3±3.5) h and (10.8±2.9) h, respectively; and the TAT 2% were (65.2±21.7) h and (57.0±17.9) h, respectively. ③In the Kogenate FS group, the drug half-lives in patients aged ≥12 and <12 years old were (12.7±3.7) h and (11.1±2.5) h, respectively; the TAT 2% were (68.6±22.9) h and (55.8±14.6) h, respectively. In the Advate group, the drug half-lives in patients aged ≥12 and <12 years old were (11.4±3.1) h and (9.4±1.8) h, respectively; and the TAT 2% were (61.1±18.0) h and (45.2±11.3) h, respectively. ④In the Kogenate FS group, the drug half-lives in <20 IU/kg, (20-29) IU/kg, (30-39) IU/kg and ≥40 IU/kg groups were (13.3±4.0) h, (12.3±3.6) h, (12.2±3.5) h and (11.6±2.6) h, respectively; and the TAT 2% were (61.5±21.4) h, (63.9±22.4) h, (67.0±24.3) h and (68.0±19.5) h, respectively. In the Advate group, the drug half-lives in <20 IU/kg, (20-29) IU/kg, (30-39) IU/kg and <40 IU/kg groups were (11.5±3.8) h, (11.4±3.7) h, (11.0±2.9) h and (10.4±2.3) h, respectively; and the TAT 2% were (50.8±19.2) h, (56.7±21.0) h, (58.2±18.8) h and (58.1±15.8) h, respectively. Conclusion: The PK parameters of Kogenate FS are superior to those of Advate among different age and dose subgroups.


Assuntos
Fator VIII/uso terapêutico , Hemofilia A , Adolescente , Adulto , Testes de Coagulação Sanguínea , Criança , Hemofilia A/tratamento farmacológico , Humanos , Masculino , Proteínas Recombinantes , Adulto Jovem
4.
Adv Exp Med Biol ; 1148: 1-24, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482492

RESUMO

The use of therapeutic enzymes embraces currently a vast array of applications, abridging from diggestive disorders to cancer therapy, cardiovascular and lysosomal storage diseases. Enzyme drugs bind and act on their targets with great affinity and specificity, converting substrates to desired products in a reduced time frame with minimal side reactions. These characteristics have resulted in the development of a multitude of enzyme biopharmaceuticals for a wide range of human disorders.The advances in genetic engineering and DNA recombination techniques facilitated the production of therapeutical human-like enzymes, using different cells as host organisms. The selection of hosts generally privileges those that secrete the enzyme into the culture medium, as this eases the purification process, and those that are able to express complex glycoproteins, with glycosylation patterns and other post-translational modifications close to human proteins. Moreover, engineering approaches such as pegylation, encapsulation in micro- and nanocarriers, and mutation of amino acid residues of the native enzyme molecule to yield variants with improved therapeutic activity, half-life and/or stability, have been also addressed. Engineered enzyme products have been designed to display enhanced delivery to target sites and reduced adverse side-effects (e.g., immunogenicity) upon continuous drug administration.Irrespectively of the production method, the final formulation of therapeutic enzymes must display high purity and specificity, and they are often marketed as lyophilized pure preparations with biocompatible buffering salts and diluents to prepare the reconstituted aqueous solution before treatment.


Assuntos
Enzimas/biossíntese , Enzimas/isolamento & purificação , Enzimas/farmacologia , Produtos Biológicos , Meios de Cultura , Engenharia Genética , Glicosilação , Humanos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes
5.
Bratisl Lek Listy ; 120(9): 668-672, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31475551

RESUMO

Restrospective study to evaluate the efficacy of early vs. delayed initiation of G-CSF after autologous hematopoietic stem cell transplantation (AHSCT) in patients with lymphoid malignancies. BACKGROUND: Granulocyte colony stimulating factor (G-CSF) is commonly used after AHSCT to accelerate stem cell engraftment to minimize the morbidity and mortality associated with prolonged neutropenia. However, there is no consensus on the optimal timing of G-CSF after HSCT. METHODS: A total of 117 patients with lymphoid malignancies who underwent AHSCT were included. All patients received G-CSF (filgrastim 5 µg/kg s.c.) daily after AHSCT (43 patients on day 6-8 and 74 patients on day 3 or 4). All patients received standard conditioning regimen for the underlying disease, and standard supportive treatment, including treatment of febrile neutropenia. RESULTS: The incidence of severe neutropenia was 81 % vs 17 %, and very severe neutropenia 61 % vs 4 % in the delayed and early G-CSF groups, respectively (p < 0.0001). The rate of fungal infection was higher in the group of patients who received delayed G-CSF (p < 0.005). CONCLUSION: An early administration of G-CSF after AHSCT (on day 3 or 4) accelerates neutophil engraftment; decreases the incidence of severe neutropenia and the risk of infectious complications (especially fungal infections) (Tab. 1, Fig. 3, Ref. 22).


Assuntos
Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Filgrastim/administração & dosagem , Humanos , Neutropenia/terapia , Neutrófilos/citologia , Proteínas Recombinantes/administração & dosagem , Condicionamento Pré-Transplante , Transplante Autólogo
6.
J Biol Regul Homeost Agents ; 33(4): 1085-1095, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31389223

RESUMO

The purpose of this study was to explore the effect of Allograft Inflammatory Factor 1 (AIF-1) on the regulation of proliferation of breast cancer cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), cell culture and counting, and mass spectrometry were performed. The biologically active high-purity recombinant protein rhAIF-1 was obtained by optimizing the rhAIF-1 protein purification system, and MDA-MB-231 and MDA-MB-361 breast cancer cell lines were used. After adding to the culture medium, rhAIF-1 was found to promote cell proliferation in dose-dependent and time-dependent manners. The purified protein rhAIF-1 was marked with rhodamine and incubated with the cells. Confocal imaging analysis revealed that the foreign protein was localized in the cytoplasm, and rhAIF-1 was unevenly distributed in the cytoplasm. Although AIF-1 accumulates around the nucleus, it can not enter the nucleus, suggesting that other factors might be involved in the regulation of cell proliferation. In order to find the possible interacting protein of rhAIF-1, protein immunoprecipitation technique and mass spectrometry were employed, and it was indicated that ADAM28m was the possible interacting protein of rhAIF-1. The interaction between rhAIF-1 and ADAM28m was validated by immunoprecipitation along with Western blotting. It was found that rhAIF-1 could precipitate ADAM28m protein by immunoprecipitation. The results indicated that IF-1 participates in the development of breast cancer by interacting with ADAM28m and activating downstream signaling pathways. It was concluded that AIF-1 provides a new idea for the molecular mechanism of breast cancer cell proliferation and acts as a new target for the prevention and treatment of breast cancer in the future.


Assuntos
Neoplasias da Mama/patologia , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais , Proteínas ADAM/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas Recombinantes/metabolismo
7.
Medicine (Baltimore) ; 98(32): e16592, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31393358

RESUMO

RATIONALE: Refractory nasopharyngeal carcinoma is challenging to treat and at present there is no standard treatment or any good choice. PATIENT CONCERNS: Although the three patients in our case reports had already underwent multiple treatments before, they still suffered from disease recurrence of nasopharyngeal carcinoma. DIAGNOSIS: They were diagnosed as refractory nasopharyngeal carcinoma. INTERVENTIONS: A continuous infusion of Endostar, an antiangiogenic agent, combined with chemotherapy and radiation therapy was given to treat the patients. OUTCOMES: Patients showed complete or partial response to the combined therapy as evidenced by regression of tumors and decrease in plasma Epstein-Barr virus (EBV) DNA load. LESSONS: Continuous infusions of Endostar in combination with chemotherapy and/or radiation therapy showed promising efficacy and safety. The combination therapy indicates a new approach to treat refractory nasopharyngeal carcinoma.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Endostatinas/uso terapêutico , Carcinoma Nasofaríngeo/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Adulto , Quimiorradioterapia/métodos , Feminino , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/terapia
8.
Life Sci ; 234: 116743, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31408660

RESUMO

AIMS: The present study aimed to investigate the mechanism of bone repair mediated by recombination BMP-2 (rhBMP-2)/recombination CXC chemokine ligand-13 (rhCXCL13)-loaded hollow hydroxyapatite (HA) microspheres/chitosan (CS) composite. MATERIALS AND METHODS: Firstly, the biological activity of rhBMP-2 and rhCXCL13 released from the complex was investigated. Secondly, the effect of rhBMP-2 sustained release solution on ALP activity and rhCXCL13 sustained release solution on cell migration of rat bone marrow mesenchyme stem cells was tested. Thirdly, osteoblasts differentiation test, X-ray scoring and three-point bending test were performed. Finally, the mRNAs expression of osteogenic marker genes and the protein expression of Runx2 was tested by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting (WB), respectively. KEY FINDINGS: RhBMP-2 could significantly promote the proliferation and differentiation, and RhCXCL13 could promote the migration of rat bone marrow MSCs. Detection of ALP activity and calcium salt deposition showed that rhBMP-2 and rhCXCL13 could significantly improve the biological activity and promote cell differentiation ability. X-ray scoring of radius and flexural strength test showed that rhBMP-2 and rhCXCL13 could promote bone healing and improve the bending resistance of bone tissue. The in vitro molecular experiments including RT-PCR and WB further demonstrated the roles of rhBMP-2 and rhCXCL13 in bone formation and bone repair. SIGNIFICANCE: Our results indicated that the hollow HA microspheres/CS composite could be effective as a delivery vehicle for rhBMP-2 and rhCXCL13 in bone regeneration and bone repair. In this process, rhBMP-2 may promote bone regeneration by regulating bone marrow MSCs cells recruited by rhCXCL13.


Assuntos
Proteína Morfogenética Óssea 2/administração & dosagem , Quimiocina CXCL13/administração & dosagem , Quitosana/análogos & derivados , Preparações de Ação Retardada/química , Durapatita/química , Osteogênese/efeitos dos fármacos , Tecidos Suporte/química , Fator de Crescimento Transformador beta/administração & dosagem , Animais , Materiais Biocompatíveis/química , Proteína Morfogenética Óssea 2/farmacologia , Regeneração Óssea/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL13/farmacologia , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Coelhos , Ratos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta/farmacologia
9.
Arch Virol ; 164(10): 2505-2513, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377888

RESUMO

Infectious hematopoietic necrosis virus (IHNV) was developed as a vector to aid the construction of vaccines against viral diseases such as viral hemorrhagic septicemia virus, spring viremia of carp virus, and influenza virus H1N1. However, the optimal site for foreign gene expression in the IHNV vector has not been determined. In the present study, five recombinant viruses with the green fluorescence protein (GFP) gene inserted into different genomic junction regions of the IHNV genomic sequence were generated using reverse genetics technology. Viral growth was severely delayed when the GFP gene was inserted into the intergenic region between the N and P genes. Real-time fluorescence quantitative PCR assays showed that the closer the GFP gene was inserted towards the 3' end, the higher the GFP mRNA levels. Measurement of the GFP fluorescence intensity, which is the most direct method to determine the GFP protein expression level, showed that the highest GFP protein level was obtained when the gene was inserted into the intergenic region between the P and M genes. The results of this study suggest that the P and M gene junction region is the optimal site within the IHNV vector to express foreign genes, providing valuable information for the future development of live vector vaccines.


Assuntos
Expressão Gênica , Vetores Genéticos , Vírus da Necrose Hematopoética Infecciosa/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Fluorometria , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Reação em Cadeia da Polimerase em Tempo Real , Genética Reversa
10.
World J Microbiol Biotechnol ; 35(9): 135, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432264

RESUMO

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.


Assuntos
Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Thermoactinomyces/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Thermoactinomyces/genética
11.
Sheng Wu Gong Cheng Xue Bao ; 35(8): 1478-1490, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31441619

RESUMO

Bacillus subtilis can be widely used as an important microorganism for metabolic engineering and recombinant proteins expression in industrial biotechnology and synthetic biology. However, it is difficult to make accurate regulation of exogenous gene by biological tools in B. subtilis, which limits the application of B. subtilis in synthetic biology. The purpose of this study is to develop regulatory tools for precise control of gene expression by using non-coding RNAs, by which the activation of heterologous gene could be achieved without the auxiliary protein factors. We constructed the synthetic riboswitch E and aptazyme AZ using the theophylline aptamer. Six different native promoters from B. subtilis were functionally adapted with the E and AZ to fabricate an array of novel regulatory elements activated by theophylline. Then, we determined the performance of these elements using green fluorescence protein as reporter, and then further verified using red fluorescence protein and pullulanase as cargo proteins. Results showed that the same kind of RNA elements with different promoters showed different levels of efficiency. Promoter PsigW and E combination (sigWE) had the highest induction rate in B. subtilis. Compared with the control group, it can produce the induction rate of 16.8. Promoter PrpoB and AZ combination (rpoBAZ) showed the highest induction rate of 6.2. SigWE mediated mCherry induction rate was 9.2, and P43E mediated pullulanase induction rate was 32.8, in which enzyme activity reached 81 U/mL. This study confirmed that GFP, mCherry and pullulan can all be regulated by riboswitch and aptazyme, but there were differences between different combinations of promoters with RNA regulators.


Assuntos
Bacillus subtilis , Regiões Promotoras Genéticas , RNA , Proteínas Recombinantes , Teofilina
12.
Gene ; 717: 144043, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31400407

RESUMO

Genes involved in the repair of DNA damage are emerging as playing important roles during the disease processes caused by pathogenic fungi. However, there are potentially hundreds of genes involved in DNA repair in a fungus and some of those genes can play additional roles within the cell. One such gene is RAD23, required for virulence of the human pathogenic fungus Cryptococcus neoformans, that encodes a protein involved in the nucleotide excision repair (NER) pathway. However, Rad23 is a dual function protein, with a role in either repair of damaged DNA or protein turn over by directing proteins to the proteasome. Here, these two functions of Rad23 were tested by the creation of a series of domain deletion alleles of RAD23 and the assessment of the strains for DNA repair, proteasome functions, and virulence properties. Deletion of the different domains was able to uncouple the two functions of Rad23, and the phenotypes of strains carrying such forms indicated that the role of RAD23 in virulence is due to its function in proteasomal-mediated protein degradation rather than NER.


Assuntos
Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Reparo do DNA/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Animais , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Larva/microbiologia , Mariposas/microbiologia , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico/genética , Virulência
13.
Pharm Res ; 36(10): 146, 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31396727

RESUMO

PURPOSE: CTB-001, a recently developed generic version of bivalirudin, an FDA-approved anticoagulant used for prophylaxis and treatment of cardiovascular diseases, has shown good efficacy and safety in clinical trials. We characterized the pharmacokinetics (PK) and pharmacodynamics (PD) of CTB-001 by modeling and simulation analysis. METHODS: PK/PD data were collected from a randomized, double-blind, placebo-controlled, single-dose, dose-escalation phase 1 study conducted in 24 healthy Korean male subjects. PK/PD analysis was conducted sequentially by nonlinear mixed-effects modeling implemented in NONMEM®. Monte-Carlo simulations were conducted for PK, activated partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin time (TT). RESULTS: The CTB-101 PK was best described by a three-compartment linear model with a saturable binding peripheral compartment. All PD endpoints showed dose-response relationship, and their changes over time paralleled those of CTB-101 concentrations. A simple maximum effect model best described the aPTT, PT in INR, PT in seconds, and TT, whereas an inhibitory simple maximum effect model best described PT in percentages. The maximum duration of effect of CTB-001 on aPTT prolongation was 52.1 s. CONCLUSIONS: The modeling and simulation analysis well-characterized the PK and PD of CTB-001 in healthy Koreans, which will be valuable for identifying optimal dosing regimens of CBT-001.


Assuntos
Anticoagulantes/farmacologia , Hirudinas/farmacologia , Modelos Biológicos , Fragmentos de Peptídeos/farmacologia , Adulto , Anticoagulantes/farmacocinética , Simulação por Computador , Relação Dose-Resposta a Droga , Método Duplo-Cego , Medicamentos Genéricos , Hirudinas/farmacocinética , Humanos , Masculino , Método de Monte Carlo , Fragmentos de Peptídeos/farmacocinética , Tempo de Protrombina , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologia , Resultado do Tratamento , Adulto Jovem
15.
N Engl J Med ; 381(8): 716-726, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31433919

RESUMO

BACKGROUND: Serelaxin is a recombinant form of human relaxin-2, a vasodilator hormone that contributes to cardiovascular and renal adaptations during pregnancy. Previous studies have suggested that treatment with serelaxin may result in relief of symptoms and in better outcomes in patients with acute heart failure. METHODS: In this multicenter, double-blind, placebo-controlled, event-driven trial, we enrolled patients who were hospitalized for acute heart failure and had dyspnea, vascular congestion on chest radiography, increased plasma concentrations of natriuretic peptides, mild-to-moderate renal insufficiency, and a systolic blood pressure of at least 125 mm Hg, and we randomly assigned them within 16 hours after presentation to receive either a 48-hour intravenous infusion of serelaxin (30 µg per kilogram of body weight per day) or placebo, in addition to standard care. The two primary end points were death from cardiovascular causes at 180 days and worsening heart failure at 5 days. RESULTS: A total of 6545 patients were included in the intention-to-treat analysis. At day 180, death from cardiovascular causes had occurred in 285 of the 3274 patients (8.7%) in the serelaxin group and in 290 of the 3271 patients (8.9%) in the placebo group (hazard ratio, 0.98; 95% confidence interval [CI], 0.83 to 1.15; P = 0.77). At day 5, worsening heart failure had occurred in 227 patients (6.9%) in the serelaxin group and in 252 (7.7%) in the placebo group (hazard ratio, 0.89; 95% CI, 0.75 to 1.07; P = 0.19). There were no significant differences between the groups in the incidence of death from any cause at 180 days, the incidence of death from cardiovascular causes or rehospitalization for heart failure or renal failure at 180 days, or the length of the index hospital stay. The incidence of adverse events was similar in the two groups. CONCLUSIONS: In this trial involving patients who were hospitalized for acute heart failure, an infusion of serelaxin did not result in a lower incidence of death from cardiovascular causes at 180 days or worsening heart failure at 5 days than placebo. (Funded by Novartis Pharma; RELAX-AHF-2 ClinicalTrials.gov number, NCT01870778.).


Assuntos
Doenças Cardiovasculares/mortalidade , Insuficiência Cardíaca/tratamento farmacológico , Relaxina/uso terapêutico , Vasodilatadores/uso terapêutico , Doença Aguda , Idoso , Pressão Sanguínea/efeitos dos fármacos , Progressão da Doença , Método Duplo-Cego , Feminino , Insuficiência Cardíaca/mortalidade , Insuficiência Cardíaca/fisiopatologia , Hospitalização , Humanos , Incidência , Infusões Intravenosas , Masculino , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Relaxina/efeitos adversos , Relaxina/farmacologia , Falha de Tratamento , Vasodilatadores/efeitos adversos
16.
Anticancer Res ; 39(8): 4185-4190, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366504

RESUMO

BACKGROUND/AIM: Insulin-like growth factor 1 (IGF-1)-mediated molecular pathway has been implicated in non-small cell lung cancer (NSCLC) pathogenesis and progression. We aimed to evaluate serum levels of IGF-1, IGF-2 and IGF-binding protein 3 (IGF-BP3) before and after standard treatment in patients with advanced NSCLC and their prognostic and predictive correlations. PATIENTS AND METHODS: Seventy-three patients were prospectively included. Analysis and quantification of circulating levels of IGF1, IGF2, IGFBP3 were performed by total ELISA in peripheral blood samples at baseline and 3 months post-treatment. RESULTS: The median values of IGF-1 and IGF-1/IGF-BP3 ratios (125.82 vs. 133.4 ng/ml, p=0.087 and 0.01006 vs. 0.01252, p=0.011) were both decreased after treatment. Importantly, the post-treatment value of the ratio was significantly reduced only among responders to treatment (0.01044 from 0.01255, p=0.02). CONCLUSION: Reduction of IGF-1/IGF-BP3 ratio was statistically significant only among patients with NSCLC who responded to first-line treatment. If validated in larger cohorts, IGF-1/IGFBP3 might be a useful predictive tool for response to chemotherapy in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/sangue , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like I/genética , Idoso , Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Pemetrexede/administração & dosagem , Prognóstico , Proteínas Recombinantes , Transdução de Sinais/efeitos dos fármacos
17.
BMC Vet Res ; 15(1): 274, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370852

RESUMO

BACKGROUND: In Poland, the leader in goose production in Europe, goose parovirus infection, or Derzsy's disease (DD), must be reported to the veterinary administration due to the serious economic and epizootic threat to waterfowl production. Prophylactic treatment for DD includes attenuated live or inactivated vaccines. Moreover, the control of DD includes the monitoring of maternal derived antibody (MDA) levels in the offspring and antibody titers in the parent flock after vaccination. The aim of this study was to develop an ELISA for the detection of goose parvovirus (GPV) antibodies. RESULTS: Two recombinant protein fragments derived from VP3 (viral protein 3) GPV, namely VP3ep6 and VP3ep4-6 with a mass of 20.9 and 32.3 kDa, respectively, were produced using an Escherichia coli expression system. These proteins were purified by one-step nickel-affinity chromatography, which yielded protein preparations with a purity above 95%. These recombinant proteins were useful in the detection of serum anti-GPV antibodies, and this was confirmed by Western blotting. However, recombinant VP3ep4-6 protein showed a greater ability to correctly identify sera from infected geese. In the next stage of the project, a pool of 166 goose sera samples, previously examined by a virus neutralization test (VN), was tested. For further studies, one recombinant protein (VP3ep4-6) was selected for optimization of the test conditions. After optimization, the newly developed ELISA was compared to other serological tests, and demonstrated high sensitivity and specificity. CONCLUSION: In conclusion, the VP3ep4-6 ELISA method described here can be used for the detection of antibodies to GPV in serum.


Assuntos
Anticorpos Antivirais/sangue , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Parvoviridae/veterinária , Parvovirinae/imunologia , Doenças das Aves Domésticas/diagnóstico , Animais , Ensaio de Imunoadsorção Enzimática/normas , Infecções por Parvoviridae/sangue , Infecções por Parvoviridae/diagnóstico , Doenças das Aves Domésticas/sangue , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade
18.
Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi ; 33(9): 870-874;882, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31446708

RESUMO

Objective:The study aimed to investigate the role of nuocytes in allergic rhinitis (AR) murine models. Method:After intranasal administration of recombinant (rm) interleukin (IL)-33 in BALB/c mice, nuocytes were sorted and purified from the mouse nasal-associated lymphoid tissue (NALT). Then, we examined the response of nuocytes to rmIL-33 in vitro. After a murine model of AR was established using ovalbumin, we adoptively transferred the cultured NALT-derived nuocytes to mice models, and determined allergic responses in them. Result:rmIL-33 expanded nuocytes in NALT of mice compared with AR mice (t=3.66, P<0.01), and increased production of IL-13 from these cells in vitro in comparison with unstimulated nuocytes (t=19.90, P<0.000 1). After adoptive transfer of nuocytes, sneezing (t=9.89, P<0.000 1) ,numbers of eosinophils(t=8.17, P<0.000 1), concentrations of IL-13 (t=40.47, P<0.000 1) and IL-33 (t=19.89, P<0.000 1) in nasal lavage fluid were all enhanced when compared with AR mice. Conclusion:Nuocytes promote allergic inflammation in a murine model of AR.


Assuntos
Interleucina-33/farmacologia , Mucosa Nasal/citologia , Rinite Alérgica/fisiopatologia , Transferência Adotiva , Animais , Modelos Animais de Doenças , Eosinófilos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Líquido da Lavagem Nasal/citologia , Ovalbumina , Proteínas Recombinantes
19.
Cancer Sci ; 110(9): 2722-2733, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31461572

RESUMO

Mesothelin (MSLN) shows increased expression in various cancer cells. For clinical application of antibodies as a positron emission tomography (PET) imaging reagent, a human shortened antibody is essential both for avoiding redundant immune responses and for providing rapid imaging. Therefore, we cloned a single-chain fragment of variable regions (scFv) from a human-derived gene sequence. This was achieved through the construction of a naïve phage library derived from human tonsil lymphocytes. Using a column with human recombinant MSLN, we carried out bio-panning of phage-variants by colony formation. We first obtained 120 clones that were subjected to selection in an ELISA using human recombinant MSLN as a solid phase antigen, and 15 phage clones of scFv with a different sequence were selected and investigated by flow cytometry (FCM). Then, six variants were selected and the individual scFv gene was synthesized in the VL and VH domains and expressed in Chinese hamster ovary cells. Mammalian cell-derived human-origin scFv clones were analyzed by FCM again, and one MSLN highly specific scFv clone was established. PET imaging by 89 Zr-labeled scFv was done in mice bearing xenografts with MSLN-expressing cancer cells, and tumor legions were successfully visualized. The scFv variant established in the present study may be potentially useful for cancer diagnosis by PET imaging.


Assuntos
Proteínas Ligadas por GPI/imunologia , Neoplasias/diagnóstico por imagem , Compostos Radiofarmacêuticos/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Células CHO , Linhagem Celular Tumoral , Clonagem Molecular , Cricetulus , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/isolamento & purificação , Proteínas Ligadas por GPI/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Imagem Molecular/métodos , Neoplasias/patologia , Biblioteca de Peptídeos , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons/métodos , Radioisótopos , Compostos Radiofarmacêuticos/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Cadeia Única/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto , Zircônio
20.
Cancer Radiother ; 23(5): 449-465, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400956

RESUMO

Nowadays, ionizing radiations have numerous applications, especially in medicine for diagnosis and therapy. Pharmacological radioprotection aims at increasing detoxification of free radicals. Radiomitigation aims at improving survival and proliferation of damaged cells. Both strategies are essential research area, as non-contained radiation can lead to harmful effects. Some advances allowing the comprehension of normal tissue injury mechanisms, and the discovery of related predictive biomarkers, have led to developing several highly promising radioprotector or radiomitigator drugs. Next to these drugs, a growing interest does exist for biotherapy in this field, including gene therapy and cell therapy through mesenchymal stem cells. In this review article, we provide an overview of the management of radiation damages to healthy tissues via gene or cell therapy in the context of radiotherapy. The early management aims at preventing the occurrence of these damages before exposure or just after exposure. The late management offers promises in the reversion of constituted late damages following irradiation.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Genética/métodos , Lesões por Radiação/prevenção & controle , Proteção Radiológica/métodos , Amifostina/uso terapêutico , Animais , Antioxidantes/uso terapêutico , Ensaios Clínicos como Assunto , Fracionamento da Dose de Radiação , Edição de Genes , Vetores Genéticos/uso terapêutico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Transplante de Células-Tronco Mesenquimais , Camundongos , Oxirredutases/genética , Oxirredutases/uso terapêutico , Lesões por Radiação/etiologia , Lesões por Radiação/terapia , Lesões Experimentais por Radiação/prevenção & controle , Lesões Experimentais por Radiação/terapia , Protetores contra Radiação/farmacologia , Protetores contra Radiação/uso terapêutico , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Fatores de Tempo
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