RESUMO
The need to increase agricultural yield has led to an extensive use of antibiotics against plant pathogens, which has resulted in the emergence of resistant strains. Therefore, there is an increasing demand for new methods, preferably with lower chances of developing resistant strains and a lower risk to the environment or public health. Many Gram-negative bacterial pathogens use quorum sensing, a population-density-dependent regulatory mechanism, to monitor the secretion of N-acyl-homoserine lactones (AHLs) and pathogenicity. Therefore, quorum sensing represents an attractive antivirulence target. AHL lactonases hydrolyze AHLs and have potential antibacterial properties; however, their use is limited by thermal instability and durability, or low activity. Here, we demonstrate that an AHL lactonase from the phosphotriesterase-like lactonase family exhibits high activity with the AHL secreted from the plant pathogen Erwinia amylovora and attenuates infection in planta. Using directed enzyme evolution, we were able to increase the enzyme's temperature resistance (T50, the temperature at which 50% of the activity is retained) by 8 °C. Then, by performing enzyme encapsulation in nanospherical capsules composed of tertbutoxycarbonyl-Phe-Phe-OH peptide, the shelf life was extended for more than 5 weeks. Furthermore, the encapsulated and free mutant were able to significantly inhibit up to 70% blossom's infection in the field, achieving the same efficacy as seen with antibiotics commonly used today to treat the plant pathogen. We conclude that specific AHL lactonase can inhibit E. amylovora infection in the field, as it degrades the AHL secreted by this plant pathogen. The combination of directed enzyme evolution and peptide nanostructure encapsulation significantly improved the thermal resistance and shelf life of the enzyme, respectively, increasing its potential in future development as antibacterial treatment.
Assuntos
Hidrolases de Éster Carboxílico/farmacologia , Erwinia amylovora/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Nanosferas/química , Doenças das Plantas/prevenção & controle , Percepção de Quorum/efeitos dos fármacos , Acil-Butirolactonas/metabolismo , Hidrolases de Éster Carboxílico/administração & dosagem , Hidrolases de Éster Carboxílico/genética , Evolução Molecular Direcionada/métodos , Enzimas Imobilizadas/administração & dosagem , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/farmacologia , Erwinia amylovora/fisiologia , Modelos Moleculares , Peptídeos/química , Doenças das Plantas/microbiologia , Pyrus/microbiologiaRESUMO
Dense colonization of mucoid Pseudomonas aeruginosa within the self-secreted extracellular matrix (mainly alginate), called biofilm, is a principal reason for the failure of antimicrobial therapy in cystic fibrotic patients. Alginate is a key component in the biofilm of mucoid P. aeruginosa and responsible for surface adhesion and stabilization of biofilm. To overcome this problem, alginate lyase functionalized chitosan nanoparticles of ciprofloxacin were developed for the effective treatment of P. aeruginosa infection in cystic fibrosis patients. The developed nanoparticles were found to have desired quality attributes and demonstrated sustained release following the Higuchi release kinetics. Drug compatibility with the chitosan was confirmed by FTIR while powder X-ray diffraction analysis confirmed the entrapment of drug within the nanoparticle matrix. Lactose adsorbed NPs showed promising aerodynamic property. Nanoparticles showed prolonged MIC and significant reduction in biofilm aggregation and formation in planktonic bacterial suspension. Nanoparticles exhibited significantly higher inhibitory effect against biofilm of P. aeruginosa and reduced the biomass, thickness and density confirmed by confocal microscopy. Furthermore, developed nanoparticles were haemocompatible and did not exhibit any toxicity in vitro MTT assay and in vivo on lungs male Wistar rats. The data in hand collectively suggest the proposed strategy a better alternative for the effective treatment of cystic fibrosis infections.
Assuntos
Antibacterianos/administração & dosagem , Quitosana/administração & dosagem , Ciprofloxacina/administração & dosagem , Enzimas Imobilizadas/administração & dosagem , Nanopartículas/administração & dosagem , Polissacarídeo-Liases/administração & dosagem , Animais , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Quitosana/química , Ciprofloxacina/química , Fibrose Cística/tratamento farmacológico , Liberação Controlada de Fármacos , Enzimas Imobilizadas/química , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Pulmão/efeitos dos fármacos , Masculino , Nanopartículas/química , Agregação Plaquetária/efeitos dos fármacos , Polissacarídeo-Liases/química , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Ratos WistarRESUMO
We describe an innovative strategy to generate catalytic compartments with triggered functionality at the nanoscale level by combining pH-reversible biovalves and enzyme-loaded synthetic compartments. The biovalve has been engineered by the attachment of stimuli-responsive peptides to a genetically modified channel porin, enabling a reversible change of the molecular flow through the pores of the porin in response to a pH change in the local environment. The biovalve functionality triggers the reaction inside the cavity of the enzyme-loaded compartments by switching the in situ activity of the enzymes on/off based on a reversible change of the permeability of the membrane, which blocks or allows the passage of substrates and products. The complex functionality of our catalytic compartments is based on the preservation of the integrity of the compartments to protect encapsulated enzymes. An increase of the in situ activity compared to that of the free enzyme and a reversible on/off switch of the activity upon the presence of a specific stimulus is achieved. This strategy provides straightforward solutions for the development of catalytic nanocompartments efficiently producing desired molecules in a controlled, stimuli-responsive manner with high potential in areas, such as medicine, analytical chemistry, and catalysis.
Assuntos
Preparações de Ação Retardada/química , Escherichia coli/química , Membranas Artificiais , Peptídeos/química , Polímeros/química , Porinas/química , Sequência de Aminoácidos , Biomimética , Catálise , Enzimas Imobilizadas/administração & dosagem , Enzimas Imobilizadas/química , Peroxidase do Rábano Silvestre/administração & dosagem , Peroxidase do Rábano Silvestre/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , PermeabilidadeRESUMO
High intensity focused ultrasound (HIFU) surgery generally suffers from poor precision and low efficiency in clinical application, especially for cancer therapy. Herein, a multiscale hybrid catalytic nanoreactor (catalase@MONs, abbreviated as C@M) has been developed as a tumor-sensitive contrast and synergistic agent (C&SA) for ultrasound-guided HIFU cancer surgery, by integrating dendritic-structured mesoporous organosilica nanoparticles (MONs) and catalase immobilized in the large open pore channels of MONs. Such a hybrid nanoreactor exhibited sensitive catalytic activity toward H2O2, facilitating the continuous O2 gas generation in a relatively mild manner even if incubated with 10 µM H2O2, which finally led to enhanced ablation in the tissue-mimicking PAA gel model after HIFU exposure mainly resulting from intensified cavitation effect. The C@M nanoparticles could be accumulated within the H2O2-enriched tumor region through enhanced permeability and retention effect, enabling durable contrast enhancement of ultrasound imaging, and highly efficient tumor ablation under relatively low power of HIFU exposure in vivo. Very different from the traditional perfluorocarbon-based C&SA, such an on-demand catalytic nanoreactor could realize the accurate positioning of tumor without HIFU prestimulation and efficient HIFU ablation with a much safer power output, which is highly desired in clinical HIFU application.
Assuntos
Catalase/administração & dosagem , Meios de Contraste/administração & dosagem , Ablação por Ultrassom Focalizado de Alta Intensidade/métodos , Nanopartículas/química , Neoplasias/cirurgia , Compostos de Organossilício/química , Animais , Catalase/farmacologia , Meios de Contraste/farmacologia , Enzimas Imobilizadas/administração & dosagem , Enzimas Imobilizadas/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Camundongos Nus , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Oxigênio/metabolismo , Ultrassonografia de Intervenção/métodosAssuntos
Asparaginase , Drenagem/métodos , Endoscopia Gastrointestinal/métodos , Pseudocisto Pancreático , Pancreatite Necrosante Aguda , Polietilenoglicóis , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Asparaginase/administração & dosagem , Asparaginase/efeitos adversos , Pré-Escolar , Enzimas Imobilizadas/administração & dosagem , Enzimas Imobilizadas/efeitos adversos , Humanos , Masculino , Pseudocisto Pancreático/diagnóstico , Pseudocisto Pancreático/etiologia , Pseudocisto Pancreático/fisiopatologia , Pseudocisto Pancreático/cirurgia , Pancreatite Necrosante Aguda/induzido quimicamente , Pancreatite Necrosante Aguda/complicações , Pancreatite Necrosante Aguda/diagnóstico , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/efeitos adversos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Resultado do TratamentoRESUMO
Fabrication of various efficient enzyme reactors has triggered increasing interests for its extensive applications in biological and clinical research. In this study, magnetic nanoparticles were functionalized by a biocompatible reactive polymer, poly(2-vinyl-4,4-dimethylazlactone), which was synthesized by reversible addition-fragmentation chain transfer polymerization. Then, the prepared polymer-modified magnetic nanoparticles were employed as favorable carriers for enzyme immobilization. l-Asparaginase was selected as the model enzyme to fabricate the enzyme reactor, and the prepared enzyme reactor exhibited high loading capacity of 318.0 µg mg(-1) magnetic nanoparticle. Interestingly, it has been observed that the enzymolysis efficiency increased slightly with the lengthened polymer chain, resulting from the increased immobilization amount of enzyme. Meanwhile, the immobilized enzyme could retain more than 95.7% activity after 10 repeated uses and maintain more than 72.6% activity after 10 weeks storage. Moreover, an extracorporeal shunt system was simulated to estimate the potential application capability of the prepared l-asparaginase reactor in acute lymphoblastic leukemia treatment.
Assuntos
Enzimas Imobilizadas/química , Lactonas/química , Nanopartículas de Magnetita/química , Polivinil/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Asparaginase/administração & dosagem , Asparaginase/química , Reatores Biológicos , Enzimas Imobilizadas/administração & dosagem , HumanosRESUMO
The effect of immobilized hyaluronidase on stem and progenitor cells of the lungs was studied on the model of partially reversible toxic bleomycin-induced pulmonary fibrosis in C57Bl/6 mice. During the inflammation phase, immobilized hyaluronidase reduced infiltration of alveolar interstitium with hemopoietic stem cells Sca-1(+), c-Kit(+), CD34(-), (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b (Mac1), TER-119)(-). Improvement of histological parameters of bleomycin lungs during the phase of collagen fiber deposition after the treatment was accompanied by accumulation of mesenchymal multipotent stromal cells (CD31(-), CD34(-), CD45(-), CD44(+), CD73(+), CD90(+), CD106(+)decrease in the population of pan-hemopoietic cells (CD45(+)), accelerated restoration of the content of endothelial cells, and inhibition of clonal activity of fibroblast precursors (CD45(-)).
Assuntos
Enzimas Imobilizadas/administração & dosagem , Hialuronoglucosaminidase/administração & dosagem , Fibrose Pulmonar/patologia , Células-Tronco/metabolismo , Animais , Antígenos CD/metabolismo , Medula Óssea/imunologia , Medula Óssea/patologia , Pulmão/patologia , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/imunologia , Células-Tronco/efeitos dos fármacosRESUMO
Antifibrotic activity of testicular hyaluronidase, immobilized on polyethylenoxide and obtained by electron beam synthesis, was studied on the model of bleomycin injuries to the alveolar epithelium (irreversible pneumofibrosis) in C57Bl/6 mice and compared to the effect of testicular hyaluronidase. Intranasal therapy with immobilized and testicular hyaluronidases prevented the deposition of fibrotic mass in the parenchyma of "bleomycin" lungs. The effect of immobilized hyaluronidase was more pronounced than that of testicular hyaluronidase. The studied compounds were virtually inessential for infiltration of the alveolar interstitium and alveolar tracts by lymphocytes, macrophages, neutrophils, and plasma cells. Unchanged histoarchitectonics of bleomycin-damaged lungs in immobilized hyaluronidase therapy was due to suppression of the progenitor fibroblast cells (CD45(-)).
Assuntos
Anti-Inflamatórios/administração & dosagem , Hialuronoglucosaminidase/administração & dosagem , Fibrose Pulmonar Idiopática/tratamento farmacológico , Animais , Anti-Inflamatórios/química , Bleomicina , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Enzimas Imobilizadas/administração & dosagem , Enzimas Imobilizadas/química , Hialuronoglucosaminidase/química , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/patologia , Camundongos , Camundongos Endogâmicos C57BL , Polietilenos/química , Alvéolos Pulmonares/patologia , Mucosa Respiratória/patologiaRESUMO
This study was carried out to determine the optimum conditions for water-in-oil-in-water (W/O/W) microencapsulated lactase (ß-galactosidase) in order to prevent the intolerance of lactose in milk. The core material was lactase and the coating materials were medium-chain triglyceride for W/O phase, and whey protein isolate (WPI), maltodextrin, gum arabic, and its mixtures for W/O/W phase. Polyglycerol polyricinoleate (PGPR) was used as a primary emulsifier, and polyoxyethylene sorbitan monolaurate (PSML) was selected as a secondary emulsifier based on emulsion stability index. To determine the most efficient conditions for the W/O/W-lactase microencapsulation, the ratio of core to coating materials and amounts of emulsifiers were investigated by response surface methodology. The optimum ratio of core to coating materials in W/O, amount of PGPR, ratio of core to coating material in W/O/W, and amount of PSML were found to be 0.5-9.5, 0.75% (w/v), 1.7-8.3, and 0.25% (w/v), respectively. The average size of the microcapsules was about 10 µm under optimum conditions. Microcapsules of 30% (w/v) WPI as a secondary coating material could evenly distribute the pocket of lactase. Based on the data obtained from this study, lactase microcapsules could effectively be produced by the method of W/O/W double emulsion.
Assuntos
Emulsões/química , Enzimas Imobilizadas/administração & dosagem , beta-Galactosidase/administração & dosagem , Cápsulas , Composição de Medicamentos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glicerol/análogos & derivados , Glicerol/química , Goma Arábica/química , Humanos , Lactose/metabolismo , Polissacarídeos/química , Polissorbatos/química , Ácidos Ricinoleicos/química , Triglicerídeos/química , Água/química , beta-Galactosidase/química , beta-Galactosidase/metabolismoRESUMO
We report an efficient strategy to conjugate methacrylamide moieties to the lysine units of lysozyme for co-polymerization and subsequent triggered release from hydrogels. Two novel linker molecules, containing an ester bond and/or a disulfide bond for temporary immobilization, were synthesized and conjugated to lysozyme. Lysozyme was successfully modified with on average 2.5 linker molecules per protein molecule, as evidenced by MALDI-TOF and by titration of the free amine groups, while spectral analysis verified the preservation of the protein structure. Next, methacrylated dextran (Dex-MA) was polymerized in presence of native or modified lysozyme to yield hydrogels. The release of native and modified lysozyme from Dex-MA hydrogels was studied in acetate buffer (pH 5, in absence of any trigger) and only a minor fraction (~15%) of the modified lysozyme was released, whereas ~74% of the native lysozyme was released. This indicates successful immobilization of the majority of the modified lysozyme in the hydrogel network. Upon hydrolysis of the ester bonds or incubation with glutathione to reduce disulfide bonds of the linker molecules that conjugate the lysozyme to the gel network, the modified lysozyme was mobilized and released from the hydrogel to the same extent as native lysozyme. These data were confirmed by fluorescence recovery after photobleaching experiments. This approach appeared to be highly interesting for temporary immobilization and subsequent glutathione triggered intracellular delivery of proteins from hydrogels.
Assuntos
Preparações de Ação Retardada/química , Dextranos/química , Enzimas Imobilizadas/administração & dosagem , Glutationa/química , Hidrogéis/química , Metacrilatos/química , Muramidase/administração & dosagem , Acrilamidas/química , Animais , Galinhas , Enzimas Imobilizadas/química , Hidrólise , Muramidase/química , Oxirredução , Polimerização , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
CONTEXT: Patients with chronic disabling gout refractory to conventional urate-lowering therapy need timely treatment to control disease manifestations related to tissue urate crystal deposition. Pegloticase, monomethoxypoly(ethylene glycol)-conjugated mammalian recombinant uricase, was developed to fulfill this need. OBJECTIVE: To assess the efficacy and tolerability of pegloticase in managing refractory chronic gout. DESIGN, SETTING, AND PATIENTS: Two replicate, randomized, double-blind, placebo-controlled trials (C0405 and C0406) were conducted between June 2006 and October 2007 at 56 rheumatology practices in the United States, Canada, and Mexico in patients with severe gout, allopurinol intolerance or refractoriness, and serum uric acid concentration of 8.0 mg/dL or greater. A total of 225 patients participated: 109 in trial C0405 and 116 in trial C0406. INTERVENTION: Twelve biweekly intravenous infusions containing either pegloticase 8 mg at each infusion (biweekly treatment group), pegloticase alternating with placebo at successive infusions (monthly treatment group), or placebo (placebo group). MAIN OUTCOME MEASURE: Primary end point was plasma uric acid levels of less than 6.0 mg/dL in months 3 and 6. RESULTS: In trial C0405 the primary end point was reached in 20 of 43 patients in the biweekly group (47%; 95% CI, 31%-62%), 8 of 41 patients in the monthly group (20%; 95% CI, 9%-35%), and in 0 patients treated with placebo (0/20; 95% CI, 0%-17%; P < .001 and <.04 for comparisons between biweekly and monthly groups vs placebo, respectively). Among patients treated with pegloticase in trial C0406, 16 of 42 in the biweekly group (38%; 95% CI, 24%-54%) and 21 of 43 in the monthly group (49%; 95% CI, 33%-65%) achieved the primary end point; no placebo-treated patients reached the primary end point (0/23; 95% CI, 0%-15%; P = .001 and < .001, respectively). When data in the 2 trials were pooled, the primary end point was achieved in 36 of 85 patients in the biweekly group (42%; 95% CI, 32%-54%), 29 of 84 patients in the monthly group (35%; 95% CI, 24%-46%), and 0 of 43 patients in the placebo group (0%; 95% CI, 0%-8%; P < .001 for each comparison). Seven deaths (4 in patients receiving pegloticase and 3 in the placebo group) occurred between randomization and closure of the study database (February 15, 2008). CONCLUSION: Among patients with chronic gout, elevated serum uric acid level, and allopurinol intolerance or refractoriness, the use of pegloticase 8 mg either every 2 weeks or every 4 weeks for 6 months resulted in lower uric acid levels compared with placebo. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00325195.
Assuntos
Enzimas Imobilizadas/administração & dosagem , Gota/tratamento farmacológico , Polietilenoglicóis/administração & dosagem , Urato Oxidase/administração & dosagem , Ácido Úrico/sangue , Alopurinol/uso terapêutico , Doença Crônica , Método Duplo-Cego , Esquema de Medicação , Resistência a Medicamentos , Feminino , Supressores da Gota/uso terapêutico , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
Immobilized hyaluronidase (nanotechnology method of electron-beam synthesis) exhibited high hepatoprotective activity on the model of Cl4-induced hepatitis. This agent produced anticholestatic, anti-inflammatory, and antisclerotic effects. These effects were shown to accompany stimulation of multipotent bone marrow precursors, mobilization of these cells into the peripheral blood, and cell migration to the target organ increasing the number of parenchymal progenitor cells in the liver. The mechanisms for targeted migration of progenitor cells suggest a decrease in SDF-1 production by bone marrow stromal cells and increase in the synthesis of this factor by microenvironmental cells of the liver tissue.
Assuntos
Citoproteção , Enzimas Imobilizadas/uso terapêutico , Hepatite Animal/tratamento farmacológico , Hialuronoglucosaminidase/uso terapêutico , Fígado/efeitos dos fármacos , Células-Tronco Multipotentes/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Tetracloreto de Carbono , Movimento Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Enzimas Imobilizadas/administração & dosagem , Enzimas Imobilizadas/química , Hepatite Animal/metabolismo , Hepatite Animal/patologia , Hialuronoglucosaminidase/administração & dosagem , Hialuronoglucosaminidase/química , Fígado/metabolismo , Fígado/patologia , Camundongos , Células-Tronco Multipotentes/citologia , Nanotecnologia , Ratos , Células Estromais/citologia , Células Estromais/efeitos dos fármacosAssuntos
Enzimas Imobilizadas , Cirurgia Geral/história , Peptídeo Hidrolases , Procedimentos Cirúrgicos Operatórios , Ferimentos e Lesões , Bandagens/história , Bandagens/normas , Enzimas Imobilizadas/administração & dosagem , Enzimas Imobilizadas/história , Enzimas Imobilizadas/uso terapêutico , História do Século XX , História do Século XXI , Humanos , Peptídeo Hidrolases/administração & dosagem , Peptídeo Hidrolases/história , Peptídeo Hidrolases/uso terapêutico , Federação Russa , Procedimentos Cirúrgicos Operatórios/métodos , Procedimentos Cirúrgicos Operatórios/tendências , Ferimentos e Lesões/história , Ferimentos e Lesões/cirurgiaRESUMO
Adenosine deaminase (ADA) deficiency is an inherited disorder which leads to elevated cellular levels of deoxyadenosine triphosphate (dATP) and systemic accumulation of its precursor, 2-deoxyadenosine. These metabolites impair lymphocyte function, and inactivate S-adenosylhomocysteine hydrolase (SAHH) respectively, leading to severe immunodeficiency. Enzyme replacement therapy with polyethylene glycol-conjugated ADA is available, but its efficacy is reduced by anti-ADA neutralising antibody formation. We report here carrier erythrocyte encapsulated native ADA therapy in an adult-type ADA deficient patient. Encapsulated enzyme is protected from antigenic responses and therapeutic activities are sustained. ADA-loaded autologous carrier erythrocytes were prepared using a hypo-osmotic dialysis procedure. Over a 9-yr period 225 treatment cycles were administered at 2-3 weekly intervals. Therapeutic efficacy was determined by monitoring immunological and metabolic parameters. After 9 yr of therapy, erythrocyte dATP concentration ranged between 24 and 44 micromol/L (diagnosis, 234) and SAHH activity between 1.69 and 2.29 nmol/h/mg haemoglobin (diagnosis, 0.34). Erythrocyte ADA activities were above the reference range of 40-100 nmol/h/mg haemoglobin (0 at diagnosis). Initial increases in absolute lymphocyte counts were not sustained; however, despite subnormal circulating CD20(+) cell numbers, serum immunoglobulin levels were normal. The patient tolerated the treatment well. The frequency of respiratory problems was reduced and the decline in the forced expiratory volume in 1 s and vital capacity reduced compared with the 4 yr preceding carrier erythrocyte therapy. Carrier erythrocyte-ADA therapy in an adult patient with ADA deficiency was shown to be metabolically and clinically effective.
Assuntos
Adenosina Desaminase/administração & dosagem , Adenosina Desaminase/deficiência , Enzimas Imobilizadas/administração & dosagem , Imunodeficiência Combinada Severa/tratamento farmacológico , Imunodeficiência Combinada Severa/enzimologia , Adenosina Desaminase/imunologia , Adenosil-Homocisteinase/imunologia , Adenosil-Homocisteinase/metabolismo , Adulto , Antígenos CD20/sangue , Antígenos CD20/imunologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Nucleotídeos de Desoxiadenina/imunologia , Nucleotídeos de Desoxiadenina/metabolismo , Eritrócitos/enzimologia , Eritrócitos/imunologia , Feminino , Fluxo Expiratório Forçado/efeitos dos fármacos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Pneumopatias/enzimologia , Pneumopatias/imunologia , Pneumopatias/fisiopatologia , Contagem de Linfócitos , Polietilenoglicóis/administração & dosagem , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/fisiopatologia , Fatores de TempoRESUMO
We describe the effects of polyethylene glycol-conjugated adenosine deaminase (ADA) replacement therapy on lymphocyte counts, activation, apoptosis, proliferation, and cytokine secretion in a 14-month-old girl with "delayed-onset" ADA deficiency and marked immunodysregulation. Pretreatment lymphopenia affected T cells (CD4, 150/microl; CD8, 459/microl), B cells (16/microl), and NK cells (55/microl). T cells were uniformly activated and largely apoptotic (CD4, 59%; CD8, 82%); and T-cell-dependent cytokine levels in plasma were elevated, including the levels of interleukin 2 (IL-2; 26 pg/ml), IL-4 (81 pg/ml), IL-5 (46 pg/ml), gamma interferon (1,430 pg/ml), tumor necrosis factor alpha (210 pg/ml), and IL-10 (168 pg/ml). Mitogen-stimulated peripheral blood mononuclear cells show reduced IL-2 secretion and proliferation. During the first 5 months of therapy there was clinical improvement and partial immune reconstitution, with nearly normal lymphocyte subset numbers, reduced T-cell activation and CD4-cell apoptosis, and decreased plasma cytokine levels. In parallel, IL-2 secretion and the lymphocyte mitogenic response improved. Between 4 and 7 months, immunoglobulin G antibodies to bovine ADA developed and resulted in the complete reversal of immune recovery.
Assuntos
Adenosina Desaminase/administração & dosagem , Enzimas Imobilizadas/administração & dosagem , Polietilenoglicóis/administração & dosagem , Erros Inatos do Metabolismo da Purina-Pirimidina/tratamento farmacológico , Adenosina Desaminase/deficiência , Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Citocinas/sangue , Feminino , Humanos , Lactente , Contagem de Leucócitos , Ativação Linfocitária/efeitos dos fármacos , Erros Inatos do Metabolismo da Purina-Pirimidina/sangue , Subpopulações de Linfócitos T/metabolismoRESUMO
Cardosin A is extracted from the pistils of the plant Cynara cardunculus L. and chitosan is a polysaccharide derived from chitin with valuable properties as a biomaterial. In this work we report our experiments on the synthesis of chitosan sponges and immobilisation of cardosin A, by entrapment. We observed that 10-15% of the incorporated cardosin A were released over 6 days of incubation. In addition, we could also note that this immobilisation procedure did not induce any specificity alterations on cardosin A. The specificity study of the enzyme, using beta-chain of oxidised insulin, showed that the immobilised and released enzymes have the same hydrolysis pattern as the free enzyme. The ability of this enzyme to hydrolyse type I collagen was maintained, after the immobilisation procedure. The biocompatibility in vivo of these sponges was evaluated by histological staining after implantation in rats submitted to abdominal surgery. Results of this study demonstrated that these chitosan sponges are very promising vehicles for the application of cardosin A, in abdominal cavity for prevention and reduction of the adhesions formation.
Assuntos
Ácido Aspártico Endopeptidases/administração & dosagem , Quitosana/administração & dosagem , Sistemas de Liberação de Medicamentos , Implantes de Medicamento , Enzimas Imobilizadas/administração & dosagem , Proteínas de Plantas/administração & dosagem , Animais , Ácido Aspártico Endopeptidases/química , Biodegradação Ambiental , Colágeno Tipo I/química , Enzimas Imobilizadas/química , Feminino , Hidrólise , Insulina/química , Proteínas de Plantas/química , Ratos , Ratos WistarRESUMO
BACKGROUND: The purpose of this study is to report the incidence of pancreatitis in patients treated with pegaspargase in our hospital during a 2-year period. PROCEDURE: We identified episodes of pancreatitis related to the intramuscular administration of pegaspargase 2,500 IU/m(2) for the treatment of childhood hematological malignancies during a 2-year period (May 1996-April 1998). Patients were evaluated clinically and by sequential serum amylase and lipase determinations and radiographic examinations. For comparison, episodes of pancreatitis in patients who only received native Escherichia coli L-asparaginase were examined during the same time period. RESULTS: Nine children with acute lymphoblastic leukemia (ALL) of 50 (18%) patients who received pegaspargase were diagnosed to have pancreatitis. All had prior therapy with native L-asparaginase. These children developed symptoms consisting of abdominal pain, nausea, vomiting, and decreased appetite within a median of 15 days from the onset of pegaspargase administration. Six patients became symptomatic after their initial dose. Seven patients developed severe or unacceptable toxicity (grades 3 and 4), measured by increased amylase (>2 times normal) and lipase levels or radiographic evidence of pancreatic inflammation or pseudocyst. One patient also developed hyperammonemia and encephalopathy. In contrast, only one out of 52 (1.9%) ALL patients who received native E. coli L-asparaginase during the same time period developed pancreatitis (P= 0.007). CONCLUSION: Clinicians should be aware of a possible higher incidence of pancreatitis associated with pegaspargase.
Assuntos
Antineoplásicos/efeitos adversos , Asparaginase/efeitos adversos , Enzimas Imobilizadas/efeitos adversos , Pancreatite/induzido quimicamente , Polietilenoglicóis/efeitos adversos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adolescente , Antineoplásicos/administração & dosagem , Asparaginase/administração & dosagem , Criança , Pré-Escolar , Enzimas Imobilizadas/administração & dosagem , Feminino , Humanos , Injeções Intramusculares , Masculino , Pancreatite/diagnóstico por imagem , Pancreatite/enzimologia , Polietilenoglicóis/administração & dosagem , RadiografiaRESUMO
BACKGROUND: L-asparaginase in combination with methotrexate has synergistic antileukemic activity in a schedule-dependent fashion. A new preparation of L-asparaginase, polyethylene-glycol conjugated (PEG)-asparaginase, is a pharmacologically different formulation of L-asparaginase with distinct properties including a longer half-life and less immunogenic properties. METHODS: Patients with refractory or recurrent acute lymphoblastic leukemia (ALL) were treated with a combination of methotrexate (MTX), vincristine, PEG-asparaginase, and prednisone (MOAP). The treatment was comprised of MTX, 100 mg/m(2) intravenously (i.v.), over 15 minutes on Days 1 and 14; PEG-asparaginase, 2500 IU/m(2), with a maximum dose of 3750 IU i.v. approximately 4-6 hours after MTX on Days 1 and 14; vincristine, 1.4 mg/m(2) (maximum dose, 2 mg) i.v., over 15 minutes on Days 1, 7, and 14; and prednisone, 200 mg daily orally, on Days 1-5 and 14-19. RESULTS: Thirty-two patients with a median age of 34 years (range, 20-74 years) were treated. Eight patients (25%) had ALL that was refractory to prior therapy and 24 patients (75%) had recurrent disease. Seven patients (22%) achieved a complete remission (CR). Five patients (16%) died early due to infections. Features associated with a poor response were high pretreatment lactate dehydrogenase levels and Philadelphia chromosome positive disease. The median duration of CR was 16 weeks and the overall median survival after MOAP therapy was 12 weeks. Anaphylactic reactions were not observed during MOAP combination therapy. CONCLUSIONS: MOAP is an active regimen in patients with refractory or recurrent ALL. This regimen is well tolerated and is not associated with allergic reactions. However, further studies regarding the pharmacologic interaction of MTX with PEG-asparaginase are needed to optimize this regimen.