RESUMO
Interleukin-2 (IL-2) engineered versions, with biased immunological functions, have emerged from yeast display and rational design. Here we reshaped the human IL-2 interface with the IL-2 receptor beta chain through the screening of phage-displayed libraries. Multiple beta super-binders were obtained, having increased receptor binding ability and improved developability profiles. Selected variants exhibit an accumulation of negatively charged residues at the interface, which provides a better electrostatic complementarity to the beta chain, and faster association kinetics. These findings point to mechanistic differences with the already reported superkines, characterized by a conformational switch due to the rearrangement of the hydrophobic core. The molecular bases of the favourable developability profile were tracked to a single residue: L92. Recombinant Fc-fusion proteins including our variants are superior to those based on H9 superkine in terms of expression levels in mammalian cells, aggregation resistance, stability, in vivo enhancement of immune effector responses, and anti-tumour effect.
Assuntos
Evolução Molecular Direcionada , Subunidade beta de Receptor de Interleucina-2 , Interleucina-2 , Biblioteca de Peptídeos , Humanos , Subunidade beta de Receptor de Interleucina-2/química , Interleucina-2/química , Interleucina-2/genética , Interleucina-2/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Evolução Molecular Direcionada/métodos , Domínios Proteicos , Animais , Camundongos , Linhagem Celular TumoralRESUMO
High doses of interleukin-2 (IL-2) have been used for the treatment of melanoma and renal cell carcinoma, but this therapy has limited efficacy, with a ~15% response rate. Remarkably, 7%-9% of patients achieve complete or long-lasting responses. Many patients treated with IL-2 experienced an expansion of regulatory T cells (Tregs), specifically the expansion of ICOS+ highly suppressive Tregs, which correlate with worse clinical outcomes. This partial efficacy together with the high toxicity associated with the therapy has limited the use of IL-2-based therapy. Taking into account the understanding of IL-2 structure, signaling, and in vivo functions, some efforts to improve the cytokine properties are currently under study. In previous work, we described an IL-2 mutein with higher antitumor activity and less toxicity than wtIL-2. Mutein was in silico designed for losing the binding capacity to CD25 and for preferential stimulation of effector cells CD8+ and NK cells but not Tregs. Mutein induces a higher anti-metastatic effect than wtIL-2, but the extent of the in vivo antitumor activity was still unexplored. In this work, it is shown that mutein induces a strong antitumor effect on four primary tumor models, being effective even in those models where wtIL-2 does not work. Furthermore, mutein can change the in vivo balance between Tregs and T CD8+ memory/activated cells toward immune activation, in both healthy and tumor-bearing mice. This change reaches the tumor microenvironment and seems to be the major explanation for mutein efficacy in vivo.
Assuntos
Linfócitos T CD8-Positivos , Interleucina-2 , Neoplasias , Linfócitos T Reguladores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Imunoterapia , Interleucina-2/genética , Interleucina-2/imunologia , Melanoma , Camundongos , Mutação , Neoplasias/tratamento farmacológico , Linfócitos T Reguladores/imunologia , Microambiente TumoralRESUMO
Elevated levels of immunoglobulin E (IgE) are associated with allergies and other immunological disorders. Sensitization with alum adjuvant favours IgE production while CpG-ODN adjuvant, a synthetic toll-like receptor 9 (TLR9) agonist, inhibits it. The cellular mechanisms underlying in vivo TLR regulation of immunoglobulin production, specially IgE, are still controversial. Specifically, TLR-mediated IgE regulation in vivo is not yet known. In this study we showed that augmented levels of IgE induced by sensitizations to OVA with or without alum adjuvant or with OVA-pulsed dendritic cells (DCs) were inhibited by co-administration of CpG. Notably, CpG-mediated suppression of IgE production required MyD88-expression on DCs but not on B-cells. This finding contrasts with previous in vitro studies reporting regulation of IgE by a direct action of CpG on B cells via MyD88 pathway. In addition, we showed that CpG also inhibited IgE production in a MyD88-dependent manner when sensitization was performed with OVA-pulsed DCs. Finally, CpG signalling through MyD88 pathway was also necessary and sufficient to prevent anaphylactic antibody production involved in active cutaneous anaphylaxis.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Células Dendríticas/imunologia , Imunoglobulina E/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Oligodesoxirribonucleotídeos/administração & dosagem , Ovalbumina/efeitos adversos , Hipersensibilidade Respiratória/tratamento farmacológico , Adjuvantes Imunológicos/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoglobulina E/efeitos dos fármacos , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Oligodesoxirribonucleotídeos/farmacologia , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Selection from a phage display library derived from human Interleukin-2 (IL-2) yielded mutated variants with greatly enhanced display levels of the functional cytokine on filamentous phages. Introduction of a single amino acid replacement selected that way (K35E) increased the secretion levels of IL-2-containing fusion proteins from human transfected host cells up to 20-fold. Super-secreted (K35E) IL-2/Fc is biologically active in vitro and in vivo, has anti-tumor activity and exhibits a remarkable reduction in its aggregation propensity- the major manufacturability issue limiting IL-2 usefulness up to now. Improvement of secretion was also shown for a panel of IL-2-engineered variants with altered receptor binding properties, including a selective agonist and a super agonist that kept their unique properties. Our findings will improve developability of the growing family of IL-2-derived immunotherapeutic agents and could have a broader impact on the engineering of structurally related four-alpha-helix bundle cytokines.
Assuntos
Substituição de Aminoácidos , Antineoplásicos/farmacologia , Interleucina-2/genética , Receptores Fc/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Visualização da Superfície Celular , Sobrevivência Celular/efeitos dos fármacos , Evolução Molecular , Humanos , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Engenharia de Proteínas , Receptores Fc/genéticaRESUMO
OBJECTIVE: To evaluate the accuracy of fetal weight prediction by ultrasonography labor employing a formula including the linear measurements of femur length (FL) and mid-thigh soft-tissue thickness (STT). METHODS: We conducted a prospective study involving singleton uncomplicated term pregnancies within 48 hours of delivery. Only pregnancies with a cephalic fetus admitted in the labor ward for elective cesarean section, induction of labor or spontaneous labor were included. We excluded all non-Caucasian women, the ones previously diagnosed with gestational diabetes and the ones with evidence of ruptured membranes. Fetal weight estimates were calculated using a previously proposed formula [estimated fetal weight = 1687.47 + (54.1 x FL) + (76.68 x STT). The relationship between actual birth weight and estimated fetal weight was analyzed using Pearson's correlation. The formula's performance was assessed by calculating the signed and absolute errors. Mean weight difference and signed percentage error were calculated for birth weight divided into three subgroups: < 3000 g; 3000-4000 g; and > 4000 g. RESULTS: We included for analysis 145 cases and found a significant, yet low, linear relationship between birth weight and estimated fetal weight (p < 0.001; R2 = 0.197) with an absolute mean error of 10.6%. The lowest mean percentage error (0.3%) corresponded to the subgroup with birth weight between 3000 g and 4000 g. CONCLUSIONS: This study demonstrates a poor correlation between actual birth weight and the estimated fetal weight using a formula based on femur length and mid-thigh soft-tissue thickness, both linear parameters. Although avoidance of circumferential ultrasound measurements might prove to be beneficial, it is still yet to be found a fetal estimation formula that can be both accurate and simple to perform.
Assuntos
Peso Fetal , Ultrassonografia Pré-Natal , Peso ao Nascer , Feminino , Humanos , Recém-Nascido , Trabalho de Parto , Gravidez , Estudos ProspectivosRESUMO
Objective To evaluate the accuracy of fetal weight prediction by ultrasonography labor employing a formula including the linear measurements of femur length (FL) and mid-thigh soft-tissue thickness (STT). Methods We conducted a prospective study involving singleton uncomplicated term pregnancies within 48 hours of delivery. Only pregnancies with a cephalic fetus admitted in the labor ward for elective cesarean section, induction of labor or spontaneous labor were included. We excluded all non-Caucasian women, the ones previously diagnosed with gestational diabetes and the ones with evidence of ruptured membranes. Fetal weight estimates were calculated using a previously proposed formula [estimated fetal weight = [1] 1687.47 + (54.1 x FL) + (76.68 x STT). The relationship between actual birth weight and estimated fetal weight was analyzed using Pearson's correlation. The formula's performance was assessed by calculating the signed and absolute errors. Mean weight difference and signed percentage error were calculated for birth weight divided into three subgroups: < 3000 g; 3000-4000g; and > 4000 g. Results We included for analysis 145 cases and found a significant, yet low, linear relationship between birth weight and estimated fetal weight (p < 0.001; R2 = 0.197) with an absolute mean error of 10.6%. The lowest mean percentage error (0.3%) corresponded to the subgroup with birth weight between 3000 g and 4000 g. Conclusions This study demonstrates a poor correlation between actual birth weight and the estimated fetal weight using a formula based on femur length and mid-thigh soft-tissue thickness, both linear parameters. Although avoidance of circumferential ultrasound measurements might prove to be beneficial, it is still yet to be found a fetal estimation formula that can be both accurate and simple to perform.
Objetivo Avaliar a precisão da determinação ultrassonográfica da estimativa de peso fetal recorrendo apenas a parâmetros lineares (comprimento do fémur - FL - e espessura de tecido mole a meio da coxa fetal - STT), no período precedente ao parto. Métodos Realizamos umestudo prospectivo que incluiu gestações simples de termo, comfeto cefálico, nas quais o parto ocorreu nas 48h seguintes à avaliação ecográfica. A inclusão no estudo foi feita nomomento de admissão ao bloco de partos para cesariana eletiva, indução do trabalho de parto ou trabalho de parto espontâneo. Foram excluídas todas as grávidas não caucasianas, com diagnóstico de diabetes gestacional ou evidência de rotura de membranas. A estimativa de peso fetal foi calculada através de uma fórmula previamente publicada [estimativa de peso fetal = [1]1687,47 + (54,1 x FL) + (76,68 x STT). A relação entre o peso real e o peso estimado foi analisada através da correlação de Pearson. O desempenho desta fórmula foi avaliado através do cálculo da percentagem de erro absoluto e não absoluto. Os recém-nascidos foram divididos em3 grupos consoante o peso real: < 3000 g; 3000 g - 4000 g; e > 4000 g; para cada grupo foi calculada diferençamédia entre a estimativa de peso e o peso real e a percentagem de erro associada. Resultados Incluímos 145 casos no estudo, cuja estimativa de peso e peso real se correlacionaram significativamente, apesar do valor de correlação ser pouco elevado (p < 0,001; R2 = 0,197). Globalmente, a percentagem de erro absoluto foi 10,6%. A percentagem de erro mais baixa correspondeu ao grupo com peso real entre 3000 g e 4000 g. Conclusões Comeste estudo demonstramos uma correlação fraca entre o peso real e a estimativa de peso fetal ultrassonográfica, quando calculada combase numa fórmula que usa o comprimento do fémur e a espessura de tecido mole a meio da coxa fetal, ambos parâmetros lineares. Ainda que a exclusão de parâmetros circunferenciais no cálculo da estimativa de peso fetal se venha a provar benéfica, esta não parece ser uma fórmula simultaneamente simples e precisa no cálculo da estimativa de peso fetal.
Assuntos
Humanos , Feminino , Gravidez , Recém-Nascido , Peso Fetal , Ultrassonografia Pré-Natal , Peso ao Nascer , Trabalho de Parto , Estudos ProspectivosRESUMO
This study aimed to analyze the contributions of the Primary Healthcare nursing interventions, with primiparae in the promotion of breastfeeding. This is a quasi-experimental, longitudinal study, with a sample consisting of 151 primiparae, who had less than 28 weeks of pregnancy, with the child living for at least six months after the birth, performed between 15 October 2007 and 29 February 2008. Almost all the women initiated breastfeeding, with a sharp decline verified in the prevalence at six months. The mean duration of breastfeeding was 123.8±68.9 days. The intervention that began in the prepartum and continued into the postpartum period, using various strategies (individual consultation, preparation courses for parenting/childbirth, and domicile visits) and intervention contexts (health services and domicile) had significant effects on the duration of breastfeeding, which was not verified in the prevalence.
Assuntos
Aleitamento Materno , Papel do Profissional de Enfermagem , Enfermagem Obstétrica , Adolescente , Adulto , Feminino , Promoção da Saúde , Humanos , Estudos Longitudinais , Gravidez , Atenção Primária à Saúde , Adulto JovemRESUMO
This study aimed to analyze the contributions of the Primary Healthcare nursing interventions, with primiparae in the promotion of breastfeeding. This is a quasi-experimental, longitudinal study, with a sample consisting of 151 primiparae, who had less than 28 weeks of pregnancy, with the child living for at least six months after the birth, performed between 15 October 2007 and 29 February 2008. Almost all the women initiated breastfeeding, with a sharp decline verified in the prevalence at six months. The mean duration of breastfeeding was 123.8±68.9 days. The intervention that began in the prepartum and continued into the postpartum period, using various strategies (individual consultation, preparation courses for parenting/childbirth, and domicile visits) and intervention contexts (health services and domicile) had significant effects on the duration of breastfeeding, which was not verified in the prevalence.
O presente estudo teve como objetivo analisar os contributos das intervenções de enfermeiras de Cuidados de Saúde Primários, com primíparas, na promoção do aleitamento materno. Trata-se de um desenho quase-experimental, longitudinal, com amostra de 151 primíparas, com menos de 28 semanas de gravidez, entre 15 de outubro de 2007 e 29 de fevereiro de 2008, com filhos vivos aos seis meses após o parto. A quase totalidade das mulheres iniciou o aleitamento materno, verificando-se quebra acentuada da prevalência até os seis meses. A duração média do aleitamento materno foi 123,8±68,9 dias. A intervenção que se iniciou no pré-parto e se prolongou para o pós-parto, com diversidade de estratégias (consulta individual; curso de preparação para a parentalidade/parto e visita domiciliária) e contextos de intervenção (serviços de saúde e domicílio), teve efeitos significativos na duração do aleitamento materno, não se verificando na prevalência.
El presente estudio tuvo como objetivo analizar las contribuciones de las intervenciones de enfermeras de Cuidados de Salud Primarios, con primíparas, en la promoción del amamantamiento materno. Se trata de un estudio casi experimental, longitudinal, con una muestra de 151 primíparas, con menos de 28 semanas de embarazo entre 15 de Octubre de 2.007 y 29 de Febrero de 2.008, con hijos vivos después de seis meses del parto. La casi totalidad de las mujeres inició el amamantamiento materno, verificándose una quiebra acentuada de la prevalencia a los seis meses. La duración promedio del amamantamiento materno fue 123,8±68,9 días. La intervención se inició en el preparto y se prolongó para el posparto, con diversidad de estrategias (consulta individual, curso de preparación para la paternidad/parto, y visita domiciliaria) y contextos de intervención (servicios de salud y domicilio) tuvo efectos significativos en la duración del amamantamiento materno, lo que no fue verificando en la prevalencia.