RESUMEN
Background: Monoclonal antibodies (mAbs) and their derivatives are the fastest expanding category of pharmaceuticals. Efficient screening and generation of appropriate therapeutic human antibodies are important and urgent issues in the field of medicine. The successful in vitro biopanning method for antibody screening largely depends on the highly diverse, reliable and humanized CDR library. To rapidly obtain potent human antibodies, we designed and constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library greater than a giga in size by phage display. Herein, the novel TIM-3-neutralizing antibodies with immunomodulatory functions derived from this library serve as an example to demonstrate the library's potential for biomedical applications. Methods: The library was designed with high stability scaffolds and six complementarity determining regions (CDRs) tailored to mimic human composition. The engineered antibody sequences were optimized for codon usage and subjected to synthesis. The six CDRs with variable length CDR-H3s were individually subjected to ß-lactamase selection and then recombined for library construction. Five therapeutic target antigens were used for human antibody generation via phage library biopanning. TIM-3 antibody activity was verified by immunoactivity assays. Results: We have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5 × 1010 phage clones. Three selected TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity by TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Furthermore, clone DCBT3-22 was exceptionally superior with good physicochemical property and a purity of more than 98% without aggregation. Conclusion: The promising results illustrate not only the potential of the DSyn-1 library for biomedical research applications, but also the therapeutic potential of the three novel fully human TIM-3-neutralizing antibodies.
Asunto(s)
Bacteriófagos , Anticuerpos de Cadena Única , Humanos , Biblioteca de Péptidos , Receptor 2 Celular del Virus de la Hepatitis A , Regiones Determinantes de Complementariedad/química , Anticuerpos Monoclonales , Anticuerpos de Cadena Única/genética , Anticuerpos NeutralizantesRESUMEN
BACKGROUNDS: Previous reports revealed that balance and stability are essential for human physical activity. The present study aimed to evaluate the comparative outcome of a novel beginning movement load training (BMLT) versus conventional leg press training in terms of improvement of lower-extremity stability. RESEARCH QUESTION: Does the leg press training improve the stability of lower extremities? METHODS: Twenty adult, asymptomatic and active amateur basketball players were enrolled in this study and randomized in equal numbers into a BMLT group (study group) and a conventional leg press training group (control group). RESULTS: The results after eight-week course of training showed that conventional leg press training had an opposite effect on dynamic stability in the left leg (p = 0.015) and backward movement of the right leg (p = 0.038). The athletes in the study group revealed superior dynamic stability maintenance in both legs backwards (p = 0.001), the left leg (p = 0.013), the right leg backwards (p = 0.022) and overall stability (p = 0.002). SIGNIFICANCE: The study group showed better postural stability in terms of the medial/lateral index (p = 0.017), and the athletes in the control group exhibited a significant inability to maintain postural stability in the medial/lateral aspect after the training (p = 0.016). Rotational BMLT lower-extremity press training yielded superior enhancement of dynamic stability and maintenance of center of gravity and postural stability.
Asunto(s)
Baloncesto , Pierna , Adulto , Humanos , Equilibrio Postural , Extremidad Inferior , AtletasRESUMEN
Promyelocytic leukemia protein (PML) is a tumor suppressor possessing multiple modes of action, including induction of apoptosis. We unexpectedly find that PML promotes necroptosis in addition to apoptosis, with Pml-/- macrophages being more resistant to TNF-mediated necroptosis than wild-type counterparts and PML-deficient mice displaying resistance to TNF-induced systemic inflammatory response syndrome. Reduced necroptosis in PML-deficient cells is associated with attenuated receptor-interacting protein kinase 1 (RIPK1) activation, as revealed by reduced RIPK1[S166] phosphorylation, and attenuated RIPK1-RIPK3-MLKL necrosome complex formation. We show that PML deficiency leads to enhanced TNF-induced MAPK-activated kinase 2 (MK2) activation and elevated RIPK1[S321] phosphorylation, which suppresses necrosome formation. MK2 inhibitor treatment or MK2 knockout abrogates resistance to cell death induction in PML-null cells and mice. PML binds MK2 and p38 MAPK, thereby inhibiting p38-MK2 interaction and MK2 activation. Moreover, PML participates in autocrine production of TNF induced by cellular inhibitors of apoptosis 1 (cIAP1)/cIAP2 degradation, since PML-knockout attenuates autocrine TNF. Thus, by targeting MK2 activation and autocrine TNF, PML promotes necroptosis and apoptosis, representing a novel tumor-suppressive activity for PML.
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Péptidos y Proteínas de Señalización Intracelular , Proteínas Serina-Treonina Quinasas , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Transducción de Señal , Animales , Apoptosis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Necroptosis , Fosforilación , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The functional activities of the tumor suppressor promyelocytic leukemia protein (PML) are mostly associated with its nuclear location. In the present study, we discovered an unexpected role of PML in NLRP3 inflammasome activation. In PML-deficient macrophages, the production of IL-1ß was strongly impaired. The expression of pro-IL-1ß, NLRP3, ASC, and procaspase-1 was not affected in Pml(-/-) macrophages. PML deficiency selectively reduced the processing of procaspase-1. We further showed that PML is required for the assembly of the NLRP3 inflammasome in reconstitution experiment. All PML isoforms were capable of stimulating NLRP3 inflammasome activation. In Pml(-/-) macrophages, the generation of reactive oxygen species and release of mitochondrial DNA were decreased. The involvement of PML in inflammasome activation constitutes an important activity of PML and reveals a new mechanism underlying the inflammasome activation. In addition, downregulation of PML by arsenic trioxide suppressed monosodium urate (MSU)-induced IL-1ß production, suggesting that targeting to PML could be used to treat NLRP3 inflammasome-associated diseases.
Asunto(s)
Proteínas Portadoras/inmunología , Inflamasomas/inmunología , Proteínas Nucleares/inmunología , Factores de Transcripción/inmunología , Proteínas Supresoras de Tumor/inmunología , Animales , Trióxido de Arsénico , Arsenicales/farmacología , Proteínas Portadoras/genética , Caspasa 1/inmunología , Línea Celular , Células Cultivadas , ADN Mitocondrial/inmunología , Regulación hacia Abajo/efectos de los fármacos , Eliminación de Gen , Inhibidores de Crecimiento/farmacología , Humanos , Interleucina-1beta/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas Nucleares/genética , Óxidos/farmacología , Proteína de la Leucemia Promielocítica , Especies Reactivas de Oxígeno/inmunología , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
The molecular process underlying T cell anergy is incompletely understood. Deltex1 (DTX1) is a Notch target with unknown physiological function. Here we show that Dtx1 was a transcription target of nuclear factor of activated T cells (NFAT) and participated in T cell anergy. DTX1 protein was upregulated during T cell anergy, and transgenic expression of Dtx1 attenuated T cell activation. DTX1 inhibited T cell activation by both E3-dependent and E3-independent mechanisms. In addition, DTX1 suppressed T cell activation in the absence of its Notch-binding domain. Importantly, DTX1 regulated the expression of two anergy-associated molecules, growth arrest and DNA-damage-inducible 45 beta (Gadd45 beta) and Cbl-b. DTX1 interacted with early growth response 2 (Egr-2) for optimum expression of Cbl-b. Furthermore, deficiency of DTX1 augmented T cell activation, conferred resistance to anergy induction, enhanced autoantibody generation, and increased inflammation. DTX1 therefore represents a component downstream of calcium-NFAT signaling that regulates T cell anergy.