RESUMEN
Spatial analysis of microbiomes at single cell resolution with high multiplexity and accuracy has remained challenging. Here we present spatial profiling of a microbiome using sequential error-robust fluorescence in situ hybridization (SEER-FISH), a highly multiplexed and accurate imaging method that allows mapping of microbial communities at micron-scale. We show that multiplexity of RNA profiling in microbiomes can be increased significantly by sequential rounds of probe hybridization and dissociation. Combined with error-correction strategies, we demonstrate that SEER-FISH enables accurate taxonomic identification in complex microbial communities. Using microbial communities composed of diverse bacterial taxa isolated from plant rhizospheres, we apply SEER-FISH to quantify the abundance of each taxon and map microbial biogeography on roots. At micron-scale, we identify clustering of microbial cells from multiple species on the rhizoplane. Under treatment of plant metabolites, we find spatial re-organization of microbial colonization along the root and alterations in spatial association among microbial taxa. Taken together, SEER-FISH provides a useful method for profiling the spatial ecology of complex microbial communities in situ.
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Microbiota , Hibridación Fluorescente in Situ/métodos , Microbiota/genética , Bacterias , ARNRESUMEN
Neutrophils are predominant leukocytes in the circulation, which are essential for killing invading pathogens via the activation of effector responses and the production of reactive oxygen species (ROS), also named as "oxidative burst." When infected, activated neutrophils fight bacteria, fungi, and viruses through oxidative burst, phagocytosis, degranulation, and the production of neutrophil extracellular traps (NETs) in a neutrophil death process named as "NETosis" (Mutua and Gershwin, 2021). NETs, consisting of DNA fibers decorated with modified histones and numerous antimicrobial proteins from cytoplasmic granules and the nucleus, can either be beneficial or detrimental (Mutua and Gershwin, 2021). Several pathways can lead to this death process. In response to various stimuli, NETosis traps and clears pathogens, facilitating phagocytosis by other neutrophils and phagocytes. However, excessive NETosis often results in disease due to increasing the pro-inflammatory response and perpetuating the inflammatory condition (Hellebrekers et al., 2018; Hidalgo et al., 2019; Klopf et al., 2021). Accordingly, inhibiting aberrant NETosis may alleviate the severity of various autoimmune and inflammatory diseases.
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Trampas Extracelulares , Estallido Respiratorio , ADN , Trampas Extracelulares/metabolismo , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Oxidative stress and endoplasmic reticulum (ER) stress play crucial roles in pancreatic ß cell destruction, leading to the development and progression of type 1 diabetes mellitus (T1DM). Curcumin, extracted from plant turmeric, possesses multiple bioactivities such as antioxidant, anti-inflammatory and anti-apoptosis properties and . However, it remains unknown whether curcumin improves ER stress to prevent ß cells from apoptosis. In this study, we aim to investigate the role and mechanism of curcumin in ameliorating HO-induced injury in MIN6 (a mouse insulinoma cell line) cells. Cell viability is examined by CCK8 assay. Hoechst 33258 staining, TUNEL and flow cytometric assay are performed to detect cell apoptosis. The relative amounts of reactive oxygen species (ROS) are measured by DCFH-DA. WST-8 is used to determine the total superoxide dismutase (SOD) activity. Protein expressions are determined by western blot analysis and immunofluorescence staining. Pretreatment with curcumin prevents MIN6 cells from HO-induced cell apoptosis. Curcumin decreases ROS generation and inhibits protein kinase like ER kinase (PERK)-C/EBP homologous protein (CHOP) signaling axis, one of the critical branches of ER stress pathway. Moreover, incubation with curcumin activates silent information regulator 1 (SIRT1) expression and subsequently decreases the expression of CHOP. Additionally, EX527, a specific inhibitor of SIRT1, blocks the protective effect of curcumin on MIN6 cells exposed to HO. In sum, curcumin inhibits the PERK-CHOP pathway of ER stress mediated by SIRT1 and thus ameliorates HO-induced MIN6 cell apoptosis, suggesting that curcumin and SIRT1 may provide a potential therapeutic approach for T1DM.
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Curcumina , Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Animales , Apoptosis , Curcumina/farmacología , Curcumina/uso terapéutico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Estrés del Retículo Endoplásmico , Células Secretoras de Insulina/metabolismo , Ratones , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 1/metabolismo , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismoRESUMEN
Automatic classification of retinal arteries and veins plays an important role in assisting clinicians to diagnosis cardiovascular and eye-related diseases. However, due to the high degree of anatomical variation across the population, and the presence of inconsistent labels by the subjective judgment of annotators in available training data, most of existing methods generally suffer from blood vessel discontinuity and arteriovenous confusion, the artery/vein (A/V) classification task still faces great challenges. In this work, we propose a multi-scale interactive network with A/V discriminator for retinal artery and vein recognition, which can reduce the arteriovenous confusion and alleviate the disturbance of noisy label. A multi-scale interaction (MI) module is designed in encoder for realizing the cross-space multi-scale features interaction of fundus images, effectively integrate high-level and low-level context information. In particular, we also design an ingenious A/V discriminator (AVD) that utilizes the independent and shared information between arteries and veins, and combine with topology loss, to further strengthen the learning ability of model to resolve the arteriovenous confusion. In addition, we adopt a sample re-weighting (SW) strategy to effectively alleviate the disturbance from data labeling errors. The proposed model is verified on three publicly available fundus image datasets (AV-DRIVE, HRF, LES-AV) and a private dataset. We achieve the accuracy of 97.47%, 96.91%, 97.79%, and 98.18% respectively on these four datasets. Extensive experimental results demonstrate that our method achieves competitive performance compared with state-of-the-art methods for A/V classification. To address the problem of training data scarcity, we publicly release 100 fundus images with A/V annotations to promote relevant research in the community.
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Arteria Retiniana , Vasos Retinianos , Fondo de Ojo , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Vasos Retinianos/diagnóstico por imagenRESUMEN
Pyroptosis is identified as a pro-inflammatory programmed cell death, mediated by gasdermins (GSDMs) family of proteins accompanied by pro-inflammatory signals release. As essential players in innate immunity, inflammasomes are intracellular protein complexes which cleave gasdermin D (GSDMD), forming structurally stable pores in the cell membrane, subsequently inducing pyroptosis. Extensive evidence indicates that inflammasomes and pyroptosis contributes to tumors, nerve injury, inflammatory diseases and metabolic disorders. As a metabolic disorder, diabetes is characterized with hyperglycemia, insulin resistance and chronic inflammation. Meanwhile, aberrant pyroptosis exerts a key role in the occurrence and progression of diabetes and its common complication, diabetic nephropathy (DN). Furthermore, evidence has shown that DN patients and animal models exhibit increased circulating IL-1ß and inflammasome, while decreasing the expression of key components of the inflammasome mitigates kidney damage and delays progression. Current research has reported that non-coding RNAs (ncRNAs) are involved in activation of inflammasomes and play a crucial role in the control of pyroptosis in DN pathogenesis. In addition, studies have indicated that some natural plant compounds have therapeutic potential via regulation of inflammasomes and pyroptosis to prevent and potentially treat DN. This mini-review examines the molecular mechanism of inflammasome activation and pyroptosis, highlights the critical roles of ncRNA and explores potential therapeutics to regulate pyroptosis in DN.
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Diabetes Mellitus , Nefropatías Diabéticas , Animales , Humanos , Inflamasomas/metabolismo , Inflamación/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Piroptosis/genéticaRESUMEN
Type 1 diabetes (T1D) is an autoimmune and inflammatory disease with excessive loss of pancreatic islet [Formula: see text]-cells. Accumulating evidence indicated that endoplasmic reticulum (ER) stress played a critical role in [Formula: see text]-cells loss, leading to T1D. Therefore, promoting the survival of pancreatic [Formula: see text]cells would be beneficial for patients with T1D. Puerarin is a natural isoflavone that has been demonstrated to be able to decrease blood glucose in patients with T1D. However, it remains unknown whether puerarin improves ER stress to prevent [Formula: see text]-cells from apoptosis. Here, we sought to investigate the role of puerarin in ER stress-associated apoptosis and explore its underlying mechanism in the mouse insulinoma cell line (MIN6). Flow cytometry and cell counting kit-8 (CCK8) experiments showed that puerarin caused a significant increase in the viability of MIN6 cells injured by H2O2. Furthermore, the protein kinase R-like ER kinase (PERK) signal pathway, a critical branch of ER stress response, was found to be involved in this process. Puerarin inhibited the phosphorylation of PERK, subsequently suppressed the phosphorylation of eukaryotic initiation factor 2[Formula: see text] (eIF2[Formula: see text], then decreased the activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression, ultimately attenuating ER stress to prevent MIN6 cells from apoptosis. In addition, puerarin inhibited the activation of Janus kinase 2 (JAK2)/signal transducer and activators of transcription 3 (STAT3), which suppressed the PERK signal cascade with decreased ATF4 and CHOP levels. Taken together, our results firstly demonstrated that puerarin could prevent MIN6 cells from apoptosis at least in part by inhibiting the PERK-eIF2[Formula: see text]-ATF4-CHOP axis under ER stress conditions, which might be mediated by inactivation of the JAK2/STAT3 signal pathway. Therefore, investigating the mechanism underlying the effects of puerarin might highlight the potential roles of puerarin developing into an antidiabetic drug.
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Células Secretoras de Insulina/efectos de los fármacos , Isoflavonas/farmacología , Factor de Transcripción Activador 4/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Janus Quinasa 2/metabolismo , Ratones , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción CHOP/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
Retinal blood vessel morphological abnormalities are generally associated with cardiovascular, cerebrovascular, and systemic diseases, automatic artery/vein (A/V) classification is particularly important for medical image analysis and clinical decision making. However, the current method still has some limitations in A/V classification, especially the blood vessel edge and end error problems caused by the single scale and the blurred boundary of the A/V. To alleviate these problems, in this work, we propose a vessel-constraint network (VC-Net) that utilizes the information of vessel distribution and edge to enhance A/V classification, which is a high-precision A/V classification model based on data fusion. Particularly, the VC-Net introduces a vessel-constraint (VC) module that combines local and global vessel information to generate a weight map to constrain the A/V features, which suppresses the background-prone features and enhances the edge and end features of blood vessels. In addition, the VC-Net employs a multiscale feature (MSF) module to extract blood vessel information with different scales to improve the feature extraction capability and robustness of the model. And the VC-Net can get vessel segmentation results simultaneously. The proposed method is tested on publicly available fundus image datasets with different scales, namely, DRIVE, LES, and HRF, and validated on two newly created multicenter datasets: Tongren and Kailuan. We achieve a balance accuracy of 0.9554 and F1 scores of 0.7616 and 0.7971 for the arteries and veins, respectively, on the DRIVE dataset. The experimental results prove that the proposed model achieves competitive performance in A/V classification and vessel segmentation tasks compared with state-of-the-art methods. Finally, we test the Kailuan dataset with other trained fusion datasets, the results also show good robustness. To promote research in this area, the Tongren dataset and source code will be made publicly available. The dataset and code will be made available at https://github.com/huawang123/VC-Net.
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Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Humanos , Células Secretoras de Insulina/patología , Proteínas de Transporte de Membrana/genética , Mitocondrias/genéticaRESUMEN
Endoplasmic reticulum (ER) stress plays a critical role in pancreatic ß cell destruction which leads to the pathogenesis of type 1 diabetes mellitus (T1DM). Vitamin D (VD) has been reported to reduce the risk of T1DM; however, it remains unknown whether VD affects ER stress in pancreatic ß cells. In this study, we investigated the role of the active form of VD, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], in ER stress-induced ß cell apoptosis and explored its potential mechanism in mouse insulinoma cell line mouse insulinoma 6 (MIN6). The results of cell counting kit-8 (CCK8) and flow cytometric analyses showed that 1,25-(OH)2D3 caused a significant increase in the viability of MIN6 cells injured by H2O2. The protein kinase like ER kinase (PERK) signal pathway, one of the most conserved branches of ER stress, was found to be involved in this process. H2O2 activated the phosphorylation of PERK, upregulated the activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression, and subsequently initiated cell apoptosis, which were significantly reversed by 1,25-(OH)2D3 pretreatment. In addition, GSK2606414, a specific inhibitor of PERK, suppressed PERK phosphorylation and reduced the expressions of ATF4 and CHOP, leading to a significant decrease in ß cell apoptosis induced by H2O2. Taken together, the present findings firstly demonstrated that 1,25-(OH)2D3 could prevent MIN6 cells against ER stress-associated apoptosis by inhibiting the PERK-ATF4-CHOP pathway. Therefore, our results suggested that 1,25-(OH)2D3 might serve as a potential therapeutic target for preventing pancreatic ß cell destruction in T1DM.
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Factor de Transcripción Activador 4/antagonistas & inhibidores , Calcitriol/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Sustancias Protectoras/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Transcripción CHOP/antagonistas & inhibidores , eIF-2 Quinasa/antagonistas & inhibidores , Adenina/análogos & derivados , Adenina/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Indoles/farmacología , Células Secretoras de Insulina/citología , RatonesRESUMEN
Type 1 diabetes (T1D) results from dysfunction of pancreatic islets ß cells. Recent studies supported that endoplasmic reticulum (ER) stress takes an important role in pancreatic ß cell excessive loss, resulting in T1D. Here, we aimed to review the relationship between ER stress and T1D. Additionally, we also reviewed the potential mechanisms underlying ER stress mediated T1D. Studies have shown that severe ER stress is directly involved in the pancreatic ß cells destruction and pathogenesis of T1D. ER stress plays a key part in pancreatic ß cells and T1D, which will help in developing new effective therapeutics for T1D.
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Diabetes Mellitus Tipo 1/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Células Secretoras de Insulina/metabolismo , Animales , HumanosRESUMEN
ABSTRACT Obesity is defined as a chronic and excessive growth of adipose tissue. It has been associated with a high risk for development and progression of obesity-associated malignancies, while adipokines may mediate this association. Adiponectin is an adipose tissue-derived adipokines, with significant anti-diabetic, anti-inflammatory, anti-atherosclerotic and anti-proliferative properties. Plasma adiponectin levels are decreased in obese individuals, and this feature is closely correlated with development of several metabolic, immunological and neoplastic diseases. Recent studies have shown that prostate cancer patients have lower serum adiponectin levels and decreased expression of adiponectin receptors in tumor tissues, which suggests plasma adiponectin level is a risk factor for prostate cancer. Furthermore, exogenous adiponectin has exhibited therapeutic potential in animal models. In this review, we focus on the potential role of adiponectin and the underlying mechanism of adiponectin in the development and progression of prostate cancer. Exploring the signaling pathways linking adiponectin with tumorigenesis might provide a potential target for therapy.
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Humanos , Animales , Masculino , Neoplasias de la Próstata/sangre , Adiponectina/sangre , Receptores de Adiponectina/sangre , Neoplasias de la Próstata/etiología , Neoplasias de la Próstata/patología , Tejido Adiposo , Factores de Riesgo , Progresión de la Enfermedad , Modelos Animales de Enfermedad , Obesidad/complicacionesRESUMEN
Obesity is defined as a chronic and excessive growth of adipose tissue. It has been associated with a high risk for development and progression of obesity-associated malignancies, while adipokines may mediate this association. Adiponectin is an adipose tissue-derived adipokines, with significant anti-diabetic, anti-inflammatory, anti-atherosclerotic and anti-proliferative properties. Plasma adiponectin levels are decreased in obese individuals, and this feature is closely correlated with development of several metabolic, immunological and neoplastic diseases. Recent studies have shown that prostate cancer patients have lower serum adiponectin levels and decreased expression of adiponectin receptors in tumor tissues, which suggests plasma adiponectin level is a risk factor for prostate cancer. Furthermore, exogenous adiponectin has exhibited therapeutic potential in animal models. In this review, we focus on the potential role of adiponectin and the underlying mechanism of adiponectin in the development and progression of prostate cancer. Exploring the signaling pathways linking adiponectin with tumorigenesis might provide a potential target for therapy.
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Adiponectina/sangre , Neoplasias de la Próstata/sangre , Receptores de Adiponectina/sangre , Tejido Adiposo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Masculino , Obesidad/complicaciones , Neoplasias de la Próstata/etiología , Neoplasias de la Próstata/patología , Factores de RiesgoRESUMEN
Extensive investigations have been carried out for evaluating the toxicology of various nanomaterials (e.g., carbon- and metal-based nanomaterials), which offer invaluable information for assessing the feasibility of nanomaterial-based wide-ranging applications. In recent years, sufficient efforts have been made to develop fluorescent small-sized silicon nanoparticles (SiNPs) as a novel optical material simultaneously featuring strong fluorescence and ultrahigh photostability, providing high promise for a myriad of biological, biomedical and electronic applications. It is worth pointing out that, despite the non- or low-toxicity of silicon, sufficient and objective toxicology evaluation of SiNPs is urgently required at both the in vitro and in vivo levels. However, there currently exists scanty information about the intracellular behaviors of the SiNPs, particularly the underlying mechanism of entry into cells and intracellular fate. Herein, we present a report aimed at determining the uptake and intracellular transport of SiNPs of ca. 4 nm diameter. Taking advantage of the strong and stable fluorescent signals of SiNPs, we reveal that these small-sized SiNPs accumulate in the plasma membrane prior to internalization, and are further internalized predominantly by clathrin-mediated and caveolae-dependent endocytosis. After endocytosis, the SiNPs are localized in early endosomes within a short time (â¼1 h), while in up to 24 h of incubation the SiNPs are mainly transported to lysosomes in a microtubule-dependent way; and interestingly, to a smaller extent are sorted to the Golgi apparatus. Moreover, we demonstrate that there are no toxic effects of SiNPs on the cell metabolic activity and integrity of the plasma membrane.
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Endocitosis , Fluorescencia , Nanopartículas/toxicidad , Silicio/toxicidad , Membrana Celular , Aparato de Golgi , Células HeLa , Humanos , Lisosomas , Silicio/farmacocinéticaRESUMEN
A phytochemical study on the whole plant of Spermacoce latifolia led to the isolation of a new anthraquinone, 1,2,6-trihydroxy-5-methoxy-9,10-anthraquinone (1), and a new naphthoquinone, (2R)-6-hydroxy-7-methoxy-dehydroiso-α-lapachone (2), together with three known anthraquinones (3-5). Their structures were established on the basis of detailed spectroscopic analysis, including one- and two-dimensional NMR, ESI-MS, and HR-ESI-MS techniques. All the compounds were isolated from S. latifolia for the first time. Compounds 1, 2, 4, and 5 showed significant antibacterial activity toward Bacillus subtilis with MIC values ranging from 0.9 to 31.2 µg/ml, and compound 4 aslo exhibited antibacterial activity against Bacillus cereus with a MIC value 62.5 µg/ml. Compound 1 was further revealed to show significant in vitro α-glucosidase inhibitory activity with IC50 value of 0.653 mM.
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Antraquinonas/aislamiento & purificación , Antibacterianos/aislamiento & purificación , Medicamentos Herbarios Chinos/aislamiento & purificación , Inhibidores de Glicósido Hidrolasas/aislamiento & purificación , Naftoquinonas/aislamiento & purificación , Rubiaceae/química , Antraquinonas/química , Antraquinonas/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Bacillus cereus/efectos de los fármacos , Bacillus subtilis/efectos de los fármacos , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Escherichia coli/efectos de los fármacos , Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/farmacología , Concentración 50 Inhibidora , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Naftoquinonas/química , Naftoquinonas/farmacología , Salmonella typhimurium/efectos de los fármacos , Shigella dysenteriae/efectos de los fármacos , alfa-Glucosidasas/efectos de los fármacosRESUMEN
LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN-γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ-induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ - induced pancreatic beta cell destruction. LIGHT and IFN-γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V(+) cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF-κB activation, the combination of LIGHT and IFN-γ caused an obvious decrease in expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, but an increase in expression of the pro-apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF-κB activation and Bak expression, and peri-insulitis in non-obese diabetes mice. Inhibition of NF-κB activation with the specific NF-κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl-xL down-regulation and Bax up-regulation, and led to a significant increase in LIGHT- and IFN-γ-treated cell viability. Moreover, cleaved caspase-9, -3, and PARP (poly (ADP-ribose) polymerase) were observed after LIGHT and IFN-γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT- and IFNγ-induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN-γ induces beta cells apoptosis via an NF-κB/Bcl2-dependent mitochondrial pathway.
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Apoptosis/efectos de los fármacos , Interferón gamma/farmacología , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/farmacología , Animales , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citocromos c/metabolismo , Activación Enzimática/efectos de los fármacos , Femenino , Células Secretoras de Insulina/metabolismo , Ratones Endogámicos NOD , Mitocondrias/efectos de los fármacos , Modelos Biológicos , Proteínas Recombinantes de Fusión/metabolismo , Estrés Fisiológico/efectos de los fármacosRESUMEN
Recombinant immunotoxin HA22, composed of an anti- CD22 Fv fragment fused to PE38, a truncated portion of Pseudomonas Exotoxin A (PE), has been developed for targeted treatment of various B-cell malignancies. As a foreign, internalized macromolecule, PE38 often induces lysosomal degradation and neutralizing antibodies to limit the efficacy of treating B-cell malignancies. The region of PE38 containing lysosomal protease cleavage sites deleted, leaving only furin processing site. The resulting immunotoxin HA22-LR (lysosome resistant) retains excellent biologic activity and removes immunogenic epitopes as an additional benefit. Another approach for avoiding immunogenicity is to identify B-cell epitopes and remove them by mutagenesis. Previously, to determine B-cell epitopes on PE38, murine Ab as a model, 7 major mouse-specific B-cell epitope groups with 13 subgroups were identified and located through a series of point mutations. Two new mutants, HA22-8X and HA22-LR-8X, were prepared, containing 8 epitope-silencing mutations which greatly reduced immunogenicity in mice. Later, by phage-display assay, human Fvs against PE toxin were isolated and human-specific B-cell epitopes were located by alanine scanning mutagenesis. HA22-LR as a scaffold, HA22-LR-LO10 with 7 point mutations was constructed, has low reactivity with human antisera, yet has high cytotoxic and antitumor activity. In this review, theoretical aspects and experimental evidence for the removal of B-cell epitope is discussed.