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Optimization of aptamers in length and chemistry is crucial for industrial applications. Here, we developed aptamers against the SARS-CoV-2 spike protein and achieved optimization with a deep-learning-based algorithm, RaptGen. We conducted a primer-less SELEX against the receptor binding domain (RBD) of the spike with an RNA/DNA hybrid library, and the resulting sequences were subjected to RaptGen analysis. Based on the sequence profiling by RaptGen, a short truncation aptamer of 26 nucleotides was obtained and further optimized by a chemical modification of relevant nucleotides. The resulting aptamer is bound to RBD not only of SARS-CoV-2 wildtype but also of its variants, SARS-CoV-1, and Middle East respiratory syndrome coronavirus (MERS-CoV). We concluded that the RaptGen-assisted discovery is efficient for developing optimized aptamers.
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Aptâmeros de Nucleotídeos , SARS-CoV-2 , Humanos , COVID-19/prevenção & controle , DNA , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Atopic dermatitis (AD) is a chronic inflammatory skin disease where Th2-type immune responses are dominant. In the lesional skin of AD, keratinocytes show differentiation defects and secrete proinflammatory cytokines and chemokines, amplifying Th2-type responses in AD. We previously reported that inducible loss of B-cell lymphoma 6 (Bcl6), a transcription repressor and a master transcriptional regulator of follicular helper T cells and germinal center B cells, in the whole body results in upregulation of Th2-related cytokines in mouse skin. However, the role of Bcl6 in keratinocytes remains to be clarified. Here, we observed that BCL6 positively regulates the expression of keratinocyte differentiation markers and plasma membrane localization of adherence junctional proteins in keratinocyte cell culture. Although keratinocyte-specific loss of Bcl6 alone did not induce AD-like skin inflammation, it aggravates MC903-induced AD-like skin inflammation in mice. In addition, Bcl6 expression is decreased in the epidermis of lesional skin from MC903-induced AD-like skin inflammation in mice. These results strongly suggest that Bcl6 downregulation in keratinocytes contributes to the development and aggravation of AD-like skin inflammation in mice.
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Calcitriol/análogos & derivados , Dermatite Atópica , Camundongos , Animais , Epiderme/metabolismo , Pele/metabolismo , Queratinócitos/metabolismo , Citocinas/metabolismo , Inflamação/patologia , Proteínas Proto-Oncogênicas c-bcl-6/metabolismoRESUMO
Atopic dermatitis (AD) is a common chronic skin disease caused by immune dysfunction, specifically the hyperactivation of Th2 immunity. AD is a complex disease with multiple factors contributing to its development; however, the interaction between these factors is not fully understood. In this study, we demonstrated that the conditional deletion of both the forkhead box p3 (Foxp3) and B-cell lymphoma 6 (Bcl6) genes induced the spontaneous development of AD-like skin inflammation with hyperactivation of type 2 immunity, skin barrier dysfunction, and pruritus, which were not induced by the single deletion of each gene. Furthermore, the development of AD-like skin inflammation was largely dependent on IL-4/13 signaling but not on immunoglobulin E (IgE). Interestingly, we found that the loss of Bcl6 alone increased the expression of thymic stromal lymphopoietin (TSLP) and interleukin (IL)-33 in the skin, suggesting that Bcl6 controls Th2 responses by suppressing TSLP and IL-33 expression in epithelial cells. Our results suggest that Foxp3 and Bcl6 cooperatively suppress the pathogenesis of AD. Furthermore, these results revealed an unexpected role of Bcl6 in suppressing Th2 responses in the skin.
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Dermatite Atópica , Humanos , Citocinas/metabolismo , Pele , Prurido , Linfopoietina do Estroma do Timo , Inflamação/metabolismoRESUMO
(+)-Plakevulin A (1), an oxylipin isolated from an Okinawan sponge Plakortis sp. inhibits enzymatic inhibition of DNA polymerases (pols) α and δ and exhibits cytotoxicity against murine leukemia (L1210) and human cervix carcinoma (KB) cell lines. However, the half-maximal inhibitory concentration (IC50) value for cytotoxicity significantly differed from those observed for the enzymatic inhibition of pols α and ß, indicating the presence of target protein(s) other than pols. This study demonstrated cytotoxicity against human promyelocytic leukemia (HL60), human cervix epithelioid carcinoma (HeLa), mouse calvaria-derived pre-osteoblast (MC3T3-E1), and human normal lung fibroblast (MRC-5) cell lines. This compound had selectivity to cancer cells over normal ones. Among these cell lines, HL60 exhibited the highest sensitivity to (+)-plakevulin A. (+)-Plakevulin A induced DNA fragmentation and caspase-3 activation in HL60 cells, indicating its role in apoptosis induction. Additionally, hydroxysteroid 17-ß dehydrogenase 4 (HSD17B4) was isolated from the HL60 lysate as one of its binding proteins through pull-down experiments using its biotinylated derivative and neutravidin-coated beads. Moreover, (+)-plakevulin A suppressed the activation of interleukin 6 (IL-6)-induced signal transducer and activator of transcription 3 (STAT3). Because the knockdown or inhibition of STAT3 induces apoptosis and HSD17B4 regulates STAT3 activation, (+)-plakevulin A may induce apoptosis in HL60 cell lines by suppressing STAT3 activation, potentially by binding to HSD17B4. The present findings provide valuable information for the mechanism of its action.
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Apoptose , Interleucina-6 , Fator de Transcrição STAT3 , Humanos , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Células HL-60 , Interleucina-6/metabolismo , Animais , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Ensaios de Seleção de Medicamentos Antitumorais , Relação Dose-Resposta a Droga , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
G protein-coupled receptors (GPCRs) play diverse roles in physiological processes, and hence the ligands to modulate GPCRs have served as important molecules in biological and pharmacological approaches. However, the exploration of novel ligands for GPCR still remains an arduous challenge. In this study, we report a method for the discovery of nucleic acid ligands against GPCRs by an advanced RNA aptamer screening technology that employs a virus-like particle (VLP), exposing the GPCR of interest. An array of biochemical analyses coupled with a cell-based assay revealed that one of the aptamers raised against purinergic receptor P2Y2 (P2RY2), a GPCR, exhibits an activation potency to unliganded receptor and prohibits a further receptor activation by endogenous ligand, behaving like a partial agonist. However, the aptamer enhances the activity of intrinsic ligand-binding P2RY2, thereby acting as a positive allosteric modulator (PAM) to liganded receptor. Our findings demonstrate that the nucleic acid aptamer conditionally exerts PAM and agonist effects on GPCRs, depending on their intrinsic ligand binding state. These results indicate the validity of our VLP-based aptamer screening targeting GPCR and reemphasize the great potential of nucleic acid ligands for exploring the GPCR activation mechanism and therapeutic applications.
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Aptâmeros de Nucleotídeos/genética , Ácidos Nucleicos/genética , Receptores Acoplados a Proteínas G/genética , Receptores Purinérgicos P2Y2/genética , Regulação Alostérica/genética , Sítios de Ligação/genética , Humanos , LigantesRESUMO
Polyarteritis nodosa (PAN) is a systemic necrotizing vasculitis that predominantly affects medium-sized arteries. With advances in our understanding of the pathogenesis and classification of vasculitis, PAN and microscopic polyangiitis (MPA), a disease of anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV), were separated from the group of diseases previously diagnosed as periarteritis nodosa (PN) at the Chapel Hill Consensus Conference (CHCC) in 1994 (1).
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This study investigated the effect of anti-autotaxin (ATX) aptamers on the development of proliferative vitreoretinopathy (PVR) in both in vivo and in vitro PVR swine models. For the in vitro study, primary retinal pigment epithelial (RPE) cells were obtained from porcine eyes and cultured for cell proliferation and migration assays. For the in vivo study, a swine PVR model was established by inducing retinal detachment and injecting cultured RPE cells (2.0 × 106). Concurrently, 1 week after RPE cell injection, the anti-ATX aptamer, RBM-006 (10 mg/mL, 0.1 mL), was injected twice into the vitreous cavity. Post-injection effects of the anti-ATX aptamer on PVR development in the in vivo swine PVR model were investigated. For the in vitro evaluation, the cultured RPE cell proliferation and migration were significantly reduced at anti-ATX aptamer concentrations of 0.5-0.05 mg and at only 0.5 mg, respectively. Intravitreal administration of the anti-ATX aptamer also prevented tractional retinal detachment caused by PVR in the in vivo PVR model. We observed that the anti-ATX aptamer, RBM-006, inhibited PVR-related RPE cell proliferation and migration in vitro and inhibited the progression of PVR in the in vivo model, suggesting that the anti-ATX aptamer may be effective in preventing PVR.
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Descolamento Retiniano , Vitreorretinopatia Proliferativa , Animais , Suínos , Vitreorretinopatia Proliferativa/tratamento farmacológico , Epitélio Pigmentado da Retina , Proliferação de Células , Células CultivadasRESUMO
The magnocellular neurosecretory cells (MNCs) of the hypothalamus play a vital role in osmoregulation, but the mechanisms underlying MNC osmosensitivity are not fully understood. We showed previously that high osmolality activates phospholipase C (PLC) in rat MNCs in a Ca2+-dependent manner and that PLC activation is necessary for full osmotic activation of an N-terminal variant of the TRPV1 (ΔN-TRPV1) channel. We therefore hypothesized that the Ca2+-dependent δ1 isoform of PLC contributes to ΔN-TRPV1 activation and tested whether MNC function is defective in a transgenic PLCδ1 KO mouse. Water deprivation for 24 h caused greater increases in serum osmolality and losses in body weight in PLCδ1 KO mice than it did in control mice. Action potentials and ΔN-TRPV1 currents were measured in acutely isolated mouse MNCs using whole-cell patch clamp before and after exposure to hypertonic solutions. This treatment elicited a significant activation of ΔN-TRPV1 currents and an increase in firing rate in MNCs isolated from control mice, but not from PLCδ1 KO mice. Submembranous filamentous actin was measured in isolated MNCs before and after treatment with angiotensin II and hypertonic solution. Both treatments caused an increase in filamentous actin fluorescence in MNCs isolated from control mice, but both responses were significantly attenuated in MNCs from PLCδ1 KO mice. Our data demonstrate that the PLCδ1 isoform plays a key role in the activation of ΔN-TRPV1 channels and in osmosensory transduction in MNCs. This study advances our understanding of the molecular mechanisms underlying mammalian osmoregulation.SIGNIFICANCE STATEMENT Magnocellular neurosecretory cells (MNCs) of the hypothalamus play a central role in osmoregulation. We have identified a key role for the PLCδ1 isoform in the activation of ΔN-TRPV1 channels and osmosensory transduction in MNCs. The data indicate that the PLCδ1 isoform is activated by the Ca2+ influx occurring during MNC action potentials and exerts a positive feedback on ΔN-TRPV1 channels to enhance MNC excitability. This study provides evidence that PLCδ1 is a key molecule underlying osmosensory transduction, the regulation of VP release, and osmoregulation.
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Neurônios/metabolismo , Osmorregulação/fisiologia , Fosfolipase C delta/fisiologia , Núcleo Supraóptico/metabolismo , Canais de Cátion TRPV/metabolismo , Actinas/metabolismo , Potenciais de Ação/fisiologia , Angiotensina II/farmacologia , Animais , Fenômenos Eletrofisiológicos , Soluções Hipertônicas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sistemas Neurossecretores/metabolismo , Osmose , Fosfolipase C delta/genética , Canais de Cátion TRPV/genética , Privação de ÁguaRESUMO
Aptamers are short single-stranded RNA/DNA molecules that bind to specific target molecules. Aptamers with high binding-affinity and target specificity are identified using an in vitro procedure called high throughput systematic evolution of ligands by exponential enrichment (HT-SELEX). However, the development of aptamer affinity reagents takes a considerable amount of time and is costly because HT-SELEX produces a large dataset of candidate sequences, some of which have insufficient binding-affinity. Here, we present RNA aptamer Ranker (RaptRanker), a novel in silico method for identifying high binding-affinity aptamers from HT-SELEX data by scoring and ranking. RaptRanker analyzes HT-SELEX data by evaluating the nucleotide sequence and secondary structure simultaneously, and by ranking according to scores reflecting local structure and sequence frequencies. To evaluate the performance of RaptRanker, we performed two new HT-SELEX experiments, and evaluated binding affinities of a part of sequences that include aptamers with low binding-affinity. In both datasets, the performance of RaptRanker was superior to Frequency, Enrichment and MPBind. We also confirmed that the consideration of secondary structures is effective in HT-SELEX data analysis, and that RaptRanker successfully predicted the essential subsequence motifs in each identified sequence.
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Aptâmeros de Nucleotídeos/química , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/isolamento & purificação , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Simulação por Computador , Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Curva ROCRESUMO
BACKGROUND: The treatment guidelines for acute Kawasaki disease (KD) have been revised several times. Moreover, the criterion used to define coronary artery abnormalities (CAAs) has changed from the coronary artery's internal diameter to the Z-score. Treatment for KD and methods for evaluating CAAs vary between hospitals, so we investigated the actual status of acute KD treatment and development of CAAs under the 2012 Japanese treatment guidelines for acute KD. METHODS: The 24th Japanese Nationwide Survey on Kawasaki Disease yielded 2618 patients who developed KD in the Kinki area in 2016. We sent a secondary questionnaire to each participating hospital and used the resulting data to investigate the frequency of CAAs according to Z-score, treatment by KD treatment stage, and predictors of CAAs. RESULTS: The response rate was 80.0%. The data for 1426 patients without major data deficiencies were examined. The frequency of CAAs was 3.0% when based on coronary artery internal diameters and 8.8% when based on Z-scores. Intravenous immunoglobulins combined with corticosteroids were administered as an initial treatment in 12.8% of cases and as a second-line treatment in 16.8% of cases. Corticosteroids, cyclosporine A, infliximab, and plasma exchange were used at similar frequencies for third-line treatment. A pretreatment maximum coronary artery Z-score of ≥1.9 and age <1 year were associated with significantly higher incidences of CAAs. CONCLUSIONS: Using the Z-score resulted in a threefold increase in the number of patients diagnosed with CAAs. A pretreatment maximum coronary artery Z-score of ≥1.9 and age <1 year are useful predictors of CAAs.
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Doença da Artéria Coronariana , Síndrome de Linfonodos Mucocutâneos , Humanos , Lactente , Síndrome de Linfonodos Mucocutâneos/diagnóstico , Síndrome de Linfonodos Mucocutâneos/epidemiologia , Síndrome de Linfonodos Mucocutâneos/terapia , Japão/epidemiologia , Doença da Artéria Coronariana/epidemiologia , Imunoglobulinas Intravenosas/uso terapêutico , Resultado do Tratamento , Estudos RetrospectivosRESUMO
INTRODUCTION: Lysosomal storage disorders and peroxisomal disorders are rare diseases caused by the accumulation of substrates of the metabolic pathway within lysosomes and peroxisomes, respectively. Owing to the rarity of these diseases, the prevalence of lysosomal storage disorders and peroxisomal disorders in Japan is unknown. Therefore, we conducted a nationwide survey to estimate the number of patients with lysosomal storage disorders and peroxisomal disorders in Japan. METHODS: A nationwide survey was conducted following the "Manual of nationwide epidemiological survey for understanding patient number and clinical epidemiology of rare diseases (3rd version)". A questionnaire asking for detailed information, such as disease phenotypes and medical history, was created and sent to 504 institutions with doctors who have experience in treating patients with lysosomal storage disorders and peroxisomal disorders. Result A total of 303 completed questionnaires were collected from 504 institutions (response rate: 60.1%). The number of patients was estimated by calculating the rate/frequency of overlap. The estimated number of patients was 1658 (±264.8) for Fabry disease, 72 (±11.3) for mucopolysaccharidosis I, 275 (±49.9) for mucopolysaccharidosis II, 211 (±31.3) for Gaucher disease, 124 (±25.8) for Pompe disease, 83 (±44.3) for metachromatic leukodystrophy, 57 (±9.4) for Niemann-Pick type C, and 262 (±42.3) for adrenoleukodystrophy. In addition the birth prevalence was calculated using the estimated number of patients and birth year data for each disease, and was 1.25 for Fabry disease, 0.09 for mucopolysaccharidosis I, 0.38 for mucopolysaccharidosis II, 0.19 for Gaucher disease, 0.14 for Pompe disease, 0.16 for metachromatic leukodystrophy, 0.16 for Niemann-Pick type C, and 0.20 for adrenoleukodystrophy. DISCUSSION: Among the diseases analyzed, the disease with the highest prevalence was Fabry disease, followed by mucopolysaccharidosis II, adrenoleukodystrophy, Gaucher disease and metachromatic leukodystrophy. In particular, the high prevalence of mucopolysaccharidosis II and Gaucher disease type II was a feature characteristic of Japan. CONCLUSION: We estimated the number of patients with lysosomal storage disorders and peroxisomal disorders in Japan. The details of the age at diagnosis and treatment methods for each disease were clarified, and will be useful for the early diagnosis of these patients and to provide appropriate treatments. Furthermore, our results suggest that supportive care and the development of an environment that can provide optimal medical care is important in the future.
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Monitoramento Epidemiológico , Doenças por Armazenamento dos Lisossomos/diagnóstico , Doenças por Armazenamento dos Lisossomos/epidemiologia , Transtornos Peroxissômicos/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Terapia de Reposição de Enzimas , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Lactente , Recém-Nascido , Japão/epidemiologia , Doenças por Armazenamento dos Lisossomos/classificação , Doenças por Armazenamento dos Lisossomos/terapia , Masculino , Pessoa de Meia-Idade , Triagem Neonatal , Transtornos Peroxissômicos/sangue , Transtornos Peroxissômicos/diagnóstico , Prevalência , Inquéritos e Questionários , Adulto JovemRESUMO
AIM: Down syndrome has been considered an independent risk factor for respiratory syncytial virus (RSV) infection. Palivizumab, an anti-RSV humanised monoclonal antibody, was currently approved for all children with Down syndrome in Japan. To investigate the change in RSV-associated hospitalisation (RSVH) rates before and after the universal approval of palivizumab in Japan in 2013, we conducted a nationwide retrospective survey. METHODS: We conducted a nationwide, retrospective, questionnaire survey across paediatric institutions in Japan. The recruited children with Down syndrome were divided into two groups: those born April 2010 to March 2013 (2010-2012 cohort) and those born April 2013 to March 2016 (2013-2015 cohort). RESULTS: Of the 664 institutions, 321 (48.3%) replied, and a total of 3929 children with Down syndrome were registered. The percentage of children who received palivizumab increased from 49.2% to 82.2%. The cumulative RSVH rate showed a decreased trend in the 2013-2015 cohort (OR, 0.83; 95%CI, 0.63-1.10), while the rate of these children (without CHD and born at a gestational age ≥ 36 weeks) was significantly decreased in the 2013-2015 cohort (OR, 0.56; 95%CI, 0.34-0.92). CONCLUSION: The cumulative RSVH rate tended to be decreased after approval for all children with Down syndrome although the result was not significant.
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Síndrome de Down , Infecções por Vírus Respiratório Sincicial , Antivirais/uso terapêutico , Criança , Síndrome de Down/complicações , Hospitalização , Humanos , Lactente , Japão/epidemiologia , Palivizumab/uso terapêutico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/epidemiologia , Estudos RetrospectivosRESUMO
Phospholipids are distributed asymmetrically in the plasma membrane (PM) of mammalian cells. Phosphatidylinositol (PI) and its phosphorylated forms are primarily located in the inner leaflet of the PM. Among them, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a well-known substrate for phospholipase C (PLC) or phosphoinositide-3 kinase, and is also a regulator for the actin cytoskeleton or ion channels. Although functions of PI(4,5)P2 in the inner leaflet are well characterized, those in the outer leaflet are poorly understood. Here, PI(4,5)P2 was detected in the cell surface of non-permeabilized cells by anti-PI(4,5)P2 antibodies and the pleckstrin-homology (PH) domain of PLCδ1 that specifically binds PI(4,5)P2. Cell surface PI(4,5)P2 signal was universally detected in various cell lines and freshly isolated mouse bone marrow cells and showed a punctate pattern in a cholesterol, sphingomyelin, and actin polymerization-dependent manner. Furthermore, blocking cell surface PI(4,5)P2 by the addition of anti-PI(4,5)P2 antibody or the PH domain of PLCδ1 inhibited cell attachment, spreading, and migration. Taken together, these results indicate a unique localization of PI(4,5)P2 in the outer leaflet that may have a crucial role in cell attachment, spreading, and migration.
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Adesão Celular , Membrana Celular/metabolismo , Movimento Celular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Actinas/metabolismo , Linhagem Celular , Colesterol/metabolismo , Humanos , Fosfatidilinositol 4,5-Difosfato/análise , Domínios de Homologia à Plecstrina , Esfingomielinas/metabolismo , Fosfolipases Tipo C/análise , Fosfolipases Tipo C/metabolismoRESUMO
Cancer-associated fibroblasts (CAFs) are known to promote tumourigenesis through various mechanisms. Fibroblast growth factor (FGF)/FGF receptor (FGFR)-dependent lung cancers have been described. We have developed a mouse model of lung adenocarcinoma that was constructed through the induction of Fgf9 overexpression in type 2 alveolar cells. The expression of Fgf9 in adult lungs resulted in the rapid development of multiple adenocarcinoma-like tumour nodules. Here, we have characterised the contribution of CAFs and the Fgf/Fgfr signalling pathway in maintaining the lung tumours initiated by Fgf9 overexpression. We found that CAF-secreted Fgf2 contributes to tumour cell growth. CAFs overexpressed Tgfb, Mmp7, Fgf9, and Fgf2; synthesised more collagen, and secreted inflammatory cell-recruiting cytokines. CAFs also enhanced the conversion of tumour-associated macrophages (TAMs) to the tumour-supportive M2 phenotype but did not influence angiogenesis. In vivo inhibition of Fgfrs during early lung tumour development resulted in significantly smaller and fewer tumour nodules, whereas inhibition in established lung tumours caused a significant reduction in tumour size and number. Fgfr inhibition also influenced tumour stromal cells, as it significantly abolished TAM recruitment and reduced tumour vascularity. However, the withdrawal of the inhibitor caused a significant recurrence/regrowth of Fgf/Fgfr-independent lung tumours. These recurrent tumours did not possess a higher proliferative or propagative potential. Our results provide evidence that fibroblasts associated with the Fgf9-induced lung adenocarcinoma provide multiple means of support to the tumour. Although the Fgfr blocker significantly suppressed the tumour and its stromal cells, it was not sufficient to completely eliminate the tumour, probably due to the emergence of alternative (resistance/maintenance) mechanism(s). This model represents an excellent tool to further study the complex interactions between CAFs, their related chemokines, and the progression of lung adenocarcinoma; it also provides further evidence to support the need for a combinatorial strategy to treat lung cancer. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 9 de Crescimento de Fibroblastos/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Adenocarcinoma de Pulmão/enzimologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Fibroblastos Associados a Câncer/enzimologia , Fibroblastos Associados a Câncer/patologia , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/enzimologia , Matriz Extracelular/patologia , Fator 2 de Crescimento de Fibroblastos/deficiência , Fator 2 de Crescimento de Fibroblastos/genética , Fator 9 de Crescimento de Fibroblastos/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Comunicação Parácrina , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Incomplete Kawasaki disease (iKD) showed a higher incidence of coronary artery abnormalities (CAAs) than complete KD. However, the incidence of CAAs among iKD patients may have changed recently. METHODS: We examined KD patients from recent nationwide surveys conducted between 2013 and 2016 and compared them with the results of a previous survey (2001-2002). RESULTS: Of 63 270 KD patients, 13 770 patients (22%) had iKD. They showed a higher incidence of convalescent-phase CAAs (cCAAs, 2.8%) than complete KD (2.1%). The incidence of cCAAs in patients with one or two symptoms (6.7%) was significantly higher than those with three or four symptoms (2.6%) (P < 0.0001). Intravenous immunoglobulin (IVIG) treatment was administered to 80% of iKD patients; 30% of them received IVIG before the fifth illness day (early treatment) and 12% of patients received IVIG after the seventh illness day (late treatment). In the previous survey, the incidence of cCAAs was higher in both iKD (5.9%) and cKD (4.4%). Intravenous immunoglobulin was administered to 62% of iKD patients; 26% of them received early treatment, and 16% received late treatment. CONCLUSIONS: The incidence of cCAAs remained higher among iKD patients than cKD patients but this difference was reduced by the increased proportion of iKD patients treated with IVIG and those at an earlier time point. It is important to recognize the possibility that patients may have iKD and perform echocardiography even if they present with a few principal symptoms.
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Anomalias dos Vasos Coronários/epidemiologia , Imunoglobulinas Intravenosas/administração & dosagem , Síndrome de Linfonodos Mucocutâneos/epidemiologia , Síndrome de Linfonodos Mucocutâneos/terapia , Criança , Pré-Escolar , Doença da Artéria Coronariana/epidemiologia , Anomalias dos Vasos Coronários/diagnóstico , Ecocardiografia/métodos , Feminino , Humanos , Fatores Imunológicos/administração & dosagem , Incidência , Lactente , Masculino , Inquéritos e QuestionáriosRESUMO
Irritant contact dermatitis (ICD) is one of the most common inflammatory skin diseases caused by exposure to chemical irritants. Since chemical irritants primarily damage keratinocytes, these cells play a pivotal role in ICD. One of the phosphoinositide-metabolizing enzymes, phospholipase C (PLC) δ1, is abundantly expressed in keratinocytes. However, the role of PLCδ1 in ICD remains to be clarified. Here, we found that croton oil (CrO)-induced ear swelling, a feature of ICD, was attenuated in keratinocyte-specific PLCδ1 knockout mice (PLCδ1 cKO mice). Dendritic epidermal T cells (DETCs), which have a protective role against ICD, were activated in the epidermis of the PLCδ1 cKO mice. In addition, the skin of CrO-treated PLCδ1 cKO mice showed increased infiltration of Gr1+CD11b+ myeloid cells. Of note, elimination of Gr1+CD11b+ myeloid cells restored CrO-induced ear swelling in PLCδ1 cKO mice to a similar level as that in control mice. Taken together, our results strongly suggest that epidermal loss of PLCδ1 protects mice from ICD through induction of Gr1+CD11b+ myeloid cells and activation of DETCs.
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Dermatite de Contato/genética , Fosfolipase C delta/genética , Animais , Dermatite de Contato/imunologia , Modelos Animais de Doenças , Epiderme/imunologia , Epiderme/metabolismo , Masculino , Camundongos Knockout , Células Mieloides/imunologia , Fosfolipase C delta/imunologia , Linfócitos T/imunologiaRESUMO
Differentiation and proliferation of keratinocyte are controlled by various signalling pathways. The epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Inhibition of EGFR signalling disturbs keratinocyte proliferation, differentiation and migration. Previous studies have revealed that one of the EGFR downstream signalling molecules, phospholipase Cγ1 (PLCγ1), regulates differentiation, proliferation and migration of keratinocytes in in vitro cell culture system. However, the role of PLCγ1 in the regulation of keratinocyte functions in animal epidermis remains unexplored. In this study, we generated keratinocyte-specific PLCγ1 knockout (KO) mice (PLCγ1 cKO mice). Contrary to our expectations, loss of PLCγ1 did not affect differentiation, proliferation and migration of interfollicular keratinocytes. We further examined the role of PLCγ1 in irritant contact dermatitis (ICD), in which epidermal cells play a pivotal role. Upon irritant stimulation, PLCγ1 cKO mice showed exaggerated ICD responses. Further study revealed that epidermal loss of PLCγ1 induced sebaceous gland hyperplasia, indicating that PLCγ1 regulates homeostasis of one of the epidermal appendages. Taken together, our results indicate that, although PLCγ1 is dispensable in interfollicular keratinocyte for normal differentiation, proliferation and migration, it is required for normal ICD responses. Our results also indicate that PLCγ1 regulates homeostasis of sebaceous glands.
Assuntos
Dermatite Irritante/enzimologia , Queratinócitos/enzimologia , Fosfolipase C gama/fisiologia , Glândulas Sebáceas/enzimologia , Animais , Diferenciação Celular , Movimento Celular , Proliferação de Células , Óleo de Cróton/toxicidade , Dermatite Irritante/etiologia , Epiderme/efeitos dos fármacos , Epiderme/enzimologia , Epiderme/patologia , Homeostase , Hiperplasia , Irritantes , Queratinócitos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fosfolipase C gama/deficiência , Fosfolipase C gama/genética , Glândulas Sebáceas/efeitos dos fármacos , Glândulas Sebáceas/patologiaRESUMO
Aptamers are short, single-stranded oligonucleotides that bind to specific target molecules. The shape-forming feature of single-stranded oligonucleotides provides high affinity and excellent specificity toward targets. Hence, aptamers can be used as analogs of antibodies. In December 2004, the US Food and Drug Administration approved the first aptamer-based therapeutic, pegaptanib (Macugen), targeting vascular endothelial growth factor, for the treatment of age-related macular degeneration. Since then, however, no aptamer medication for public health has appeared. During these relatively silent years, many trials and improvements of aptamer therapeutics have been performed, opening multiple novel directions for the therapeutic application of aptamers. This review summarizes the basic characteristics of aptamers and the chemical modifications available for aptamer therapeutics.
Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Descoberta de Drogas , Infecções/tratamento farmacológico , Neoplasias/tratamento farmacológico , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Humanos , Infecções/metabolismo , Neoplasias/metabolismoRESUMO
Cancers, including lung cancer, are a leading cause of death worldwide. To overcome this deadly disease, multiple modality inhibitors have been developed. These include cytotoxic agents, molecular targeted small molecules, such as tyrosine kinase inhibitors, and neutralizing antibodies. An aptamer is a short single-stranded nucleic acid molecule that is selected in vitro from a large random sequence library based on its high and specific affinity to a target molecule. Aptamers can be applied to therapeutics of various types of diseases, including cancer, due to their strong and specific neutralizing activities. However, the efficacy of aptamer-based therapy for cancer cells is not well characterized. In this study, we aimed to show that the FGF2 aptamer is effective for the treatment of FGF2 dependent lung cancer cells. We previously developed PC9GR lung cancer cells, whose proliferation is dependent on EGFR and FGF2-FGFR pathways in a cell autonomous manner. Using PC9GR cells, we demonstrate that the addition of the FGF2 aptamer induces more significant inhibition of PC9GR cell proliferation than does the addition of EGFR inhibitor alone. Furthermore, the addition of the FGF2 aptamer more significantly inhibits the downstream signals and induces apoptosis to a higher extent than does the addition of EGFR inhibitor alone. Our results show that the FGF2 aptamer inhibits the growth of FGF2-FGFR pathway-dependent lung cancer cells. The findings provide preclinical evidence that aptamers can be useful for cancer treatment.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Apoptose/efeitos dos fármacos , Aptâmeros de Nucleotídeos/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Gefitinibe/farmacologia , Humanos , Neoplasias Pulmonares/patologia , Receptores de Fatores de Crescimento de Fibroblastos/genética , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
When translating mRNAs are cleaved in protein-coding regions, 5' fragments of mRNAs are detached from stop codons (i.e., nonstop mRNAs) and protected from 3'-5' exonucleases by ribosomes stalled at the 3' termini. It has been shown in yeast that the nonstop mRNA decay (NSD) machinery triggers nonstop mRNA degradation by removing stalled ribosomes in the artificial reporter mRNAs. However, it is not known well whether NSD is involved in the degradation of endogenous nonstop mRNAs in higher eukaryotes. In this work, we addressed the question of whether 5'-nonstop-mRNA fragments generated by siRNA cleavage or nonsense-mediated-mRNA decay (NMD) are degraded by the NSD pathway in Drosophila melanogaster cells by knocking down three NSD components, Pelota (a yeast Dom34 homolog), Hbs1 and ABCE1 (a ribosome-recycling factor). We found that double, but not single, knockdown of any two of these three factors efficiently stabilized nonstop reporter mRNAs and triple knockdown of Pelota, Hbs1 and ABCE1 further stabilized nonstop mRNAs in highly ribosome-associated state. These findings demonstrated that Pelota, Hbs1 and ABCE1 are crucial for NSD in Drosophila cells as in yeast for rescuing stalled ribosomes and degrading nonstop mRNAs. To our knowledge, this is the first comprehensive report to show the involvement of the NSD machinery in the clearance of mRNA 5'-fragments produced by RNAi and NMD in eukaryotes.