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1.
Protein Expr Purif ; 190: 106003, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34688919

RESUMO

SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.


Assuntos
Vacinas contra COVID-19 , Expressão Gênica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Vacinas contra COVID-19/química , Vacinas contra COVID-19/genética , Vacinas contra COVID-19/farmacologia , Humanos , Camundongos , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , SARS-CoV-2/química , SARS-CoV-2/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/farmacologia
2.
Cell Transplant ; 30: 9636897211054481, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34757857

RESUMO

Biological and cellular interleukin-6 (IL-6)-related therapies have been used to treat severe COVID-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure, which prompted further exploration of the role of IL-6 in human umbilical cord mesenchymal stem cell (hUCMSC) therapy. Peripheral blood mononuclear cells (PBMCs) were responders cocultured with hUCMSCs or exogenous IL-6. A PBMC suppression assay was used to analyze the anti-inflammatory effects via MTT assay. The IL-6 concentration in the supernatant was measured using ELISA. The correlation between the anti-inflammatory effect of hUCMSCs and IL-6 levels and the relevant roles of IL-6 and IL-6 mRNA expression was analyzed using the MetaCore functional network constructed from gene microarray data. The location of IL-6 and IL-6 receptor (IL-6R) expression was further evaluated. We reported that hUCMSCs did not initially exert any inhibitory effect on PHA-stimulated proliferation; however, a potent inhibitory effect on PHA-stimulated proliferation was observed, and the IL-6 concentration reached approximately 1000 ng/mL after 72 hours. Exogenous 1000 ng/mL IL-6 inhibited PHA-stimulated inflammation but less so than hUCMSCs. The inhibitory effects of hUCMSCs on PHA-stimulated PBMCs disappeared after adding an IL-6 neutralizing antibody or pretreatment with tocilizumab (TCZ), an IL-6R antagonist. hUCMSCs exert excellent anti-inflammatory effects by inducing higher IL-6 levels, which is different from TCZ. High concentration of IL-6 cytokine secretion plays an important role in the anti-inflammatory effect of hUCMSC therapy. Initial hUCMSC therapy, followed by TCZ, seems to optimize the therapeutic potential to treat COVID-19-related acute respiratory distress syndrome (ARDS).


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , COVID-19/complicações , Interleucina-6/biossíntese , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Síndrome do Desconforto Respiratório/terapia , SARS-CoV-2 , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Neutralizantes/imunologia , Células Cultivadas , Técnicas de Cocultura , Terapia Combinada , DNA Complementar/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação , Interleucina-6/genética , Interleucina-6/farmacologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Fito-Hemaglutininas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/biossíntese , Receptores de Interleucina-6/genética , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/etiologia , Cordão Umbilical/citologia
4.
Nutrients ; 13(10)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34684482

RESUMO

Sleep is an essential component of overall human health but is so tightly regulated that when disrupted can cause or worsen certain ailments. An important part of this process is the presence of the well-known hormone, melatonin. This compound assists in the governing of sleep and circadian rhythms. Previous studies have postulated that dysregulation of melatonin rhythms is the driving force behind sleep and circadian disorders. A computer-aided search spanning the years of 2015-2020 using the search terms melatonin, circadian rhythm, disorder yielded 52 full text articles that were analyzed. We explored the mechanisms behind melatonin dysregulation and how it affects various disorders. Additionally, we examined associated therapeutic treatments including bright light therapy (BLT) and exogenous forms of melatonin. We found that over the past 5 years, melatonin has not been widely investigated in clinical studies thus there remains large gaps in its potential utilization as a therapy.


Assuntos
Ritmo Circadiano/fisiologia , Melatonina/metabolismo , Animais , Vias Biossintéticas , Ritmo Circadiano/efeitos da radiação , Humanos , Luz , Melatonina/biossíntese , Melatonina/química , Transcrição Genética
5.
J Gen Virol ; 102(10)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34698626

RESUMO

Human noroviruses (HuNoVs) are increasingly becoming the main cause of transmissible gastroenteritis worldwide, with hundreds of thousands of deaths recorded annually. Yet, decades after their discovery, there is still no effective treatment or vaccine. Efforts aimed at developing vaccines or treatment will benefit from a greater understanding of norovirus-host interactions, including the host response to infection. In this review, we provide a concise overview of the evidence establishing the significance of type I and type III interferon (IFN) responses in the restriction of noroviruses. We also critically examine our current understanding of the molecular mechanisms of IFN induction in norovirus-infected cells, and outline the diverse strategies deployed by noroviruses to supress and/or avoid host IFN responses. It is our hope that this review will facilitate further discussion and increase interest in this area.


Assuntos
Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Interferons/fisiologia , Norovirus/imunologia , Norovirus/patogenicidade , Animais , Linhagem Celular , Humanos , Evasão da Resposta Imune , Imunidade Inata , Interferons/biossíntese , Proteínas Virais/metabolismo , Replicação Viral
6.
Nat Commun ; 12(1): 6176, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34702840

RESUMO

Serine is a non-essential amino acid that is critical for tumour proliferation and depletion of circulating serine results in reduced tumour growth and increased survival in various cancer models. While many cancer cells cultured in a standard tissue culture medium depend on exogenous serine for optimal growth, here we report that these cells are less sensitive to serine/glycine depletion in medium containing physiological levels of metabolites. The lower requirement for exogenous serine under these culture conditions reflects both increased de novo serine synthesis and the use of hypoxanthine (not present in the standard medium) to support purine synthesis. Limiting serine availability leads to increased uptake of extracellular hypoxanthine, sparing available serine for other pathways such as glutathione synthesis. Taken together these results improve our understanding of serine metabolism in physiologically relevant nutrient conditions and allow us to predict interventions that may enhance the therapeutic response to dietary serine/glycine limitation.


Assuntos
Neoplasias/metabolismo , Serina/metabolismo , Vias Biossintéticas , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultura/química , Meios de Cultura/metabolismo , Glicina/análise , Glicina/metabolismo , Humanos , Hipoxantina/análise , Hipoxantina/metabolismo , Neoplasias/dietoterapia , Neoplasias/patologia , Purinas/biossíntese , Serina/análise , Regulação para Cima
7.
PLoS One ; 16(10): e0258029, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34618841

RESUMO

Gluten-specific CD4+ T cells drive the pathogenesis of celiac disease and circulating gluten-specific T cells can be identified by staining with HLA-DQ:gluten tetramers. In this first single-cell RNA-seq study of tetramer-sorted T cells from untreated celiac disease patients blood, we found that gluten-specific T cells showed distinct transcriptomic profiles consistent with activated effector memory T cells that shared features with Th1 and follicular helper T cells. Compared to non-specific cells, gluten-specific T cells showed differential expression of several genes involved in T-cell receptor signaling, translational processes, apoptosis, fatty acid transport, and redox potentials. Many of the gluten-specific T cells studied shared T-cell receptor with each other, indicating that circulating gluten-specific T cells belong to a limited number of clones. Moreover, the transcriptional profiles of cells that shared the same clonal origin were transcriptionally more similar compared with between clonally unrelated gluten-specific cells.


Assuntos
Doença Celíaca/genética , Linhagem da Célula/genética , Regulação da Expressão Gênica/genética , Glutens/genética , Linfócitos T/metabolismo , Doença Celíaca/patologia , Perfilação da Expressão Gênica , Glutens/biossíntese , Humanos , RNA-Seq , Receptores de Antígenos de Linfócitos T/genética , Análise de Célula Única , Linfócitos T/classificação , Células Th1/metabolismo , Células Th1/patologia
8.
FASEB J ; 35(11): e21854, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34597422

RESUMO

Ammonia is one of the major metabolites produced by intestinal microorganisms; however, its role in intestinal homeostasis is poorly understood. The present study investigated the regulation of intestinal tight junction (TJ) proteins by ammonia and the underlying mechanisms in human intestinal Caco-2 cells. Ammonia (15, 30, and 60 mM) increased the permeability of the cells in a dose-dependent manner, as indicated by reduced transepithelial electrical resistance and increased dextran flux. Immunoblot and immunofluorescence analyses revealed that the ammonia-induced increase in TJ permeability reduced the membrane localization of TJ proteins such as zonula occludens (ZO)1, ZO2, occludin, claudin-1, and claudin-3. DNA microarray analysis identified a biological pathway "response to reactive oxygen species" enriched by ammonia treatment, indicating the induction of oxidative stress in the cells. Ammonia treatment also increased the malondialdehyde content and decreased the ratio of reduced to oxidized glutathione. Meanwhile, ammonia treatment-induced mitochondrial dysfunction, as indicated by the downregulation of genes associated with the electron transport chain, reduction of the cellular ATP, NADH, and tricarboxylic acid cycle intermediate content, and suppression of the mitochondrial membrane potential. In contrast, N-acetyl cysteine reversed the ammonia-induced impairment of TJ permeability and structure without affecting the mitochondrial parameters. Collectively, ammonia impaired the TJ barrier by increasing oxidative stress in Caco-2 cells. A mitochondrial dysfunction is possibly an event preceding ammonia-induced oxidative stress. The findings of this study could potentially improve our understanding of the interplay between intestinal microorganisms and their hosts.


Assuntos
Amônia/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Trifosfato de Adenosina/metabolismo , Células CACO-2 , Glutationa/metabolismo , Humanos , Interleucina-8/biossíntese , Mucosa Intestinal/metabolismo , Malondialdeído/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , NADP/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Insuficiência Renal Crônica/metabolismo
9.
FASEB J ; 35(11): e21937, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34606628

RESUMO

Defective permeability barrier is considered to be an incentive of hyperuricemia, however, the link between them has not been proven. Here, we evaluated the potential preventive effects of Lactiplantibacillus plantarum N-1 (LPN1) on gut microbiota and intestinal barrier function in rats with hyperoxaluria-induced kidney stones. Male rats were supplied with 1% ethylene glycol (EG) dissolved in drinking water for 4 weeks to develop hyperoxaluria, and some of them were administered with LPN1 for 4 weeks before EG treatment as a preventive intervention. We found that EG not only resulted hyperoxaluria and kidney stone formation, but also promoted the intestinal inflammation, elevated intestinal permeability, and gut microbiota disorders. Supplementation of LPN1 inhibited the renal crystalline deposits through reducing urinary oxalic acid and renal osteopontin and CD44 expression and improved EG-induced intestinal inflammation and barrier function by decreasing the serum LPS and TLR4/NF-κB signaling and up-regulating tight junction Claudin-2 in the colon, as well as increasing the production of short-chain fatty acid (SCFAs) and the abundance of beneficial SCFAs-producing bacteria, mainly from the families of Lachnospiraceae and Ruminococcaceae. Probiotic LPN1 could prevent EG-induced hyperoxaluria by regulating gut microbiota and enhancing intestinal barrier function.


Assuntos
Etilenoglicol/efeitos adversos , Microbioma Gastrointestinal/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Cálculos Renais/induzido quimicamente , Cálculos Renais/prevenção & controle , Lactobacillaceae , Permeabilidade , Probióticos/administração & dosagem , Animais , Colo/metabolismo , Colo/microbiologia , Ácidos Graxos Voláteis/análise , Ácidos Graxos Voláteis/biossíntese , Fezes/química , Fezes/microbiologia , Hiperoxalúria/induzido quimicamente , Hiperoxalúria/prevenção & controle , Hiperuricemia/induzido quimicamente , Hiperuricemia/prevenção & controle , Inflamação/metabolismo , Masculino , RNA Ribossômico 16S/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Junções Íntimas/metabolismo
10.
Invest Ophthalmol Vis Sci ; 62(13): 23, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34698771

RESUMO

Purpose: Optic nerve damage leads to impairment of visual functions. We previously demonstrated that apolipoprotein E-containing lipoproteins (E-LPs) protect retinal ganglion cells (RGCs) from degeneration in a glaucoma model of glutamate/aspartate transporter-deficient mice. This study aimed to determine whether E-LPs protect RGCs from N-methyl-D-aspartate (NMDA)-induced excitotoxicity, and to investigate the details of an indirect neuroprotective mechanism of E-LPs by reducing α2-macroglobulin, which interferes with the neuroprotective effect of E-LPs, in Müller glia. Methods: Excitotoxicity was caused by intravitreal injection of NMDA, and then retinae were subjected to immunoblotting or quantitative reverse transcription-PCR. Primary cultures of mouse mixed retinal cells and mouse Müller glia were used for evaluating the effects of E-LPs on the expression of α2-macroglobulin. Results: Intravitreal injection of E-LPs protected the optic nerve from degeneration and attenuated the increase in α2-macroglobulin in aqueous humor and retina of rats. E-LPs directly decreased the expression and secretion of α2-macroglobulin in primary cultures of Müller glia; this decrease in production of α2-macroglobulin was blocked by knockdown of the low-density lipoprotein receptor-related protein 1 (LRP1) with small interfering RNA. E-LPs promoted the phosphorylation of STAT3, whereas Stattic, an inhibitor of STAT3, restored the expression of α2-macroglobulin decreased by E-LPs. Conclusions: In addition to our previous findings of the protection of RGCs by E-LPs, the new observations in Müller glia indicate that a reduction of the intraocular α2-macroglobulin, regulated by the E-LP-LRP1-STAT3 pathway, might be an additional protective mechanism against excitotoxicity in the retina.


Assuntos
Apolipoproteínas E/metabolismo , Células Ependimogliais/metabolismo , Regulação da Expressão Gênica , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , alfa 2-Macroglobulinas Associadas à Gravidez/genética , Degeneração Retiniana/genética , Células Ganglionares da Retina/patologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , N-Metilaspartato/toxicidade , Fármacos Neuroprotetores/farmacologia , alfa 2-Macroglobulinas Associadas à Gravidez/biossíntese , RNA/genética , Ratos , Ratos Sprague-Dawley , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo
11.
J Biomed Sci ; 28(1): 72, 2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34706729

RESUMO

BACKGROUND: During autophagy defense against invading microbes, certain lipid types are indispensable for generating specialized membrane-bound organelles. The lipid composition of autophagosomes remains obscure, as does the issue of how specific lipids and lipid-associated enzymes participate in autophagosome formation and maturation. Helicobacter pylori is auxotrophic for cholesterol and converts cholesterol to cholesteryl glucoside derivatives, including cholesteryl 6'-O-acyl-α-D-glucoside (CAG). We investigated how CAG and its biosynthetic acyltransferase assist H. pylori to escape host-cell autophagy. METHODS: We applied a metabolite-tagging method to obtain fluorophore-containing cholesteryl glucosides that were utilized to understand their intracellular locations. H. pylori 26695 and a cholesteryl glucosyltransferase (CGT)-deletion mutant (ΔCGT) were used as the standard strain and the negative control that contains no cholesterol-derived metabolites, respectively. Bacterial internalization and several autophagy-related assays were conducted to unravel the possible mechanism that H. pylori develops to hijack the host-cell autophagy response. Subcellular fractions of H. pylori-infected AGS cells were obtained and measured for the acyltransferase activity. RESULTS: The imaging studies of fluorophore-labeled cholesteryl glucosides pinpointed their intracellular localization in AGS cells. The result indicated that CAG enhances the internalization of H. pylori in AGS cells. Particularly, CAG, instead of CG and CPG, is able to augment the autophagy response induced by H. pylori. How CAG participates in the autophagy process is multifaceted. CAG was found to intervene in the degradation of autophagosomes and reduce lysosomal biogenesis, supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Furthermore, we performed the enzyme activity assay of subcellular fractions of H. pylori-infected AGS cells. The analysis showed that the acyltransferase is mainly distributed in autophago-lysosomal compartments. CONCLUSIONS: Our results support the idea that the acyltransferase is mainly distributed in the subcellular compartment consisting of autophagosomes, late endosomes, and lysosomes, in which the acidic environment is beneficial for the maximal acyltransferase activity. The resulting elevated level of CAG can facilitate bacterial internalization, interfere with the autophagy flux, and causes reduced lysosomal biogenesis.


Assuntos
Aciltransferases/metabolismo , Colesterol/análogos & derivados , Infecções por Helicobacter/fisiopatologia , Helicobacter pylori/fisiologia , Lisossomos/fisiologia , Animais , Colesterol/biossíntese , Infecções por Helicobacter/enzimologia , Infecções por Helicobacter/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organismos Livres de Patógenos Específicos
12.
J Immunol ; 207(10): 2561-2569, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34635585

RESUMO

PGs are important proinflammatory lipid mediators, the significance of which is highlighted by the widespread and efficacious use of nonsteroidal anti-inflammatory drugs in the treatment of inflammation. 4-Octyl itaconate (4-OI), a derivative of the Krebs cycle-derived metabolite itaconate, has recently garnered much interest as an anti-inflammatory agent. In this article, we show that 4-OI limits PG production in murine macrophages stimulated with the TLR1/2 ligand Pam3CSK4. This decrease in PG secretion is due to a robust suppression of cyclooxygenase 2 (COX2) expression by 4-OI, with both mRNA and protein levels decreased. Dimethyl fumarate, a fumarate derivative used in the treatment of multiple sclerosis, with properties similar to itaconate, replicated the phenotype observed with 4-OI. We also demonstrate that the decrease in COX2 expression and inhibition of downstream PG production occurs in an NRF2-independent manner. Our findings provide a new insight into the potential of 4-OI as an anti-inflammatory agent and also identifies a novel anti-inflammatory function of dimethyl fumarate.


Assuntos
Anti-Inflamatórios/farmacologia , Fumarato de Dimetilo/farmacologia , Macrófagos/efeitos dos fármacos , Prostaglandinas/metabolismo , Succinatos/farmacologia , Animais , Ciclo-Oxigenase 2/biossíntese , Humanos , Macrófagos/metabolismo , Camundongos
13.
Invest Ophthalmol Vis Sci ; 62(13): 3, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34617961

RESUMO

Purpose: Myoblast determination protein 1 (MYOD) is a critical myogenic regulatory factor in muscle development, differentiation, myofiber repair, and regeneration. As the extraocular muscles significantly remodel their myofibers throughout life compared with limb skeletal muscles, we hypothesized that the absence of MYOD would result in their abnormal structure and function. To assess structural and functional changes in the extraocular muscles in MyoD-/- mice, fiber size and number and optokinetic nystagmus reflex (OKN) responses were examined. Methods: OKN was measured in MyoD-/- mice and littermate wild-type controls at 3, 6, and 12 months. The extraocular muscles were examined histologically for changes in mean myofiber cross-sectional area, total myofiber number, and nuclei immunostained for PAX7 and PITX2, markers of myogenic precursor cells. Results: The MyoD-/- mice developed nystagmus, with both jerk and pendular waveforms, in the absence and in the presence of moving visual stimulation. At 12 months, there were significant losses in mean myofiber cross-sectional area and in total number of orbital layer fibers in all rectus muscles, as well as in global layer fibers in the superior and inferior rectus muscles. Haploinsufficient mice showed abnormal OKN responses. PITX2-positive cell entry into myofibers of the MyoD-/- mice was significantly reduced. Conclusions: This study is the first demonstration of the development of nystagmus in the constitutive absence of expression of the muscle-specific transcription factor MYOD. We hypothesize that myofiber loss over time may alter anterograde and/or retrograde communication between the motor nerves and extraocular muscles that are critical for maintaining normalcy of extraocular muscle function.


Assuntos
Regulação da Expressão Gênica , Proteína MyoD/genética , Nistagmo Patológico/genética , Músculos Oculomotores/metabolismo , Animais , Modelos Animais de Doenças , Seguimentos , Camundongos , Proteína MyoD/biossíntese , Nistagmo Patológico/diagnóstico , Nistagmo Patológico/metabolismo , Músculos Oculomotores/diagnóstico por imagem
14.
Invest Ophthalmol Vis Sci ; 62(13): 11, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34643663

RESUMO

Purpose: This exploratory study aimed to investigate the morphological and pathological alterations of the meibomian gland (MG) with the Staphylococcus aureus crude extracts (SACEs) treatment. Methods: Mouse MG explants were cultured and differentiated with or without SACEs for 48 hours. Explant's viability and cell death were determined by thiazolyl blue tetrazolium bromide (MTT) assay and TUNEL assay. MG morphology was observed by Hematoxylin and Eosin staining. Lipid droplet production was detected by Nile Red staining and LipidTox immunostaining. The pro-inflammatory cytokines were detected by ELISA. The relative gene and protein expression in MG explants was determined via quantitative RT-PCR, immunostaining, and immunoblotting. The components of the SACEs were analyzed by immunoblotting and silver staining. Results: Our findings demonstrated that the SACEs treatment induced overexpression of keratin 1 (Krt1) in the ducts and acini of MG explants, accompanied by a decrease in viability and an increase in cell death in explants. Furthermore, the SACEs treatment dose-dependently increased the levels of TNF-α, IL-1ß, and IL-6 in MG explants. The SACEs treatment induced activation of the nuclear factor kappa B (NF-κB) and AIM2 (absent in melanoma 2)/ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) inflammasome signaling pathway in explants. Further investigation showed expression of the key adipogenesis-related molecule peroxisome proliferator-activated receptor γ was decreased after SACEs treatment. However, no change was found in the lipid synthesis of MG explants after treatment with the SACEs. Staphylococcal enterotoxins B (SEB) was detected in the SACEs. SEB induced the overexpression of Krt1 and IL-1ß in ducts and acini of MG explants. Conclusions: Our findings confirm that Staphylococcus aureus induced hyperkeratinization and pro-inflammatory cytokines expression in MG explants ducts and acini. These effects might be mediated by SEB. Activation of the NF-κB and AIM2/ASC signaling pathway is involved in this process.


Assuntos
Citocinas/genética , Infecções Oculares Bacterianas/metabolismo , Regulação da Expressão Gênica , Disfunção da Glândula Tarsal/metabolismo , Glândulas Tarsais/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/isolamento & purificação , Animais , Apoptose , Citocinas/biossíntese , Modelos Animais de Doenças , Regulação para Baixo , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/patologia , Inflamassomos/metabolismo , Masculino , Disfunção da Glândula Tarsal/microbiologia , Disfunção da Glândula Tarsal/patologia , Glândulas Tarsais/patologia , Camundongos , Transdução de Sinais , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia
15.
J Gen Virol ; 102(10)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34665110

RESUMO

Viperin has antiviral function against many viruses, including dengue virus (DENV), when studied in cells in culture. Here, the antiviral actions of viperin were defined both in vitro and in a mouse in vivo model of DENV infection. Murine embryonic fibroblasts (MEFs) derived from mice lacking viperin (vip-/-) showed enhanced DENV infection, accompanied by increased IFN-ß and induction of ISGs; IFIT1 and CXCL-10 but not IRF7, when compared to wild-type (WT) MEFs. In contrast, subcutaneous challenge of immunocompetent WT and vip-/- mice with DENV did not result in enhanced infection. Intracranial infection with DENV resulted in body weight loss and neurological disease with a moderate increase in mortality in vip-/- compared with WT mice, although this was not accompanied by altered brain morphology, immune cell infiltration or DENV RNA level in the brain. Similarly, DENV induction of IFN-ß, IFIT1, CXCL-10, IRF7 and TNF-α was not significantly different in WT and vip-/- mouse brain, although there was a modest but significant increase in DENV induction of IL-6 and IfI27la in the absence of viperin. NanoString nCounter analysis confirmed no significant difference in induction of a panel of inflammatory genes in WT compared to vip-/- DENV-infected mouse brains. Further, polyI:C stimulation of bone marrow-derived macrophages (BMDMs) induced TNF-α, IFN-ß, IL-6 and Nos-2, but responses were not different in BMDMs generated from WT or vip-/- mice. Thus, while there is significant evidence of anti-DENV actions of viperin in some cell types in vitro, for DENV infection in vivo a lack of viperin does not affect systemic or brain susceptibility to DENV or induction of innate and inflammatory responses.


Assuntos
Antivirais , Vírus da Dengue/imunologia , Vírus da Dengue/fisiologia , Dengue/imunologia , Dengue/virologia , Imunidade Inata , Proteínas/fisiologia , Animais , Encéfalo/imunologia , Encéfalo/virologia , Células Cultivadas , Inflamação , Fator Regulador 7 de Interferon/genética , Interferon beta/biossíntese , Interferon beta/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Proteínas/genética , Replicação Viral
16.
Invest Ophthalmol Vis Sci ; 62(13): 17, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34673901

RESUMO

Purpose: No lymphatic vessels have been identified in the retina. This study investigated whether pathological VEGF-A-overexpressing diabetic retina causes lymphangiogenesis. Methods: Three genetic mouse models of diabetic retinopathy (DR) (Akita [Ins2+/-], Kimba [vegfa+/+], and Akimba [Akita × Kimba] mice) were used. Retinas were examined by fundus photography, fluorescence angiography (FA), and immunostaining to detect lymphangiogenesis or angiogenesis. Lyve1-GFP (Lyve1EGFP/Cre) mice were used to examine Lyve1-expressing cells by immunostaining. Lymphatic-related factors were investigated in mouse retina and vitreous fluid from proliferative diabetic retinopathy (PDR) patients by RT-PCR and ELISA, respectively. Aged Kimba and Akimba mice were used to examine the retinal phenotype at the late phase of VEGF overexpression. Results: FA and immunostaining showed retinal neovascularization in Kimba and Akimba mice but not wild-type and Akita mice. Immunohistochemistry showed that lymphangiogenesis was not present in the retinas of Akita, Kimba, or Akimba mice despite the significant upregulation of lymphatic-related factors (Lyve1, podoplanin, VEGF-A, VEGF-C, VEGF-D, VEGFR2, and VEGFR3) in the retinas of Kimba and Akimba mice by RT-PCR (P < 0.005). Furthermore, lymphangiogenesis was not present in aged Kimba or Akimba mice. Significantly increased numbers of Lyve1-positive cells present in the retinas of Kimba and Akimba mice, especially in the peripheral areas, were CD11b positive, indicating a macrophage population (P < 0.005). VEGF-C in PDR vitreous with vitreous hemorrhage (VH) was higher than in PDR without VH or a macular hole. Conclusions: Retinal VEGF-A overexpression did not cause typical lymphangiogenesis despite upregulated lymphatic-related factors and significant Lyve1-positive macrophage infiltration.


Assuntos
Retinopatia Diabética/genética , Regulação da Expressão Gênica , Linfangiogênese/genética , Vasos Linfáticos/patologia , Retina/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Proteínas de Transporte Vesicular/genética , Animais , Diabetes Mellitus Experimental , Retinopatia Diabética/diagnóstico por imagem , Retinopatia Diabética/metabolismo , Angiofluoresceinografia/métodos , Vasos Linfáticos/metabolismo , Camundongos Endogâmicos C57BL , RNA/genética , Retina/patologia , Regulação para Cima , Fator C de Crescimento do Endotélio Vascular/biossíntese , Proteínas de Transporte Vesicular/biossíntese
17.
Molecules ; 26(20)2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34684753

RESUMO

Angelica polymorpha Maxim. (APM) is used in traditional medicine to treat chronic gastritis, rheumatic pain, and duodenal bulbar ulcers. However, it is not known whether APM has epidermis-associated biological activities. Here, we investigated the effects of APM flower absolute (APMFAb) on responses associated with skin wound healing and whitening using epidermal cells. APMFAb was obtained by solvent extraction and its composition was analyzed by GC/MS. Water-soluble tetrazolium salt, 5-bromo-2'-deoxyuridine incorporation, Boyden chamber, sprouting, and enzyme-linked immunosorbent assays and immunoblotting were used to examine the effects of APMFAb on HaCaT keratinocytes and B16BL6 melanoma cells. APMFAb contained five compounds and induced keratinocyte migration, proliferation, and type IV collagen synthesis. APMFAb also induced the phosphorylations of ERK1/2, JNK, p38 mitogen-activated protein kinase, and AKT in keratinocytes. In addition, APMFAb decreased serum-induced B16BL6 cell proliferation and inhibited tyrosinase expression, melanin contents, and microphthalmia-associated transcription factor expression in α-melanocyte-stimulating hormone-stimulated B16BL6 cells. These findings demonstrate that APMFAb has beneficial effects on skin wound healing by promoting the proliferation, migration, and collagen synthesis of keratinocytes and on skin whitening by inhibiting melanin synthesis in melanoma cells. Therefore, we suggest that APMFAb has potential use as a wound healing and skin whitening agent.


Assuntos
Angelica/metabolismo , Extratos Vegetais/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Flores/metabolismo , Humanos , Queratinócitos/efeitos dos fármacos , Melaninas/biossíntese , Melaninas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo
18.
Nat Commun ; 12(1): 6144, 2021 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-34686667

RESUMO

RIPK1 is a crucial regulator of cell death and survival. Ripk1 deficiency promotes mouse survival in the prenatal period while inhibits survival in the early postnatal period without a clear mechanism. Metabolism regulation and autophagy are critical to neonatal survival from severe starvation at birth. However, the mechanism by which RIPK1 regulates starvation resistance and survival remains unclear. Here, we address this question by discovering the metabolic regulatory role of RIPK1. First, metabolomics analysis reveals that Ripk1 deficiency specifically increases aspartate levels in both mouse neonates and mammalian cells under starvation conditions. Increased aspartate in Ripk1-/- cells enhances the TCA  flux and ATP production. The energy imbalance causes defective autophagy induction by inhibiting the AMPK/ULK1 pathway. Transcriptional analyses demonstrate that Ripk1-/- deficiency downregulates gene expression in aspartate catabolism by inactivating SP1. To summarize, this study reveals that RIPK1 serves as a metabolic regulator responsible for starvation resistance.


Assuntos
Ácido Aspártico/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Inanição/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Animais Recém-Nascidos , Ácido Aspártico/farmacologia , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Sobrevivência Celular , Ciclo do Ácido Cítrico , Humanos , Metabolômica , Camundongos , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Transdução de Sinais , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Inanição/genética , Inanição/mortalidade
19.
Int J Lab Hematol ; 43(6): 1325-1333, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34623759

RESUMO

BACKGROUND: Multiple myeloma (MM) is a hematological malignancy. Coronavirus disease 2019 (COVID-19) infection correlates with MM features. This study aimed to identify MM prognostic biomarkers with potential association with COVID-19. METHODS: Differentially expressed genes (DEGs) in five MM data sets (GSE47552, GSE16558, GSE13591, GSE6477, and GSE39754) with the same expression trends were screened out. Functional enrichment analysis and the protein-protein interaction network were performed for all DEGs. Prognosis-associated DEGs were screened using the stepwise Cox regression analysis in the cancer genome atlas (TCGA) MMRF-CoMMpass cohort and the GSE24080 data set. Prognosis-associated DEGs associated with COVID-19 infection in the GSE164805 data set were also identified. RESULTS: A total of 98 DEGs with the same expression trends in five data sets were identified, and 83 DEGs were included in the protein-protein interaction network. Cox regression analysis identified 16 DEGs were associated with MM prognosis in the TCGA cohort, and only the cytochrome c oxidase subunit 6C (COX6C) gene (HR = 1.717, 95% CI 1.231-2.428, p = .002) and the nucleotide-binding oligomerization domain containing 2 (NOD2) gene (HR = 0.882, 95% CI 0.798-0.975, p = .014) were independent factors related to MM prognosis in the GSE24080 data set. Both of them were downregulated in patients with mild COVID-19 infection compared with controls but were upregulated in patients with severe COVID-19 compared with patients with mild illness. CONCLUSIONS: The NOD2 and COX6C genes might be used as prognostic biomarkers in MM. The two genes might be associated with the development of COVID-19 infection.


Assuntos
COVID-19/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Mieloma Múltiplo/genética , SARS-CoV-2 , COVID-19/mortalidade , Conjuntos de Dados como Assunto , Complexo IV da Cadeia de Transporte de Elétrons/genética , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Ontologia Genética , Humanos , Estimativa de Kaplan-Meier , Análise em Microsséries , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteína Adaptadora de Sinalização NOD2/genética , Prognóstico , Modelos de Riscos Proporcionais , Mapas de Interação de Proteínas/genética
20.
Proc Natl Acad Sci U S A ; 118(44)2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34654739

RESUMO

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.


Assuntos
Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/farmacologia , COVID-19/imunologia , COVID-19/prevenção & controle , Pandemias , SARS-CoV-2/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/epidemiologia , Vacinas contra COVID-19/administração & dosagem , Camelídeos Americanos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunização Secundária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pandemias/prevenção & controle , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , SARS-CoV-2/genética , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
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