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1.
Curr Protoc ; 2(9): e546, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36094175

RESUMO

Expressing recombinant proteins in heterologous host cells is a prerequisite for purification and other downstream processes. Cell cultures require a protein expression test to optimize incubation time, temperature, and additives (like chemical inducers) to identify the best growth conditions with maximum recombinant protein yield. However, running SDS-PAGE followed by western blotting is cumbersome and results are not quick. Here, I describe a simple protocol to quickly check the presence of recombinant protein in cell cultures using a dot-blot experiment. The cells can be rapidly lysed and directly spotted on the nitrocellulose membrane. Then, the membrane is incubated with a horseradish peroxidase (HRP) conjugated antibody raised against the affinity tag present on the recombinant protein to confirm the protein expression by chemiluminescence. It takes less than an hour to get results. This method rapidly investigates recombinant protein expression in different cell lines and tests other variables. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Protein expression analysis for eukaryotic systems Basic Protocol 2: Protein expression analysis for bacterial systems.


Assuntos
Proteínas Recombinantes , Western Blotting , Colódio , Peroxidase do Rábano Silvestre , Immunoblotting
2.
Methods Cell Biol ; 171: 81-109, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35953207

RESUMO

Philadelphia-negative myeloproliferative neoplasms (pH-MPNs) origin from the clonal expansion of hematopoietic stem cells with acquired mutations leading to uncontrolled proliferation of differentiated myeloid cells. The main entities of Ph-MPNs are represented by Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) that are characterized by microvascular disorders, thrombosis and bleeding, splenomegaly secondary to extramedullary hematopoiesis, various degree of bone marrow fibrosis and a progressive risk of leukemic transformation. Somatic mutations in myeloid genes including JAK2, CALR, and MPL cause the constitutive activation of the Janus Kinase 2 (JAK)/signal transducer and activator of transcription (STAT) pathway that confers proliferative and differentiative advantage to mutated hematopoietic progenitors and ultimately drives the development of a Ph-MPNs phenotype. Beyond the JAK/STAT axis, a wide number of intracellular signaling pathways were found deregulated in Ph-MPNs including the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) constitutive activation. In this chapter, we provide a detailed protocol for the immunoblotting assisted assessment of Ph-MPNs pathways activation. This protocol can be easily adapted to study protein expression and phosphorylation of hematopoietic stem progenitors and differentiated cell lineages.


Assuntos
Transtornos Mieloproliferativos , Policitemia Vera , Mielofibrose Primária , Calreticulina/genética , Humanos , Immunoblotting , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/genética , Fosfatidilinositol 3-Quinases/genética , Policitemia Vera/genética , Mielofibrose Primária/genética , Proteínas Proto-Oncogênicas c-akt/genética , Células-Tronco , Serina-Treonina Quinases TOR/genética
3.
Front Immunol ; 13: 963401, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36003369

RESUMO

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease. Although the pathomechanism of BP onset has yet to be elucidated in detail, BP autoantibodies targeting two hemidesmosomal components, BP180 and BP230, are known to play a pivotal role in BP pathogenesis. Thus, the detection and measurement of BP autoantibodies are necessary for diagnosing BP and monitoring the disease activity. Immune assays such as immunofluorescence microscopy, immunoblotting, and ELISAs using BP180 and BP230 detect BP autoantibodies in most BP cases with high specificity; however, BP autoantibodies are sometimes detected in BP patients before the onset of this disease. BP autoantibodies that are detected in patients without typical tense blisters are defined as "preclinical BP autoantibodies". These preclinical BP autoantibodies are detected even in a low percentage of normal healthy individuals. Although the importance of preclinical BP autoantibodies remains elusive, these autoantibodies might be a potential risk factor for subsequent BP development. Therefore, previous comparative epidemiological studies have focused on the prevalence of preclinical BP autoantibodies in populations susceptible to BP (e.g., the elderly) or in diseases with a higher risk of comorbid BP. This mini-review summarizes the literature on the prevalence of preclinical BP autoantibodies in patients with various conditions and diseases, and we discuss the significance of preclinical BP autoantibody detection.


Assuntos
Colágenos não Fibrilares , Penfigoide Bolhoso , Idoso , Autoanticorpos , Autoantígenos , Humanos , Immunoblotting
4.
Front Immunol ; 13: 948108, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36032160

RESUMO

Pemphigoid diseases (PD) are autoimmune skin blistering diseases characterized by autoantibodies directed against proteins of the cutaneous basement membrane zone (BMZ). One of the major antigens is type XVII collagen (BP180), a transmembrane glycoprotein, which is targeted in four PDs: bullous pemphigoid, mucous membrane pemphigoid, linear IgA dermatosis, and pemphigoid gestationis. To date, different epitopes on BP180 have been described to be recognized by PD disease patients' autoantibodies. Different BP180 epitopes were associated with distinct clinical phenotypes while the underlying mechanisms are not yet fully understood. So far, the main effects of anti-BP180 reactivity are mediated by Fcγ-receptors on immune cells. More precisely, the autoantibody-antigen interaction leads to activation of complement at the BMZ and infiltration of immune cells into the upper dermis and, by the release of specific enzymes and reactive oxygen species, to the degradation of BP180 and other BMZ components, finally manifesting as blisters and erosions. On the other hand, inflammatory responses independent of Fcγ-receptors have also been reported, including the release of proinflammatory cytokines and internalization and depletion of BP180. Autoantibodies against BP180 can also be found in patients with neurological diseases. The assumption that the clinical expression of PD depends on epitope specificity in addition to target antigens, autoantibody isotypes, and antibody glycosylation is supported by the observation that epitopes of PD patients differ from those of PD patients. The aim of the present review is to describe the fine specificities of anti-BP180 autoantibodies in different PDs and highlight the associated clinical differences. Furthermore, the direct effects after binding of the autoantibodies to their target are summarized.


Assuntos
Doenças Autoimunes , Doenças do Sistema Nervoso , Penfigoide Bolhoso , Autoanticorpos , Autoantígenos , Proteínas de Transporte , Proteínas do Sistema Complemento , Epitopos , Humanos , Immunoblotting , Colágenos não Fibrilares , Receptores de IgG
5.
Methods Mol Biol ; 2523: 265-279, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35759203

RESUMO

Detection of microbes relies on the expression of germline-encoded pattern recognition receptors (PRRs). While PRRs can directly sense conserved pattern expressed by various microbes, they can also induce effector-triggered immunity (ETI) by sensing pathogenic alterations of cellular homeostasis. One consequence of ETI is the death of the infected cell through the induction of inflammasome-dependent cell death, namely, pyroptosis. Such process can be easily studied in macrophages and epithelial cells, yet neutrophils encode an arsenal of proteolytic enzymes that imped easy and reliable study of ETI-triggered inflammasome response. Here, we describe an immunoblotting methodology to study both ETI- and PRR-driven inflammasome responses in neutrophils upon bacterial infections. This method is also transposable to other microbial pathogen- and toxin-induced inflammasome response in neutrophils.


Assuntos
Inflamassomos , Neutrófilos , Bactérias/metabolismo , Immunoblotting , Inflamassomos/metabolismo , Neutrófilos/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo
6.
Exp Gerontol ; 164: 111821, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35504483

RESUMO

Adiponectin (APN), a major adipokine secreted from white adipose tissue, prevents inflammation and improves insulin sensitivity. APN exists as distinct multimeric complexes with different physiological activities, including low, middle and high molecular weight complexes (LMW, MMW and HMW, respectively) in peripheral blood. Caloric restriction (CR), an intervention that suppresses aging-related pathophysiological changes and extends lifespan, reportedly elevates the expression levels of Adipoq (encoding APN) and total circulating APN. Circulating APN levels have generally been measured using ELISA, but ELISA fails to directly and separately detect APN multimeric complexes other than HMW. Here, we aimed to evaluate the association of aging and CR with oligomerization of APN in rodent models, using immunoblotting to distinguish multimeric complexes based on molecular sizes. In mice, aging elevated plasma levels of HMW and MMW, while CR only elevated HMW. In contrast, LMW and monomeric APN levels were unchanged, suggesting that aging and CR can induce the assembly of APN oligomers in adipocytes. In rats, plasma levels of all multimeric complexes and monomeric APN were not significantly changed by aging or CR. Collectively, levels of circulating APN in mice were consistent with previous findings, whereas those of rats were partially inconsistent, probably because of experimental differences. Moreover, aging reduced Adipoq mRNA levels in mice and rats, while CR prevented this reduction only in rats. Such a discrepancy between Adipoq expression and circulating APN levels may be attributed to proteasomal regulation in adipocytes or tissue accumulation of APN. In conclusion, this study provides new findings of aging- and CR-related changes of each APN multimeric complex and underscores the importance of qualitative approaches for a greater understanding of physiological changes in APN.


Assuntos
Adiponectina , Resistência à Insulina , Envelhecimento , Animais , Restrição Calórica , Immunoblotting , Camundongos , Sobrepeso , Ratos
7.
Clin Lab ; 68(4)2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-35443596

RESUMO

BACKGROUND: Countries use national policies to screen and diagnose people infected with the hepatitis C virus to prevent transmission and eliminate the disease. In May 2016, the World Health Organization set a target of 90% diagnosis and elimination of the disease by 2030. The aim of this study was to evaluate the screening and diagnostic algorithm of hepatitis C by serological methods. METHODS: The blood samples of people referring to blood transfusion centers in Kerman province in southeastern Iran from January 2014 to January 2020 were examined with the defined algorithm for the presence of antibodies against hepatitis C virus by ELISA and confirmation test (RIBA). RESULTS: Based on the algorithm used, little/no correlations were found between the effect of age on OD in ELISA and RIBA test results, respectively (r = 0.07, p = 0.03) and (r = 0.19, p = 0.001). The correlation between the amount of OD in the ELISA test and the results of RIBA test was (r = 0.24, p = 0.01) and no significant correlation was observed between OD in ELISA and the indeterminate immunoblot test results (r = -0.04 and p = 0.2). CONCLUSIONS: The results of this study show, in low-risk populations, all samples that have reactive ELISA results should be confirmed without considering the amount of ELISA OD and the signal-to-cut-off (S/Co) ratios. The existing algorithm should be modified as soon as possible and newer technologies should be used to perform the test.


Assuntos
Doadores de Sangue , Hepatite C , Algoritmos , Ensaio de Imunoadsorção Enzimática , Hepacivirus , Anticorpos Anti-Hepatite/análise , Anticorpos Anti-Hepatite C , Humanos , Immunoblotting
8.
STAR Protoc ; 3(2): 101265, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35391936

RESUMO

Pharmacologic inhibition of the protein-protein interaction (PPI) interface of the Keap1:Nrf2 complex, which leads to Nrf2 activation and cytoprotective gene expression, offers a promising strategy for disease prevention and treatment. To facilitate identification and validation of small-molecule Keap1:Nrf2 PPI inhibitors in the cellular environment in a low- and medium-throughput manner, we detail two adapted cellular thermal shift assay (CETSA) protocols, Keap1-CETSA, an immunoblotting-based methodology for detecting endogenous Keap1, and Keap1-Glow CETSA, a microtiter plate assay of overexpressed fluorescently-tagged Keap1. For an example of the use and execution of this protocol, please refer to Dayalan Naidu et al. (2021).


Assuntos
Bioensaio , Fator 2 Relacionado a NF-E2 , Immunoblotting , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Ligação Proteica
9.
Comp Immunol Microbiol Infect Dis ; 86: 101816, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35472655

RESUMO

Cystic echinococcosis (CE) is a disease caused by Echinococcus granulosus sensu lato (s.l.), an ubiquitous worldwide zoonotic agent affecting humans and animals. Diagnosis of CE in humans is usually performed by imagine techniques along with immunoassays. The aim of our study was to evaluate and compare four commercial diagnostic kits, based on the detection of IgG antibodies against E. granulosus and E. multilocularis. The study was performed on a total of 259 sera: the positive (n = 74) and the negative (n = 185) group. The following analytic and diagnostic performances of the four kits were evaluated: operator skills, specificity, sensitivity, repeatability, reproducibility, accuracy, positive and negative predictive values. Based on the parameters evaluated, all four tests demonstrated excellent quality and proved to be reliable diagnostic tools to support the clinical evaluation of human patients suspected of having CE. The four commercial assays, in our hands, presented altogether, a range of performances from good to excellent, being immunoblotting (IB) the most reliable, used as gold standard, followed by the immunochromatographic test (ICT) and finally the two enzyme linked immunosorbent assay (ELISAs).


Assuntos
Equinococose , Echinococcus granulosus , Animais , Equinococose/diagnóstico , Equinococose/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Humanos , Immunoblotting/veterinária , Reprodutibilidade dos Testes
10.
Methods Mol Biol ; 2488: 1-12, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35347678

RESUMO

Cell signaling governs the basic functions of cells by molecular interactions that involve of many proteins. The abundance of signaling proteins can directly influence cellular responses to external signal, contributing to cellular heterogeneity. Absolute quantification of proteins is important for modeling and understanding the complex signaling network. Here, we introduce how to measure the amount of TGF-ß signaling proteins using quantitative immunoblotting. In addition, we discuss how to convert the measurements of protein abundance to the quantities of absolute molecules per cell. This method is generally applicable to the absolute quantification of other proteins.


Assuntos
Proteínas Smad , Fator de Crescimento Transformador beta , Western Blotting , Immunoblotting , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo
11.
PLoS One ; 17(3): e0265453, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35333910

RESUMO

Several SARS-CoV-2 variants emerged that harbor mutations in the surface unit of the viral spike (S) protein that enhance infectivity and transmissibility. Here, we analyzed whether ten naturally-occurring mutations found within the extended loop harboring the S1/S2 cleavage site of the S protein, a determinant of SARS-CoV-2 cell tropism and pathogenicity, impact S protein processing and function. None of the mutations increased but several decreased S protein cleavage at the S1/S2 site, including S686G and P681H, the latter of which is found in variants of concern B.1.1.7 (Alpha variant) and B.1.1.529 (Omicron variant). None of the mutations reduced ACE2 binding and cell-cell fusion although several modulated the efficiency of host cell entry. The effects of mutation S686G on viral entry were cell-type dependent and could be linked to the availability of cathepsin L for S protein activation. These results show that polymorphisms at the S1/S2 site can modulate S protein processing and host cell entry.


Assuntos
Polimorfismo Genético/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Animais , Chlorocebus aethiops , Células HEK293/virologia , Humanos , Immunoblotting , Células Vero/virologia
12.
PLoS Biol ; 20(3): e3001548, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35239649

RESUMO

Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae, is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c, renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon TORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6, which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity, and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogen conditions.


Assuntos
Fase G1/genética , Regulação Fúngica da Expressão Gênica , Nitrogênio/metabolismo , Fase S/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Divisão Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Immunoblotting , Ligação Proteica , RNA-Seq/métodos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
STAR Protoc ; 3(1): 101018, 2022 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-35243365

RESUMO

Following lysosomal damage, activation and nuclear translocation of transcription factor EB (TFEB) is the key event to maintain lysosomal homeostasis. Here, we describe steps to induce lysosomal damage in HeLa cells. This can be followed by monitoring the changes in TFEB localization using widefield fluorescence microscopy. As a complementary approach, we describe the use of immunoblotting to follow the activation and localization of TFEB in cell lysates. These protocols enable quantitative analysis of TFEB. For complete details on the use and execution of this protocol, please refer to Nakamura et al. (2020).


Assuntos
Autofagia , Microscopia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Núcleo Celular , Células HeLa , Humanos , Immunoblotting , Lisossomos
14.
Methods Mol Biol ; 2459: 93-103, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35212958

RESUMO

When characterizing posttranslational modifications like phosphorylation, using efficient screening methods to map the phospho sites is essential, especially when dealing with large multi-domain proteins. NLRP3 (the NOD, LRR, and pyrin domain-containing protein 3), which initiates the formation of an NLRP3 inflammasome complex, is regulated posttranslationally by phosphorylation at several Ser and Tyr residues. However, determining sites of modification are not straightforward. For quick and reliable screening of the candidate phospho sites in NLRP3, we use a phospho dot blot assay which we describe here. This technique employs an in vitro kinase assay with a candidate kinase, Bruton's Tyrosine Kinase (BTK), and peptides derived from the region of interest in the protein that contains the potential phosphorylation sites. The reaction containing the phosphorylated peptides is quickly screened by a dot blot where the peptides are blotted with a commercially available anti-phospho-tyrosine antibody. This method can also be adapted to detect modified Ser or Thr residues and is an ideal screening assay to map phospho residues in NLRP3 or other proteins. This can be an initial screening procedure or can be complemented by other approaches such as site directed mutagenesis and by generating phospho site-specific antibodies.


Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Tirosina Quinase da Agamaglobulinemia , Immunoblotting , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fosforilação
15.
Methods Mol Biol ; 2434: 207-215, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35213019

RESUMO

Western blot assays are not adequate for high-throughput screening of protein expression because it is an expensive and time-consuming technique. Here we demonstrate that quantitative dot blots in plate format are a better option to determine the absolute contents of a given protein in less than 48 h. The method was optimized for the detection of the Muscleblind-like 1 protein in patient-derived myoblasts treated with a collection of more than 100 experimental oligonucleotides.


Assuntos
Distrofia Miotônica , Humanos , Immunoblotting , Mioblastos/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Proteínas de Ligação a RNA/metabolismo
16.
Methods Mol Biol ; 2418: 25-39, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35119657

RESUMO

Phosphorylation of the serine residues in estrogen receptor (ER) α is important in transcriptional activation. Hence, methods to detect such posttranslational modification events are valuable. We describe, in detail, the analysis of the phosphorylated ERα by electrophoretic separation of proteins and subsequent immunoblotting techniques. In particular, phosphorylation of the ERα is one possible outcome of activation of the putative membrane estrogen receptor (mER), GPR30 or GPER1. Hence, phosphorylation represents a crosstalk event between GPR30 and ERα and may be important in estrogen-regulated physiology.


Assuntos
Receptor alfa de Estrogênio , Receptores Acoplados a Proteínas G , Estradiol , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Immunoblotting , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Serina/metabolismo
17.
Sci Rep ; 12(1): 1687, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35105907

RESUMO

The aim of this study is to evaluate the relationship between antinuclear antibody (ANA) titer and specificity, as well as the relationship between the number of positive-autoantibodies (AAbs) in antinuclear antibodies (ANAs) and specificity for systemic lupus erythematosus (SLE), so as to explore their significance in the diagnosis of SLE. A total of 1297 patients with ANA results was enrolled in this study, including 148 patients with SLE patients. The sensitivity, specificity, sensitive likelihood ratio and specific likelihood ratio of indicators in SLE were determined by receiver-operator characteristic (ROC) curve after measurement of ANA and ANAs by indirect immunofluorescence (IIF) and immunoblotting, respectively. ROC analysis showed that the specificity of ANA titer ≥ 1 +, ≥ 2 + and ≥ 3 + for SLE was estimated to be 81.29%, 90.69% and 96.52% respectively, with a increased titer-specific likelihood ratio (5.16, 9.29 and 19.60, respectively). The specificity of the number of positive-AAbs ≥ 1, ≥ 2 and ≥ 3 in ANAs for SLE was estimated to be 80.42%, 94.95% and 99.3% respectively, with a increased number-specific likelihood ratio (4.8, 15.26 and 72.48, respectively). The estimated sensitivity of the number of positive-AAbs ≥ 3, AnuA and anti-rRNP was higher than that of anti-Sm (p < 0.01) (50.68%, 41.89% and 31.76% vs. 16.89%, respectively), while there was no significant difference in their specificity (99.3%, 99.74% and 99.56% vs. 99.74%, respectively) (p > 0.05). High titers of ANA and the presence of multiple AAbs in ANAs are highly specific for SLE and highly suggestive of SLE. The likelihood of SLE can be assessed by ANA titer and the number of positive-AAbs in ANAs.


Assuntos
Anticorpos Antinucleares/imunologia , Doenças Hematológicas/imunologia , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/imunologia , Insuficiência Renal/imunologia , Doenças Reumáticas/imunologia , Transtornos Urinários/imunologia , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Técnica Indireta de Fluorescência para Anticorpo/métodos , Humanos , Immunoblotting/métodos , Masculino , Pessoa de Meia-Idade , Curva ROC , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto Jovem
18.
Front Immunol ; 13: 804037, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35154119

RESUMO

Objectives: Anti-TIF1γ is an important autoantibody in the diagnosis of cancer-associated dermatomyositis and the most common autoantibody in juvenile onset dermatomyositis. Its reliable detection is important to instigate further investigations into underlying malignancy in adults. We previously showed that commercial assays using line and dot blots do not reliably detect anti-TIF1γ. We aimed to test a new commercial ELISA and compare with previously obtained protein immunoprecipitation. Methods: Radio-labelled immunoprecipitation had previously been used to determine the autoantibody status of patients with immune-mediated inflammatory myopathies and several healthy controls. ELISA was undertaken on healthy control and anti-TIF1γ sera and compared to previous immunoprecipitation data. Results: A total of 110 serum samples were analysed: 42 myositis patients with anti- TIF1γ and 68 autoantibody negative healthy control sera. Anti-TIF1γ was detected by ELISA in 41 out of 42 of the anti-TIF1γ-positive samples by immunoprecipitation, and in none of the healthy controls, giving a sensitivity of 97.6% and specificity of 100%. The false negative rate was 2%. Conclusion: ELISA is an affordable and time-efficient method which is accurate in detecting anti-TIF1γ.


Assuntos
Autoanticorpos/imunologia , Dermatomiosite/diagnóstico , Dermatomiosite/imunologia , Testes Diagnósticos de Rotina/métodos , Testes Sorológicos/métodos , Fatores de Transcrição/imunologia , Autoanticorpos/sangue , Estudos de Casos e Controles , Confiabilidade dos Dados , Dermatomiosite/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Reações Falso-Negativas , Humanos , Immunoblotting/métodos , Imunoprecipitação/métodos , Sensibilidade e Especificidade
19.
J Orthop Surg Res ; 17(1): 70, 2022 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-35120538

RESUMO

BACKGROUND: Osteoarthritis (OA) is the most prevalent degenerative joint disease. In vitro experiments are an intuitive method used to investigate its early pathogenesis. Chondrocyte inflammation models in rats and mice are often used as in vitro models of OA. However, similarities and differences between them in the early stages of inflammation have not been reported. OBJECTIVE: This paper seeks to compare the chondrocyte phenotype of rats and mice in the early inflammatory state and identify chondrocytes suitable for the study of early OA. METHODS: Under similar conditions, chondrocytes from rats and mice were stimulated using the same IL-1ß concentration for a short period of time. The phenotypic changes of chondrocytes were observed under a microscope. The treated chondrocytes were subjected to RNA-seq to identify similarities and differences in gene expression. Chondrocytes were labelled with EdU for proliferation analysis. Cell proliferation-associated proteins, including minichromosome maintenance 2 (MCM2), minichromosome maintenance 5 (MCM5), Lamin B1, proliferating cell nuclear antigen (PCNA), and Cyclin D1, were analysed by immunocytochemical staining, cell immunofluorescence, and Western blots to verify the RNA-seq results. RESULTS: RNA-seq revealed that the expression patterns of cytokines, chemokines, matrix metalloproteinases, and collagen were similar between the rat and mouse chondrocyte inflammation models. Nonetheless, the expression of proliferation-related genes showed the opposite pattern. The RNA-seq results were further verified by subsequent experiments. The expression levels of MCM2, MCM5, Lamin B1, PCNA, and Cyclin D1 were significantly upregulated in rat chondrocytes (P < 0.05) and mouse chondrocytes (P < 0.05). CONCLUSIONS: Based on the findings, the rat chondrocyte inflammation model may help in the study of the early pathological mechanism of OA.


Assuntos
Proliferação de Células/genética , Condrócitos/metabolismo , Inflamação , Interleucina-1beta/metabolismo , Osteoartrite/metabolismo , Animais , Ciclina D1 , Modelos Animais de Doenças , Expressão Gênica , Immunoblotting , Imuno-Histoquímica , Interleucina-1beta/genética , Camundongos , Osteoartrite/genética , Antígeno Nuclear de Célula em Proliferação , RNA-Seq , Ratos
20.
Nat Commun ; 13(1): 684, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35115561

RESUMO

Loss of pancreatic beta cells is a central feature of type 1 (T1D) and type 2 (T2D) diabetes, but a therapeutic strategy to preserve beta cell mass remains to be established. Here we show that the death receptor TMEM219 is expressed on pancreatic beta cells and that signaling through its ligand insulin-like growth factor binding protein 3 (IGFBP3) leads to beta cell loss and dysfunction. Increased peripheral IGFBP3 was observed in established and at-risk T1D/T2D patients and was confirmed in T1D/T2D preclinical models, suggesting that dysfunctional IGFBP3/TMEM219 signaling is associated with abnormalities in beta cells homeostasis. In vitro and in vivo short-term IGFBP3/TMEM219 inhibition and TMEM219 genetic ablation preserved beta cells and prevented/delayed diabetes onset, while long-term IGFBP3/TMEM219 blockade allowed for beta cell expansion. Interestingly, in several patients' cohorts restoration of appropriate IGFBP3 levels was associated with improved beta cell function. The IGFBP3/TMEM219 pathway is thus shown to be a physiological regulator of beta cell homeostasis and is also demonstrated to be disrupted in T1D/T2D. IGFBP3/TMEM219 targeting may therefore serve as a therapeutic option in diabetes.


Assuntos
Regulação da Expressão Gênica , Homeostase/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Células Secretoras de Insulina/metabolismo , Proteínas de Membrana/genética , Transdução de Sinais/genética , Adulto , Animais , Células Cultivadas , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Humanos , Immunoblotting , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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