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2.
Nat Commun ; 11(1): 4124, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807787

RESUMO

In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Reparo do DNA/fisiologia , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Células HCT116 , Humanos , Immunoblotting , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Nanomedicina/métodos , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
3.
Nat Commun ; 11(1): 4053, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792481

RESUMO

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequenciamento Completo do Exoma/métodos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ciclina D1/genética , Variações do Número de Cópias de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Nus , Piperazinas/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Pteridinas/uso terapêutico , Piridinas/uso terapêutico
4.
Nat Commun ; 11(1): 4070, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792502

RESUMO

Human astroviruses are small non-enveloped viruses with positive-sense single-stranded RNA genomes. Astroviruses cause acute gastroenteritis in children worldwide and have been associated with encephalitis and meningitis in immunocompromised individuals. It is still unknown how astrovirus particles exit infected cells following replication. Through comparative genomic analysis and ribosome profiling we here identify and confirm the expression of a conserved alternative-frame ORF, encoding the protein XP. XP-knockout astroviruses are attenuated and pseudo-revert on passaging. Further investigation into the function of XP revealed plasma and trans Golgi network membrane-associated roles in virus assembly and/or release through a viroporin-like activity. XP-knockout replicons have only a minor replication defect, demonstrating the role of XP at late stages of infection. The discovery of XP advances our knowledge of these important human viruses and opens an additional direction of research into their life cycle and pathogenesis.


Assuntos
Canais Iônicos/metabolismo , Mamastrovirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Genômica/métodos , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Canais Iônicos/genética , Mamastrovirus/genética , Microscopia de Fluorescência , Plasmídeos/genética , Ribossomos , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Replicação Viral/fisiologia
5.
Nat Commun ; 11(1): 3464, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651375

RESUMO

DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCKDHR2) and membrane-associated (DOCKDHR1) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMORBD) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2-ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG-ELMO2RBD complex. The binary DOCK2-ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto-inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2DHR2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Calorimetria , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Cinética , Microscopia Eletrônica , Fosforilação , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
6.
Exp Parasitol ; 217: 107948, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32698076

RESUMO

Immunomodulation is an emerging concept to combat infection in recent years. Immunomodulators like arabinosylated-lipoarabinomannan (Ara-LAM) and glycyrrhizic-acid (GA) possess anti-leishmanial property, whereas sodium-antimony-gluconate (SAG) is still considered as the first choice for chemotherapy against leishmaniasis. During infection, invasion of Leishmania donovani needs the potential requirement of Ca2+, which is further responsible for apoptosis in intracellular amastigotes. However, suppression of elevated intracellular calcium by the activation of plasma-membrane-calcium-ATPase (PMCA4) facilitates survival of L. donovani in the host. In the present study, SAG, Ara-LAM, and GA were found to evoke significant increase in intracellular Ca2+ in L. donovani infected macrophages by inhibiting PMCA4. Moreover, PMCA4 inhibition by TFP or PMCA4 siRNA elevated the level of PKCß, whereas calcium-independent upregulation of PKCζ remained unchanged in infected macrophages. Furthermore, application of immunomodulators in infected macrophages resulted in down-regulation of PKCζ, conversion of anti-inflammatory to pro-inflammatory cytokines and inhibition of PMCA4. Plasma membrane-associated ceramide which is known to be elevated during leishmaniasis, triggered upregulation of PMCA4 via PKCζ activation. Interestingly, immunomodulators attenuated ceramide generation, which resulted into reduced PKCζ activation leading to the decreased PMCA expression in infected macrophages. Therefore, our study elucidated the efficacy of SAG, Ara-LAM, and GA in the reduction of parasite burden in macrophages by suppressing PMCA activation through inhibition of ceramide mediated upregulation of PKCζ.


Assuntos
Antiprotozoários/uso terapêutico , ATPases Transportadoras de Cálcio/sangue , Membrana Celular/enzimologia , Fatores Imunológicos/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Animais , Gluconato de Antimônio e Sódio/farmacologia , Gluconato de Antimônio e Sódio/uso terapêutico , Antiprotozoários/farmacologia , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Ceramidas/metabolismo , Meios de Cultura Livres de Soro , Densitometria , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Ácido Glicirrízico/farmacologia , Ácido Glicirrízico/uso terapêutico , Imipramina/farmacologia , Immunoblotting , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/uso terapêutico , Macrófagos/fisiologia , Camundongos , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa , Transfecção
7.
Autoimmun Rev ; 19(8): 102588, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32540447

RESUMO

The commercial tests currently available as second-level tests to detect ANA sub-specificities are generally used independently from the ANA immunofluorescence (IIF) pattern. The aim of this study was to evaluate the efficacy of the use of a customizable pattern-oriented antigenic panel by immunoblot (IB) using the International Consensus on ANA Patterns (ICAP) classification scheme, in order to introduce a novel and updated autoimmune diagnostic flowchart. 710 sera referred for routine ANA testing were selected on the basis of the ANA pattern according to the ICAP nomenclature (nuclear speckled AC-2,4,5; nucleolar AC-8,9,10,29; cytoplasmic speckled AC-18,19,20) and on an IIF titer ≥1:320. They were then assayed by three experimental IB assays using a panel of selected antigens. ICAP-oriented IB detected 515 antibody reactivities vs. 457 of traditional anti-ENA in the nuclear speckled pattern group, 108 vs. 28 in the nucleolar pattern group, and 43 vs. 34 in the cytoplasmic speckled pattern. This pilot study may lead the way for a new approach introducing an ICAP pattern-oriented follow up testing as a valid alternative to the existing standard panels, thus enabling more patients with autoimmune rheumatic disease to be accurately diagnosed.


Assuntos
Algoritmos , Anticorpos Antinucleares , Doenças Autoimunes , Técnicas e Procedimentos Diagnósticos , Immunoblotting , Anticorpos Antinucleares/sangue , Doenças Autoimunes/sangue , Doenças Autoimunes/diagnóstico , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Immunoblotting/normas , Projetos Piloto
8.
Ann Rheum Dis ; 79(9): 1194-1202, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32532752

RESUMO

OBJECTIVES: Porphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007 (RACH2007-PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA. METHODS: Recombinant RACH2007-PPAD was cloned and expressed in Escherichia coli. Purified RACH2007-PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated. RESULTS: RACH2007-PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RACH2007-PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57-71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RACH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RACH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RACH2007-PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018). CONCLUSIONS: RACH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RACH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry.


Assuntos
Anticorpos Anti-Proteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Infecções por Bacteroidaceae/imunologia , Epitopos/imunologia , Porphyromonas gingivalis/imunologia , Artrite Reumatoide/microbiologia , Infecções por Bacteroidaceae/microbiologia , Citrulinação/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Peptídeos/imunologia , Desiminases de Arginina em Proteínas/imunologia
9.
Nat Commun ; 11(1): 2319, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385234

RESUMO

Bruton's tyrosine kinase (Btk) is critical for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, Btk signaling sustains growth of several B-cell neoplasms which may be treated with tyrosine kinase inhibitors (TKIs). Here, we uncovered the structural mechanism by which certain XLA mutations in the SH2 domain strongly perturb Btk activation. Using a combination of molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS), we discovered an allosteric interface between the SH2 and kinase domain required for Btk activation and to which multiple XLA mutations map. As allosteric interactions provide unique targeting opportunities, we developed an engineered repebody protein binding to the SH2 domain and able to disrupt the SH2-kinase interaction. The repebody prevents activation of wild-type and TKI-resistant Btk, inhibiting Btk-dependent signaling and proliferation of malignant B-cells. Therefore, the SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition.


Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Cristalografia por Raios X/métodos , Linfoma/metabolismo , Tirosina Quinase da Agamaglobulinemia/genética , Western Blotting , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Dicroísmo Circular , Citometria de Fluxo , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Linfoma/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação/genética
10.
Infect Dis Poverty ; 9(1): 38, 2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-32299480

RESUMO

BACKGROUND: Drug resistant tuberculosis poses a great challenge for tuberculosis control worldwide. Timely determination of drug resistance and effective individual treatment are essential for blocking the transmission of drug resistant Mycobacterium tuberculosis. We aimed to establish and evaluate the accuracy of a reverse dot blot hybridization (RDBH) assay to simultaneously detect the resistance of four anti-tuberculosis drugs in M. tuberculosis isolated in China. METHODS: In this study, we applied a RDBH assay to simultaneously detect the resistance of rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB) in 320 clinical M. tuberculosis isolates and compared the results to that from phenotypic drug susceptibility testing (DST) and sequencing. The RDBH assay was designed to test up to 42 samples at a time. Pearson's chi-square test was used to compute the statistical measures of the RDBH assay using the phenotypic DST or sequencing as the gold standard method, and Kappa identity test was used to determine the consistency between the RDBH assay and the phenotypic DST or sequencing. RESULTS: The results showed that the concordances between phenotypic DST and RDBH assay were 95% for RIF, 92.8% for INH, 84.7% for SM, 77.2% for EMB and the concordances between sequencing and RDBH assay were 97.8% for RIF, 98.8% for INH, 99.1% for SM, 93.4% for EMB. Compared to the phenotypic DST results, the sensitivity and specificity of the RDBH assay for resistance detection were 92.4 and 98.5% for RIF, 90.3 and 97.3% for INH, 77.4 and 91.5% for SM, 61.4 and 85.7% for EMB, respectively; compared to sequencing, the sensitivity and specificity of the RDBH assay were 97.7 and 97.9% for RIF, 97.9 and 100.0% for INH, 97.8 and 100.0% for SM, 82.6 and 99.1% for EMB, respectively. The turnaround time of the RDBH assay was 7 h for testing 42 samples. CONCLUSIONS: Our data suggested that the RDBH assay could serve as a rapid and efficient method for testing the resistance of M. tuberculosis against RIF, INH, SM and EMB, enabling early administration of appropriate treatment regimens to the affected drug resistant tuberculosis patients.


Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Immunoblotting/métodos , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/microbiologia , China , Etambutol/farmacologia , Humanos , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana/instrumentação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Rifampina/farmacologia , Sensibilidade e Especificidade , Estreptomicina/farmacologia
11.
Life Sci ; 246: 117397, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32032646

RESUMO

BACKGROUND: Paraoxonase (PON), an antioxidant enzyme, comprises of three members (PON1, 2 and 3). Hyperglycemia accelerates formation of AGE in diabetes which mediates endothelial dysfunction. PON1 is studied in diabetes due to its association with HDL, lipid peroxidation and vascular complications but PON2 is not explored much. Recently decreased PON2 activity is reported in monocytes of Type 2 diabetes mellitus (T2DM) patients but its regulation by factors like high glucose and AGE has not been studied. AIM: The aim of the current study is to identify the effect of AGEs on PON2 expression and activity and its implications on endothelial dysfunction in HUVECs. MAIN METHODS: HUVECs were exposed to Glycated albumin (GA)/Carboxymethyl lysine (CML) for 24 h to check for PON2 expression. The ER stress markers GRP78 and IRE1α, pro-inflammatory genes MCP-1, IL-6, IL-8, ICAM1, VCAM1 were assessed by qPCR, western blotting/ELISA. Endothelial-leukocyte adhesion and ROS were assessed using Calcein AM and DCFDA method. One-way ANOVA and student's t-test was done using Graphpad Prism. KEY FINDINGS: AGE exposure significantly decreased PON2 expression and activity with increased oxidative stress, ER stress and inflammation. PON2 overexpression significantly reduced ROS, ER stress and inflammation as well as inhibited NFκB, and ERK1/2, phosphorylation induced by GA/CML treatment. Concomitantly, silencing of PON2 accelerated AGEs induced effects. SIGNIFICANCE: This is the first study to show that both GA and CML down regulates the PON2 expression and activity in HUVECs and over expression of PON2 alleviates AGEs induced endothelial dysfunction.


Assuntos
Arildialquilfosfatase/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Inflamação/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática , Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Immunoblotting , Inflamação/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
12.
Int J Mol Sci ; 21(3)2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-32046264

RESUMO

Interleukin (IL)-33 is a member of the IL-1 family, which acts as an alarmin. Several studies suggested that IL-33 inhibited osteoclastogenesis and bone resorption. Tumor necrosis factor-α (TNF-α) is considered a direct inducer of osteoclastogenesis. However, there has been no report regarding the effect of IL-33 on TNF-α-induced osteoclastogenesis and bone resorption. The objective of this study is to investigate the role of IL-33 on TNF-α-induced osteoclastogenesis and bone resorption. In an in vitro analysis of osteoclastogenesis, osteoclast precursors, which were derived from bone marrow cells, were treated with or without IL-33 in the presence of TNF-α. Tartrate-resistant acid phosphatase (TRAP) staining solution was used to assess osteoclast formation. In an in vivo analysis of mouse calvariae, TNF-α with or without IL-33 was subcutaneously administrated into the supracalvarial region of mice daily for 5 days. Histological sections were stained for TRAP, and osteoclast numbers were determined. Using micro-CT reconstruction images, the ratio of bone destruction area on the calvariae was evaluated. The number of TRAP-positive cells induced by TNF-α was significantly decreased with IL-33 in vitro and in vivo. Bone resorption was also reduced. IL-33 inhibited IκB phosphorylation and NF-κB nuclear translocation. These results suggest that IL-33 inhibited TNF-α-induced osteoclastogenesis and bone resorption.


Assuntos
Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/tratamento farmacológico , Interleucina-33/farmacologia , Interleucina-33/uso terapêutico , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Imunofluorescência , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Osteoclastos/metabolismo , Fosforilação/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
PLoS One ; 15(2): e0229036, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084172

RESUMO

LAT molecules defective in ubiquitination have an increased half-life and induce enhanced signaling when expressed in T cells. In this study, we have examined the role of ubiquitination in regulating LAT endocytosis, recycling, and degradation in resting and stimulated T cells. By tracking and comparing plasma membrane-labeled wild type and ubiquitination-resistant 2KR LAT, we find that ubiquitination promotes the degradation of surface LAT in T cells. Activation of T cells increases LAT ubiquitination and promotes trafficking of internalized LAT to lysosomes for degradation. Ubiquitination of LAT does not change internalization rates from the cell surface, but prevents efficient recycling of LAT to the surface of T cells. Our study demonstrates that surface LAT levels are tightly controlled by ubiquitination. LAT in unstimulated cells lacks ubiquitin allowing for increased LAT stability and efficient T cell activation upon TCR triggering; ubiquitination leads to efficient removal of LAT after activation.


Assuntos
Ativação Linfocitária/fisiologia , Ubiquitinação/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Endocitose/fisiologia , Humanos , Immunoblotting , Lisossomos/metabolismo , Microscopia Confocal , Fosforilação/fisiologia , Transdução de Sinais/fisiologia
14.
Invest Ophthalmol Vis Sci ; 61(1): 3, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31995154

RESUMO

Purpose: The purpose of this study was to investigate the expression of death ligands in the lacrimal glands (LGs), identify upstream factors that regulate their expression, and determine the functional roles of these factors in the pathogenesis of dry eye disease (DED). Methods: For DED experiment, ex vivo coculture system with LG and in vivo murine model using a controlled environment chamber were utilized. C57BL/6 mice and hypoxia-inducible factor (HIF)-1α conditional knockout (CKO) mice were used. Immunohistochemical staining, polymerase chain reaction, and immunoblotting were performed to determine levels of death ligands including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in DED-induced LGs. Additionally, acinar cell and CD45+ cell apoptosis was determined with neutralizing TRAIL treatment. Results: Desiccating stress significantly increased HIF-1α expression in LG-acinar cells. Furthermore, HIF-1α deficiency significantly enhanced the infiltration of CD45+ inflammatory cells in LG and induced LG-acinar cell death. Meanwhile, only TRAIL expression was increased in DED-LG, but abrogated in HIF-1α CKO. Interestingly, the main source of TRAIL was the CD45- LG-acinar cells, but not CD45+ immune cells after DED induction. Using ex vivo coculture system, we confirmed LG-induced apoptosis of immune cells via HIF-1α-mediated TRAIL secretion following DED. Consistent with ex vivo, the insufficiency of HIF-1α and TRAIL enhanced recruitment of inflammatory cells to the LG and subsequently exacerbated ocular surface damage in DED mice. Conclusions: Our findings offer novel insight into the regulatory function of acinar cell-derived TRAIL in limiting inflammatory damage and could be implicated in the development of potential therapeutic strategies for DED.


Assuntos
Dacriocistite/metabolismo , Síndromes do Olho Seco/metabolismo , Regulação da Expressão Gênica/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Aparelho Lacrimal/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Apoptose , Técnicas de Cocultura , Dacriocistite/patologia , Modelos Animais de Doenças , Síndromes do Olho Seco/patologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Aparelho Lacrimal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase em Tempo Real
15.
Arterioscler Thromb Vasc Biol ; 40(2): 335-349, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31941383

RESUMO

OBJECTIVE: Cardiovascular disease is a major public health problem. Among cardiovascular disease's risk factors, tobacco smoking is considered the single most preventable cause of death, with thrombosis being the main mechanism of cardiovascular disease mortality in smokers. While tobacco smoking has been on the decline, the use of waterpipes/hookah has been rising, mainly due to the perception that they are less harmful than regular cigarettes. Strikingly, there are few studies on the negative effects of waterpipes on the cardiovascular system, and none regarding their direct contribution to thrombus formation. Approach and Results: We used a waterpipe whole-body exposure protocol that mimics real-life human exposure scenarios and investigated its effects, relative to clean air, on platelet function, hemostasis, and thrombogenesis. We found that waterpipe smoke (WPS)-exposed mice exhibited both shortened thrombus occlusion and bleeding times. Further, our results show that platelets from WPS-exposed mice are hyperactive, with enhanced agonist-induced aggregation, dense and α-granule secretion, αIIbß3 integrin activation, phosphatidylserine expression, and platelet spreading, when compared with clean air-exposed platelets. Finally, at the molecular level, it was found that Akt (protein kinase B) and ERK (extracellular signal-regulated kinases) phosphorylation are enhanced in the WPS and in nicotine-treated platelets. CONCLUSIONS: Our findings demonstrate that WPS exposure directly modulates hemostasis and increases the risk of thrombosis and that this is mediated, in part, via a state of platelet hyperactivity. The negative health impact of WPS/hookah, therefore, should not be underestimated. Moreover, this study should also help in raising public awareness of the toxic effects of waterpipe/hookah.


Assuntos
Plaquetas/efeitos dos fármacos , Artérias Carótidas/efeitos dos fármacos , Ativação Plaquetária/fisiologia , Cachimbos de Água , Fumar/efeitos adversos , Trombose/metabolismo , Animais , Plaquetas/metabolismo , Artérias Carótidas/patologia , Cotinina/toxicidade , Modelos Animais de Doenças , Citometria de Fluxo , Seguimentos , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nicotina/toxicidade , Contagem de Plaquetas , Fumaça/efeitos adversos , Trombose/induzido quimicamente , Fatores de Tempo
16.
Exp Eye Res ; 191: 107933, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31935380

RESUMO

The interaction of keratocytes with extracellular matrix components plays an important role in the maintenance of corneal transparency and shape as well as in the healing of corneal wounds. In particular, the interaction of these cells with collagen and cell-mediated collagen contraction contribute to wound closure. Endo180 is a receptor for collagen that mediates its cellular internalization. We have now examined the role of Endo180 in collagen contraction mediated by corneal fibroblasts (activated keratocytes). Antibodies to Endo180 inhibited the contractile activity of mouse corneal fibroblasts embedded in a three-dimensional collagen gel and cultured in the presence of serum, with this effect being both concentration and time dependent and essentially complete at an antibody concentration of 0.2 µg/ml. Whereas corneal fibroblasts cultured in a collagen gel manifested a flattened morphology with prominent stress fibers under control conditions, they showed a spindlelike shape with few stress fibers in the presence of antibodies to Endo180. Antibodies to Endo180 had no effect on the expression of α-smooth muscle actin or the extent of collagen degradation in collagen gel cultures of corneal fibroblasts. Immunohistofluorescence analysis did not detect the expression of Endo180 in the unwounded mouse cornea. However, Endo180 expression was detected in keratocytes migrating into the wound area at 3 days after a corneal incisional injury. Together, our results suggest that Endo180 is required for the contraction of collagen matrix mediated by corneal fibroblasts and that its expression in these cells may contribute to the healing of corneal stromal wounds.


Assuntos
Colágeno/metabolismo , Ceratócitos da Córnea/metabolismo , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Cicatrização/fisiologia , Actinas/metabolismo , Animais , Anticorpos/farmacologia , Células Cultivadas , Ceratócitos da Córnea/citologia , Ceratócitos da Córnea/efeitos dos fármacos , Substância Própria/citologia , Immunoblotting , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/imunologia , Fator de Crescimento Transformador beta/farmacologia
17.
Monoclon Antib Immunodiagn Immunother ; 39(1): 23-26, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31916900

RESUMO

Stromal cell-derived factor-2 (SDF-2) is reportedly involved in multiple endoplasmic reticulum (ER) functions, including the misfolded protein catabolic process, protein glycosylation, and ER protein quality control. However, the precise molecular and cellular functions of SDF-2 remain unknown. Previously, we discovered that SDF-2 mediates acquired resistance to oxaliplatin in human gastric cancer cells. In this study, we have generated SDF-2-specific monoclonal antibodies (mAbs), using the rat medial iliac lymph node method, as a tool to explore novel mechanisms of oxaliplatin resistance. The antibodies detected endogenous human SDF-2 in immunoblotting analyses. In addition, immunoprecipitation analyses revealed the availability of these antibodies for human SDF-2. Thus, these mAbs will be available to elucidate molecular and cellular functions of SDF-2 in cancer cells.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas/imunologia , Animais , Immunoblotting , Imunoprecipitação , Linfonodos/imunologia , Linfonodos/metabolismo , Oxaliplatina , Ratos
18.
Dtsch Med Wochenschr ; 145(1): 29-34, 2020 01.
Artigo em Alemão | MEDLINE | ID: mdl-31914466

RESUMO

Lyme borreliosis caused by at least six Borrelia burgdorferi species, is the most important tick-borne disease in the northern hemisphere. With a wide spectrum of possible symptoms, the microbiological diagnosis of this disease is associated with a wide variety of ideas, divergent approaches and much uncertainty. The aim of the article is to introduce the treating physicians to a meaningful microbiological diagnostic procedure. Most important messages include that (I) the suspected diagnosis of Lyme borreliosis is first based on anamnestic data and clinical symptoms (compare "case definitions") which is substantiated by microbiological examinations; (II) microbiological diagnostics - primarily antibody detection, downstream PCR and culture - are only indicated if there are indicative symptoms, with typical erythema migrans not requiring microbiological diagnostics; (III) keep in mind: The more unspecific the symptoms are the lower the positive and the higher the negative predictive value of microbiological testing; (IV) serological diagnosis should follow a two-step procedure: a sensitive ELISA as first step, if reactive followed by immunoblot (IgM and IgG). Detection rates are ca. 50 % in localized, 70- > 90 % in disseminated early and nearly 100 % (only IgG relevant) in late disease; (V) in the immunoblot early forms of disease show a narrow band spectrum, late forms show a broad spectrum; (VI) methods that are not recommended for diagnostic purposes include lymphocyte activation or transformation tests (LTT, MELISA, ELISPOT), PCR or antigen detection from urine or blood, lymphocyte subpopulations, or direct detection of borreliae from patient material using dark field- or focus floating microscopy.


Assuntos
Doença de Lyme , Anticorpos Antibacterianos/sangue , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Doença de Lyme/diagnóstico , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Reação em Cadeia da Polimerase
19.
Arterioscler Thromb Vasc Biol ; 40(1): 159-174, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31619060

RESUMO

OBJECTIVE: In proliferative retinopathies, complications derived from neovascularization cause blindness. During early disease, pericyte's apoptosis contributes to endothelial dysfunction and leakage. Hypoxia then drives VEGF (vascular endothelial growth factor) secretion and pathological neoangiogenesis. Cardiac ANP (atrial natriuretic peptide) contributes to systemic microcirculatory homeostasis. ANP is also formed in the retina, with unclear functions. Here, we characterized whether endogenously formed ANP regulates retinal (neo)angiogenesis. Approach and Results: Retinal vascular development and ischemia-driven neovascularization were studied in mice with global deletion of GC-A (guanylyl cyclase-A), the cGMP (cyclic guanosine monophosphate)-forming ANP receptor. Mice with a floxed GC-A gene were interbred with Tie2-Cre, GFAP-Cre, or PDGF-Rß-CreERT2 lines to dissect the endothelial, astrocyte versus pericyte-mediated actions of ANP in vivo. In neonates with global GC-A deletion (KO), vascular development was mildly delayed. Moreover, such KO mice showed augmented vascular regression and exacerbated ischemia-driven neovascularization in the model of oxygen-induced retinopathy. Notably, absence of GC-A in endothelial cells did not impact retinal vascular development or pathological neovascularization. In vitro ANP/GC-A/cGMP signaling, via activation of cGMP-dependent protein kinase I, inhibited hypoxia-driven astrocyte's VEGF secretion and TGF-ß (transforming growth factor beta)-induced pericyte apoptosis. In neonates lacking ANP/GC-A signaling in astrocytes, vascular development and hyperoxia-driven vascular regression were unaltered; ischemia-induced neovascularization was modestly increased. Remarkably, inactivation of GC-A in pericytes retarded physiological retinal vascularization and markedly enhanced cell apoptosis, vascular regression, and subsequent neovascularization in oxygen-induced retinopathy. CONCLUSIONS: Protective pericyte effects of the ANP/GC-A/cGMP pathway counterregulate the initiation and progression of experimental proliferative retinopathy. Our observations indicate augmentation of endogenous pericyte ANP signaling as target for treatment of retinopathies associated with neovascularization.


Assuntos
Astrócitos/metabolismo , GMP Cíclico/genética , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos Natriuréticos/metabolismo , Pericitos/metabolismo , RNA/genética , Neovascularização Retiniana/genética , Animais , Animais Recém-Nascidos , Apoptose , Astrócitos/patologia , Células Cultivadas , GMP Cíclico/biossíntese , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Camundongos , Camundongos Transgênicos , Pericitos/patologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Transdução de Sinais
20.
Arterioscler Thromb Vasc Biol ; 40(2): 378-393, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31826650

RESUMO

OBJECTIVE: Vascular endothelial (VE)-cadherin is of dominant importance for the formation and stability of endothelial junctions, yet induced gene inactivation enhances vascular permeability in the lung but does not cause junction rupture. This study aims at identifying the junctional adhesion molecule, which is responsible for preventing endothelial junction rupture in the pulmonary vasculature in the absence of VE-cadherin. Approach and Results: We have compared the relevance of ESAM (endothelial cell-selective adhesion molecule), JAM (junctional adhesion molecule)-A, PECAM (platelet endothelial cell adhesion molecule)-1, and VE-cadherin for vascular barrier integrity in various mouse tissues. Gene inactivation of ESAM enhanced vascular permeability in the lung but not in the heart, skin, and brain. In contrast, deletion of JAM-A or PECAM-1 did not affect barrier integrity in any of these organs. Blocking VE-cadherin with antibodies caused lethality in ESAM-/- mice within 30 minutes but had no such effect in JAM-A-/-, PECAM-1-/- or wild-type mice. Likewise, induced gene inactivation of VE-cadherin caused rapid lethality only in the absence of ESAM. Ultrastructural analysis revealed that only combined interference with VE-cadherin and ESAM disrupted endothelial junctions and caused massive blood coagulation in the lung. Mechanistically, we could exclude a role of platelet ESAM in coagulation, changes in the expression of other junctional proteins or a contribution of cytoplasmic signaling domains of ESAM. CONCLUSIONS: Despite well-documented roles of JAM-A and PECAM-1 for the regulation of endothelial junctions, only for ESAM, we detected an essential role for endothelial barrier integrity in a tissue-specific way. In addition, we found that it is ESAM which prevents endothelial junction rupture in the lung when VE-cadherin is absent.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar/fisiologia , Moléculas de Adesão Celular/metabolismo , Morte Celular/fisiologia , Endotélio Vascular/metabolismo , Pulmão/metabolismo , Animais , Coagulação Sanguínea/fisiologia , Adesão Celular , Células Cultivadas , Cricetinae , Endotélio Vascular/ultraestrutura , Feminino , Immunoblotting , Pulmão/irrigação sanguínea , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Modelos Animais , Transdução de Sinais
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