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1.
Science ; 369(6507)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32855309

RESUMO

Neuronal synapses undergo structural and functional changes throughout life, which are essential for nervous system physiology. However, these changes may also perturb the excitatory-inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we designed and characterized CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1. CPTX can interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors and induced the formation of excitatory synapses both in vitro and in vivo. CPTX restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimer's disease, and spinal cord injury, respectively. Thus, CPTX represents a prototype for structure-guided biologics that can efficiently repair or remodel neuronal circuits.


Assuntos
Proteína C-Reativa/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Vias Neurais/efeitos dos fármacos , Precursores de Proteínas/farmacologia , Receptores de AMPA/metabolismo , Proteínas Recombinantes/farmacologia , Sinapses/efeitos dos fármacos , Doença de Alzheimer/terapia , Animais , Proteína C-Reativa/química , Proteína C-Reativa/uso terapêutico , Ataxia Cerebelar/terapia , Modelos Animais de Doenças , Células HEK293 , Hipocampo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/uso terapêutico , Domínios Proteicos , Precursores de Proteínas/química , Precursores de Proteínas/uso terapêutico , Receptores de Glutamato/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêutico , Coluna Vertebral/efeitos dos fármacos , Coluna Vertebral/fisiologia
2.
Subcell Biochem ; 94: 499-520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32189313

RESUMO

C-reactive protein (CRP) is an evolutionary highly conserved member of the pentraxin superfamily of proteins. CRP is widely used as a marker of inflammation, infection and for risk stratification of cardiovascular events. However, there is now a large body of evidence, that continues to evolve, detailing that CRP directly mediates inflammatory reactions and the innate immune response in the context of localised tissue injury. These data support the concept that the pentameric conformation of CRP dissociates into pro-inflammatory CRP isoforms termed pCRP* and monomeric CRP. These pro-inflammatory CRP isoforms undergo conformational changes that facilitate complement binding and immune cell activation and therefore demonstrate the ability to trigger complement activation, activate platelets, monocytes and endothelial cells. The dissociation of pCRP occurs on the surface of necrotic, apoptotic, and ischaemic cells, regular ß-sheet structures such as ß-amyloid, the membranes of activated cells (e.g., platelets, monocytes, and endothelial cells), and/or the surface of microparticles, the latter by binding to phosphocholine. Therefore, the deposition and localisation of these pro-inflammatory isoforms of CRP have been demonstrated to amplify inflammation and tissue damage in a broad range of clinical conditions including ischaemia/reperfusion injury, Alzheimer's disease, age-related macular degeneration and immune thrombocytopaenia. Given the potentially broad relevance of CRP to disease pathology, the development of inhibitors of CRP remains an area of active investigation, which may pave the way for novel therapeutics for a diverse range of inflammatory diseases.


Assuntos
Proteína C-Reativa/química , Proteína C-Reativa/metabolismo , Sequência Conservada , Evolução Molecular , Inflamação/metabolismo , Inflamação/patologia , Biomarcadores/química , Biomarcadores/metabolismo , Humanos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo
3.
Mol Immunol ; 117: 122-130, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31765841

RESUMO

The acute phase reactant C-reactive protein (CRP) binds with high affinity to fibronectin (FN), but this binding occurs only at pH 6.5 or lower, and the binding is inhibited by calcium ions at physiological pH. Since CRP in the circulating blood exists in a calcium-binding form, the interaction between CRP and FN in vivo has been uncertain. CRP can undergo a conformational rearrangement in the absence of calcium or in the local microenvironment (e.g., acidic pH) of inflamed tissue to dissociate into monomeric CRP (mCRP). Therefore, we tested whether these discrepancies can be explained by the different isoforms and locations of CRP. Surface plasmon resonance and ELISA assays showed that mCRP binds with high affinity to FN, and the binding of mCRP to FN was unaffected by calcium or pH. Peptide competition assay, deletion mutant binding assay and protein docking analyse verified that the binding site of mCRP to FN is residues a.a.35-47. Furthermore, mCRP can significantly enhance the adhesion of monocytes to FN as well as upregulate the adhesion molecules expression on endothelial cell. Colocalization of mCRP with FN was observed in mice with DSS-induced colitis, whereas there was very little signal orcolocalization of CRP. These results provide in vitro and in vivo evidence that mCRP formed by local dissociation from circulating CRP is the major isoform that interacts with FN and regulates FN-mediated monocyte adhesion, which is involved in the pro-inflammatory process.


Assuntos
Proteína C-Reativa/metabolismo , Adesão Celular/fisiologia , Fibronectinas/metabolismo , Monócitos/metabolismo , Animais , Proteína C-Reativa/química , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Monócitos/química , Ligação Proteica/fisiologia
4.
Sci Rep ; 9(1): 17131, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31748592

RESUMO

In this study, we developed a portable smartphone-based diffusometry for analyzing the C-reactive protein (CRP) concentration. An optimized fluorescence microscopic add-on system for a smartphone was used to image the 300 nm fluorescent beads. Sequential nanobead images were recorded for a period and the image data were used for fluorescence correlation spectrometric (FCS) analysis. Through the analysis, the nanobeads' diffusion coefficient was obtained. Further, the diffusion coefficients of the anti-CRP-coated nanobeads, which were suspended in the samples with various CRP concentrations, were estimated using smartphone-based diffusometry. After 10 min of reaction, the anti-CRP-coated nanobeads in a higher CRP concentration solution led to a lower diffusion coefficient. Based on the experiments, a linear sensing range of 1~8 µg/mL was found.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Proteína C-Reativa/química , Imunoensaio/instrumentação , Imunoensaio/métodos , Fluorescência , Testes Imunológicos/instrumentação , Testes Imunológicos/métodos , Nanopartículas/química , Smartphone , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos
5.
Anal Chem ; 91(21): 13377-13382, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31585029

RESUMO

Lateral flow immunoassay devices have revolutionized the style of on-site disease detection and point-of-care testing in the past few decades. The surface nanotopography of a solid substrate is a dominant parameter in the efficiency of antibody immobilization, but precise control over surface roughness has not been fully investigated. Here we presented lateral flow immunoassay platforms with nanometer-scale surface roughness, reproducibly engineered using thermal nanoimprinting lithography, and investigated the effects of surface nanotopography on immunoadsorption and immunoassay performance. We fabricated three types of imprinted polycarbonate sheets with microcone array structures having different degrees of surface roughness using three types of molds fabricated by micromachining or laser ablation. The structures fabricated by laser-ablated nickel mold exhibited numerous bumps measuring several tens of nanometers, which enhanced antibody adsorption. We performed sandwich immunoassays of C-reactive protein in serum samples and achieved highly sensitive detection with a detection limit of ∼0.01 µg mL-1 and a broad dynamic range. The present results provide useful information on the remarkable effect of nanoengineered surfaces on biomolecule adsorption, and the platforms presented here will widen the applicability and versatility of lateral flow immunoassay devices.


Assuntos
Proteína C-Reativa/química , Imunoensaio/instrumentação , Nanoestruturas , Polímeros/química , Adsorção , Bioensaio/métodos , Imunoensaio/métodos , Limite de Detecção , Propriedades de Superfície
6.
Fish Shellfish Immunol ; 94: 318-326, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31513914

RESUMO

C-reactive protein (CRP) and serum amyloid P (SAP) play essential roles in the phagocytic cell-mediated innate immune response of mammals. In-depth studies into CRP and SAP have been completed in mammals; however, such studies, particularly those relating to the functions of CRP and SAP, are rare in fish species. In this study, a homolog of CRP/SAP (BpCRP/SAP) was identified in mudskipper (Boleophthalmus pectinirostris), which had the typical characteristics of a fish short pentraxin protein. Phylogenetic tree analysis revealed that BpCRP/SAP was most closely related to mudskipper CRP/SAP-l3. BpCRP/SAP transcripts were detected in all tested tissues, with the highest level observed in the liver; transcripts in the immune tissues and protein expression in the serum were induced in response to Edwardsiella tarda infection. The active recombinant BpCRP/SAP (rBpCRP/SAP) was able to augment the mRNA expression of pro-inflammatory cytokines and attenuate the mRNA expression of anti-inflammatory cytokines in monocytes/macrophages (MO/MΦ). In addition, phagocytosis and bacterial killing of E. tarda by mudskipper MO/MΦ were boosted by rBpCRP/SAP stimulation. rBpCRP/SAP also promoted M1-type MO/MΦ polarization, but inhibited M2-type polarization. In conclusion, the present research describes the pro-inflammatory function of BpCRP/SAP in mudskipper against E. tarda infection.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteína C-Reativa/química , Proteína C-Reativa/genética , Proteína C-Reativa/imunologia , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Proteínas de Peixes/química , Perfilação da Expressão Gênica/veterinária , Macrófagos/imunologia , Monócitos/imunologia , Filogenia , Alinhamento de Sequência/veterinária , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/genética , Componente Amiloide P Sérico/imunologia
7.
Biochim Biophys Acta Gene Regul Mech ; 1862(9): 194412, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31356989

RESUMO

Non-alcoholic steatohepatitis (NASH) is one of the most predominant disorders in metabolic syndrome. Induction of pro-inflammatory mediators in hepatocytes exposed to free fatty acids represents a hallmark event during NASH pathogenesis. C-reactive protein (CRP) is a prototypical pro-inflammatory mediator. In the present study, we investigated the mechanism by which megakaryocytic leukemia 1 (MKL1) mediates palmitate (PA) induced CRP transcription in hepatocytes. We report that over-expression of MKL1, but not MKL2, activated the CRP promoter whereas depletion or inhibition of MKL1 repressed the CRP promoter. MKL1 potentiated the induction of the CRP promoter activity by PA treatment. Importantly, MKL1 knockdown by siRNA or pharmaceutical inhibition by CCG-1423 attenuated the induction of endogenous CRP expression in hepatocytes. Similarly, primary hepatocytes isolated from wild type (WT) mice produced more CRP than those isolated from MKL1 deficient (KO) mice when stimulated with PA. Mechanistically, the sequence-specific transcription factor CCAAT-enhancer-binding protein (C/EBPß) interacted with MKL1 and recruited MKL1 to activate CRP transcription. Reciprocally, MKL1 modulated C/EBPß activity by recruiting the chromatin remodeling protein BRG1 to the CRP promoter to alter histone modifications. In conclusion, our data delineate a novel epigenetic mechanism underlying augmented hepatic inflammation during NASH pathogenesis.


Assuntos
Proteína C-Reativa/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , DNA Helicases/genética , Hepatopatia Gordurosa não Alcoólica/genética , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genética , Animais , Proteína C-Reativa/química , Proteína beta Intensificadora de Ligação a CCAAT/química , Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica/genética , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/patologia , Regiões Promotoras Genéticas , Transativadores/química
8.
Inflamm Res ; 68(10): 815-823, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31312858

RESUMO

C-reactive protein (CRP) is a non-specific diagnostic marker of inflammation and an evolutionarily conserved protein with roles in innate immune signaling. Natural CRP is composed of five identical globular subunits that form a pentamer, but the role of pentameric CRP (pCRP) during inflammatory pathogenesis remains controversial. Emerging evidence suggests that pCRP can be dissociated into monomeric CRP (mCRP) that has major roles in host defenses and inflammation. Here, we discuss our current knowledge of the dissociation mechanisms of pCRP and summarize the stepwise conformational transition model to mCRP to elucidate how CRP dissociation contributes to proinflammatory activity. These discussions will evoke new understanding of this ancient protein.


Assuntos
Proteína C-Reativa/química , Proteína C-Reativa/metabolismo , Inflamação/metabolismo , Animais , Membrana Celular/metabolismo , Humanos , Conformação Proteica
9.
Brain Behav Immun ; 81: 650-654, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31175997

RESUMO

Salivary markers of immune function are increasingly commonly used in studies of human health. Yet, few studies have examined the short-term or long-term reliability or stability of these biomarkers, making their measurement properties unclear. We addressed this issue in the present study by collecting two saliva samples, two hours apart, from 426 adolescent girls during a baseline laboratory visit. Then, eighteen months later, we collected the same samples again from a subset of these participants (n = 113). The correlations between the two samples collected at each session were generally high (mean r = 0.67). In contrast, although single saliva samples were only weakly correlated across 18 month (mean rs = 0.18), averaging the two quantifications within a session considerably improved the reliability (mean r = 0.27). In short, salivary immune markers exhibited strong short-term test-retest correlations, and averaging across multiple assessments notably improved long-term test-retest correlations. Additional research is needed to establish the health relevance and mechanisms underlying these potentially useful, non-invasive biomarkers.


Assuntos
Biomarcadores/química , Reprodutibilidade dos Testes , Saliva/química , Adolescente , Proteína C-Reativa/análise , Proteína C-Reativa/química , Citocinas/análise , Citocinas/química , Feminino , Humanos
10.
Molecules ; 24(11)2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151201

RESUMO

C-reactive protein (CRP) is an intriguing protein which plays a variety of roles in either physiological or pathophysiological states. For years it has been regarded merely as a useful biomarker of infection, tissue injury and inflammation, and it was only in the early 80s that the modified isoforms (mCRP) of native CRP (nCRP) appeared. It soon became clear that the roles of native CRP should be clearly discriminated from those of the modified form and so the impacts of both isoforms were divided to a certain degree between physiological and pathophysiological states. For decades, CRP has been regarded only as a hallmark of inflammation; however, it has since been recognised as a significant predictor of future episodes of cardiovascular disease, independent of other risk factors. The existence of modified CRP isoforms and their possible relevance to various pathophysiological conditions, suggested over thirty years ago, has prompted the search for structural and functional dissimilarities between the pentameric nCRP and monomeric mCRP isoforms. New attempts to identify the possible relevance between the diversity of structures and their opposing functions have initiated a new era of research on C-reactive protein. This review discusses the biochemical aspects of CRP physiology, emphasizing the supposed relevance between the structural biology of CRP isoforms and their differentiated physiological and pathophysiological roles.


Assuntos
Proteína C-Reativa/química , Proteína C-Reativa/metabolismo , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/metabolismo , Suscetibilidade a Doenças , Animais , Biomarcadores , Glicosilação , Humanos , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Relação Estrutura-Atividade
11.
Phytother Res ; 33(8): 1991-2001, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161680

RESUMO

The present meta-analysis was performed to evaluate the efficacy of ginseng administration on serum level of inflammatory biomarkers. We performed a systematic search of all available randomized controlled trials (RCTs) conducted up to June 2018 in the following electronic databases: PubMed, Scopus, Cochrane, and Google Scholar. RCTs that investigated the effect ginseng supplementation on high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were included for final analysis. A total of seven RCTs were included in the meta-analysis. Results indicated significant reduction in IL-6 (mean difference [MD]: -0.265 pg/ml, 95% CI [-0.396, -0.135], p < .001) and TNF-α (MD: -2.471 pg/ml, 95% CI [-2.904, -2.039], p < .001) and no significant change in hs-CRP (MD: -0.125 mg/L, 95% CI [-0.597, 0.347], p = .604). Although there was publication bias across studies, trim and fill analysis showed that results from unpublished studies could not change the results for CRP. However, removing one study in sensitivity analysis did reveal a significant reduction in CRP. We conclude that ginseng supplementation significantly lowered IL-6 and TNF-α but did not significantly lower CRP. However, these findings were not robust, because they showed sensitivity for CRP and IL-6, and future long-term well-designed dose-escalating trials are required.


Assuntos
Biomarcadores/sangue , Proteína C-Reativa/química , Suplementos Nutricionais/análise , Inflamação/tratamento farmacológico , Panax/química , Humanos , Inflamação/sangue , Pessoa de Meia-Idade
12.
J Immunol ; 203(1): 225-235, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31118224

RESUMO

C-reactive protein (CRP) is an acute-phase protein produced in high quantities by the liver in response to infection and during chronic inflammatory disorders. Although CRP is known to facilitate the clearance of cell debris and bacteria by phagocytic cells, the role of CRP in additional immunological functions is less clear. This study shows that complexed CRP (phosphocholine [PC]:CRP) (formed by binding of CRP to PC moieties), but not soluble CRP, synergized with specific TLRs to posttranscriptionally amplify TNF, IL-1ß, and IL-23 production by human inflammatory macrophages. We identified FcγRI and IIa as the main receptors responsible for initiating PC:CRP-induced inflammation. In addition, we identified the underlying mechanism, which depended on signaling through kinases Syk, PI3K, and AKT2, as well as glycolytic reprogramming. These data indicate that in humans, CRP is not only a marker but also a driver of inflammation by human macrophages. Therefore, although providing host defense against bacteria, PC:CRP-induced inflammation may also exacerbate pathology in the context of disorders such as atherosclerosis.


Assuntos
Proteína C-Reativa/metabolismo , Inflamação/imunologia , Fígado/fisiologia , Receptores de IgG/metabolismo , Aterosclerose/imunologia , Proteína C-Reativa/química , Células Cultivadas , Reprogramação Celular , Citocinas/metabolismo , Glicólise , Humanos , Mediadores da Inflamação/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilcolina/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Quinase Syk/metabolismo , Receptores Toll-Like/metabolismo
13.
Front Immunol ; 10: 712, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019517

RESUMO

The innate immune system comprises a cellular and a humoral arm. Humoral pattern recognition molecules include complement components, collectins, ficolins, and pentraxins. These molecules are involved in innate immune responses by recognizing microbial moieties and damaged tissues, activating complement, exerting opsonic activity and facilitating phagocytosis, and regulating inflammation. The long pentraxin PTX3 is a prototypic humoral pattern recognition molecule that, in addition to providing defense against infectious agents, plays several functions in tissue repair and regulation of cancer-related inflammation. Characterization of the PTX3 molecular structure and biochemical properties, and insights into its interactome and multiple roles in tissue damage and remodeling support the view that microbial and matrix recognition are evolutionarily conserved functions of humoral innate immunity molecules.


Assuntos
Proteína C-Reativa/imunologia , Proteínas do Tecido Nervoso/imunologia , Componente Amiloide P Sérico/imunologia , Animais , Biomarcadores Tumorais/imunologia , Proteína C-Reativa/química , Proteína C-Reativa/genética , Progressão da Doença , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Camundongos , Modelos Imunológicos , Estrutura Molecular , Neoplasias/etiologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/genética , Cicatrização/imunologia
14.
Biosens Bioelectron ; 133: 94-99, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30913510

RESUMO

Protein-inorganic nanoflowers have been extensively used for sensing and biosensing applications by virtue of the signal enhancement of protein component---enzyme. Can the inorganic component of protein-inorganic nanoflowers be employed to amplify signal transducing for enzyme-free detection? In this work, a new kind of BSA-antibodies-copper phosphate hybrid nanoflowers (BSA-Ab2-Cu3(PO4)2) has been prepared by one-pot biomimetic mineralization process as signal enhancer for enzyme-free electrochemical immunoassay. C-reactive protein (CRP) has been chosen as a model biomarker. To the best of our knowledge, it is the first trial using the inorganic component---phosphate ions of BSA-Ab2-Cu3(PO4)2 for transducing electrochemical readout, which features the following advantages: (1) the three-dimensional hierarchical porous nanoflower morphology with a high specific surface area could load more antibodies, and BSA for blocking non-specific sites, greatly increasing the sensitivity of the fabricated immunosensors, (2) the Cu3(PO4)2 hybrid nanoflowers can supply a huge amount of phosphate anions to react with molybdate yielding molybdophosphate precipitates and generating redox currents for more robust enzyme-free electrochemical signal readout. The fabricated immunosensor has exhibited good detection performance with a linear range of 5 pg/mL-1 ng/mL and a limit of detection of 1.26 pg/mL. Moreover, our method has presented good feasibility for clinical sample analysis.


Assuntos
Técnicas Biossensoriais , Proteína C-Reativa/isolamento & purificação , Técnicas Eletroquímicas , Nanoestruturas/química , Proteína C-Reativa/química , Colorimetria , Cobre/química , Humanos , Técnicas Imunoenzimáticas/métodos , Fosfatos/química , Proteínas/química
15.
Front Immunol ; 10: 461, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30923526

RESUMO

Pentraxins and complement defense collagens are soluble recognition proteins that sense pathogens and altered-self elements, and trigger immune responses including complement activation. PTX3 has been shown to interact with the globular recognition domains (gC1q) of the C1q protein of the classical complement pathway, thereby modulating complement activity. The C1q-PTX3 interaction has been characterized previously by site-specific mutagenesis using individual gC1q domains of each of the three C1q chains. The present study is aimed at revisiting this knowledge taking advantage of full-length recombinant C1q. Four mutations targeting exposed amino acid residues in the gC1q domain of each of the C1q chains (LysA200Asp-LysA201Asp, ArgB108Asp-ArgB109Glu, TyrB175Leu, and LysC170Glu) were introduced in recombinant C1q and the interaction properties of the mutants were analyzed using surface plasmon resonance. All C1q mutants retained binding to C1r and C1s proteases and mannose-binding lectin-associated serine proteases, indicating that the mutations did not affect the function of the collagen-like regions of C1q. The effect of these mutations on the interaction of C1q with PTX3 and IgM, and both the PTX3- and IgM-mediated activation of the classical complement pathway were investigated. The LysA200Asp-LysA201Asp and LysC170Glu mutants retained partial interaction with PTX3 and IgM, however they triggered efficient complement activation. In contrast, the ArgB108Asp-ArgB109Glu mutation abolished C1q binding to PTX3 and IgM, and significantly decreased complement activation. The TyrB175Leu mutant exhibited decreased PTX3- and IgM-dependent complement activation. Therefore, we provided evidence that, in the context of the full length C1q protein, a key contribution to the interaction with both PTX3 and IgM is given by the B chain Arg residues that line the side of the gC1q heterotrimer, with a minor participation of a Lys residue located at the apex of gC1q. Furthermore, we generated recombinant forms of the human PTX3 protein bearing either D or A at position 48, a polymorphic site of clinical relevance in a number of infections, and observed that both allelic variants equally recognized C1q.


Assuntos
Proteína C-Reativa/química , Complemento C1q/química , Imunoglobulina M/química , Mutação de Sentido Incorreto , Componente Amiloide P Sérico/química , Substituição de Aminoácidos , Animais , Proteína C-Reativa/genética , Proteína C-Reativa/imunologia , Células CHO , Complemento C1q/genética , Complemento C1q/imunologia , Cricetulus , Humanos , Imunoglobulina M/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Componente Amiloide P Sérico/genética , Componente Amiloide P Sérico/imunologia
16.
Front Immunol ; 10: 166, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30863393

RESUMO

One host defense function of C-reactive protein (CRP) is to protect against Streptococcus pneumoniae infection as shown by experiments employing murine models of pneumococcal infection. The protective effect of CRP is due to reduction in bacteremia. There is a distinct relationship between the structure of CRP and its anti-pneumococcal function. CRP is functional in both native and non-native pentameric structural conformations. In the native conformation, CRP binds to pneumococci through the phosphocholine molecules present on the C-polysaccharide of the pneumococcus and the anti-pneumococcal function probably involves the known ability of ligand-complexed CRP to activate the complement system. In the native structure-function relationship, CRP is protective only when given to mice within a few hours of the administration of pneumococci. The non-native pentameric conformation of CRP is created when CRP is exposed to conditions mimicking inflammatory microenvironments, such as acidic pH and redox conditions. In the non-native conformation, CRP binds to immobilized complement inhibitor factor H in addition to being able to bind to phosphocholine. Recent data using CRP mutants suggest that the factor H-binding function of non-native CRP is beneficial: in the non-native structure-function relationship, CRP can be given to mice any time after the administration of pneumococci irrespective of whether the pneumococci became complement-resistant or not. In conclusion, while native CRP is protective only against early stage infection, non-native CRP is protective against both early stage and late stage infections. Because non-native CRP displays phosphocholine-independent anti-pneumococcal activity, it is quite possible that CRP functions as a general anti-bacterial molecule.


Assuntos
Infecções Bacterianas/metabolismo , Proteína C-Reativa/química , Proteína C-Reativa/metabolismo , Interações Hospedeiro-Patógeno , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Fosforilcolina/metabolismo , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/metabolismo , Infecções Pneumocócicas/microbiologia , Ligação Proteica , Streptococcus pneumoniae/imunologia , Relação Estrutura-Atividade
17.
Front Immunol ; 10: 29, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30740098

RESUMO

Background: Pentraxin3 (PTX3) is overexpressed in kidneys of patients developing lupus nephritis (LN). Active LN is associated with reduced anti-PTX3 antibodies. However, abnormalities of B cell differentiation against PTX3 have not been characterized in systemic lupus erythematosus (SLE). Objective: Characterization of PTX3-specific (PTX3+) B cells in peripheral blood of SLE patients with or without LN and healthy donors (HD). Patients and Methods: SLE patients without LN, biopsy-proven LN and matched HD were analyzed. Active LN was defined as proteinuria>0.5 g/day or CrCl<60 ml/min/1.73 m2 with active urinary sediment. Peripheral B cells were analyzed for direct PTX3 binding by flow cytometry using PTX3 labeled with cyanine 5 (Cy5) and phycoerythrin (PE). Results: Initially, a flow cytometry based assay to identify PTX3+ B cells was developed by demonstrating simultaneous binding of PTX3-Cy5 and PTX3-PE. Specificity of B cells was validated by blocking experiments using unlabeled PTX3. We could identify circulating PTX3+ B-cells in HD and patients. Notably, LN patients showed a significantly diminished number of PTX3+ B cells (SLE vs. LN p = 0.033; HD vs. LN p = 0.008). This decrease was identified in naïve and memory B cell compartments (naïve: SLE vs. LN p = 0.028; HD vs. LN p = 0.0001; memory: SLE vs. LN p = 0.038, HD vs. LN p = 0.011). Conclusions: Decreased PTX3+ B cells in LN within the naïve and memory compartment suggest their negative selection at early stages of B cell development potentially related to a decreased regulatory function. PTX3+ B cells could candidate for autoantigen-defined regulatory B cells as a striking abnormality of LN patients.


Assuntos
Linfócitos B/metabolismo , Proteína C-Reativa/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Nefrite Lúpica/sangue , Componente Amiloide P Sérico/metabolismo , Adulto , Autoanticorpos/sangue , Autoantígenos/metabolismo , Biomarcadores/metabolismo , Proteína C-Reativa/química , Proteína C-Reativa/imunologia , Carbocianinas/química , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Ficoeritrina/química , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/imunologia , Coloração e Rotulagem
18.
Biosens Bioelectron ; 130: 40-47, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30716591

RESUMO

This paper primarily demonstrates the approach to enhance the sensing performance on antigen C-reactive protein (CRP) and anti-CRP antibody binding event. A nanogapped electrode structure with the gap of ~100 nm was modified by the anti-CRP antibody (Probe) to capture the available CRP. In order to increase the amount of antigen to be captured, a gold nanorod with 119 nm in length and 25 nm in width was integrated, to increase the surface area. A comparative study between the existence and non-existence of gold nanorod utilization was evaluated. Analysis of the sensing surface was well-supported by atomic force microscopy, scanning electron microscopy, 3D nano-profilometry, high-power microscopy and UV-Vis spectroscopy. The dielectric voltammetric analysis was carried out from 0 V to 2 V. The sensitivity was calculated based on 3σ and attained as low as 1 pM, which is tremendously low compared to real CRP concentration (119 nM) in human blood serum. The gold nanorod conjugation with antibody has enhanced the sensitivity to 100 folds (10 fM). The specificity of the CRP detection by the proposed strategy was anchored by ELISA and failure in the detection of human blood clotting factor IX by voltammetry. Despite, CRP antigen was further detected in human serum by spiking CRP to run-through the detection with the physiologically relevant samples.


Assuntos
Técnicas Biossensoriais , Proteína C-Reativa/isolamento & purificação , Eletroquímica , Insuficiência Cardíaca/diagnóstico , Anticorpos/química , Proteína C-Reativa/química , Ouro/química , Humanos , Limite de Detecção , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Nanotubos/química
19.
Biosens Bioelectron ; 130: 389-396, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30268669

RESUMO

A novel affinity paper-based electrochemical impedance device (PEID) was first fully developed for cardiovascular risk assessment. Herein, a simple folding PEID comprising a dual screen-printed electrode was fabricated for label-free electrochemical impedance detection. The results demonstrated in a step-wise manner that the phosphocholine-modified screen-printed carbon electrodes were highly responsive to the clinically required range of C-reactive protein (CRP) (0.005 - 500 mg L-1; r2 = 0.993) levels with a detection limit (3σ/slope) of 0.001 mg L-1. The optimal binding frequency of CRP-phosphocholine interaction was determined to be 100 Hz. These results implied that our proposed system could be used for simultaneously measuring the CRP levels using a single PEID platform in combination with the specific recognition elements. When assaying two levels of CRP, the overall assay reproducibility for each concentration, expressed as relative standard error of the mean (RSE%; n = 30), was 1.21%. The variation in the measurements between individual electrodes, expressed as the relative standard deviation (RSD), was 2.82%. Using 2 measurement sites per device, the proposed sensor provided excellent precision for the simultaneous detection of CRP. Moreover, the RSD for the CRP levels measured on ten independently fabricated paper-based sheets was 2.11%, thereby offering an acceptable fabrication reproducibility. The presented folding PEIDs were used forthe determination of CRP in patient-derived blood samples with minimised bias and excellent correlation with a standard method (n = 15; CUSUM linearity test, p > 0.10), thus permitting the potential application of PEID for assessing cardiovascular risk in individuals.


Assuntos
Técnicas Biossensoriais , Proteína C-Reativa/isolamento & purificação , Doenças Cardiovasculares/diagnóstico , Técnicas Eletroquímicas , Proteína C-Reativa/química , Impedância Elétrica , Humanos , Limite de Detecção , Medição de Risco , Fatores de Risco
20.
Biosens Bioelectron ; 126: 315-323, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30448719

RESUMO

One-dimensional photonic crystal slabs are periodic optical nanostructures that produce guided-mode resonance. They couple part of the incident light into the waveguide generating bandgaps in the transmittance spectrum, whose position is sensitive to refractive index variations on their surface. In this study, we present one-dimensional photonic crystal slab biosensors based on the internal nanogrooved structure of Blu-ray disks for label-free immunosensing. We demonstrated that this polycarbonate structure coated with a critical thickness of TiO2 generates guided-mode resonance. Its optical behavior was established comparing it with other compact disk structures. The results were theoretically calculated and experimentally demonstrated, all them being in agreement. The bioanalytical performance of these photonic crystals was experimentally demonstrated in a model assay to quantify IgGs as well as in two immunoassays to determine the biomarkers C-reactive protein and lactate dehydrogenase (detection limits of 0.1, 87, and 13 nM, respectively). The results are promising towards the development of new low-cost, portable, and label-free optical biosensors that join these photonic crystals with dedicated bioanalytical scanners based on compact disk drives.


Assuntos
Técnicas Biossensoriais , Proteína C-Reativa/isolamento & purificação , Imunoensaio , L-Lactato Desidrogenase/isolamento & purificação , Biomarcadores/química , Proteína C-Reativa/química , Cristalização , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , L-Lactato Desidrogenase/química , Nanoestruturas/química , Óptica e Fotônica , Fótons , Titânio/química
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