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1.
ACS Appl Mater Interfaces ; 13(36): 43696-43707, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34470205

RESUMO

Graphene is a two-dimensional semiconducting material whose application for diagnostics has been a real game-changer in terms of sensitivity and response time, variables of paramount importance to stop the COVID-19 spreading. Nevertheless, strategies for the modification of docking recognition and antifouling elements to obtain covalent-like stability without the disruption of the graphene band structure are still needed. In this work, we conducted surface engineering of graphene through heterofunctional supramolecular-covalent scaffolds based on vinylsulfonated-polyamines (PA-VS). In these scaffolds, one side binds graphene through multivalent π-π interactions with pyrene groups, and the other side presents vinylsulfonated pending groups that can be used for covalent binding. The construction of PA-VS scaffolds was demonstrated by spectroscopic ellipsometry, Raman spectroscopy, and contact angle measurements. The covalent binding of -SH, -NH2, or -OH groups was confirmed, and it evidenced great chemical versatility. After field-effect studies, we found that the PA-VS-based scaffolds do not disrupt the semiconducting properties of graphene. Moreover, the scaffolds were covalently modified with poly(ethylene glycol) (PEG), which improved the resistance to nonspecific proteins by almost 7-fold compared to the widely used PEG-monopyrene approach. The attachment of recognition elements to PA-VS was optimized for concanavalin A (ConA), a model lectin with a high affinity to glycans. Lastly, the platform was implemented for the rapid, sensitive, and regenerable recognition of SARS-CoV-2 spike protein and human ferritin in lab-made samples. Those two are the target molecules of major importance for the rapid detection and monitoring of COVID-19-positive patients. For that purpose, monoclonal antibodies (mAbs) were bound to the scaffolds, resulting in a surface coverage of 436 ± 30 ng/cm2. KD affinity constants of 48.4 and 2.54 nM were obtained by surface plasmon resonance (SPR) spectroscopy for SARS-CoV-2 spike protein and human ferritin binding on these supramolecular scaffolds, respectively.


Assuntos
Biomarcadores/análise , COVID-19/diagnóstico , Grafite/química , Imunoensaio/métodos , Glicoproteína da Espícula de Coronavírus/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Etilenos/química , Ferritinas/imunologia , Ferritinas/metabolismo , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Poliaminas/química , Polietilenoglicóis/química , Pirenos/química , Teoria Quântica , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Semicondutores , Glicoproteína da Espícula de Coronavírus/imunologia , Ácidos Sulfônicos/química , Ressonância de Plasmônio de Superfície
2.
Viruses ; 13(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34372587

RESUMO

Distinguishing between severe and nonsevere COVID-19 to ensure adequate healthcare quality and efficiency is a challenge for the healthcare system. The aim of this study was to assess the usefulness of CBC parameters together with analysis of FLC serum concentration in risk stratification of COVID-19. MATERIALS AND METHODS: CBC was analyzed in 735 COVID ICU, COVID non-ICU, and non-COVID ICU cases. FLC concentration was analyzed in 133 of them. RESULTS: COVID ICU had neutrophils and lymphocytes with the greatest size, granularity, and nucleic acid content. Significant differences in concentrations of κ and λ FLCs were shown between COVID ICU and COVID non-ICU. However, no difference was found in the κ/λ ratio between these groups, and the ratio stayed within the reference value, which indicates the presence of polyclonal FLCs. FLC κ measurement has significant power to distinguish between severe COVID-19 and nonsevere COVID-19 (AUC = 0.7669), with a sensitivity of 86.67% and specificity of 93.33%. The κ coefficients' odds ratio of 3.0401 was estimated. CONCLUSION: It can be concluded that the results obtained from the measure of free light immunoglobulin concentration in serum are useful in distinguishing between severe and nonsevere COVID-19.


Assuntos
COVID-19/imunologia , Cadeias Leves de Imunoglobulina/sangue , SARS-CoV-2/imunologia , Idoso , Idoso de 80 Anos ou mais , Proteína C-Reativa/imunologia , COVID-19/sangue , COVID-19/diagnóstico , Teste Sorológico para COVID-19 , Feminino , Ferritinas/imunologia , Humanos , Cadeias Leves de Imunoglobulina/imunologia , Unidades de Terapia Intensiva , Interleucina-6/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e Especificidade , Índice de Gravidade de Doença
3.
PLoS One ; 16(6): e0252818, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34111164

RESUMO

Most deaths from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection occur in older subjects. We assessed the utility of serum inflammatory markers interleukin-6 (IL-6), C reactive protein (CRP), and ferritin (Roche, Indianapolis, IN), and SARS-CoV-2 immunoglobulin G (IgG), immunoglobulin M (IgM), and neutralizing antibodies (Diazyme, Poway, CA). In controls, non-hospitalized subjects, and hospitalized subjects assessed for SARS-CoV-2 RNA (n = 278), median IgG levels in arbitrary units (AU)/mL were 0.05 in negative subjects, 14.83 in positive outpatients, and 30.61 in positive hospitalized patients (P<0.0001). Neutralizing antibody levels correlated significantly with IgG (r = 0.875; P<0.0001). Having combined values of IL-6 ≥10 pg/mL and CRP ≥10 mg/L occurred in 97.7% of inpatients versus 1.8% of outpatients (odds ratio 3,861, C statistic 0.976, P = 1.00 x 10-12). Antibody or ferritin levels did not add significantly to predicting hospitalization. Antibody testing in family members and contacts of SARS-CoV-2 RNA positive cases (n = 759) was invaluable for case finding. Persistent IgM levels were associated with chronic COVID-19 symptoms. In 81,624 screened subjects, IgG levels were positive (≥1.0 AU/mL) in 5.21%, while IgM levels were positive in 2.96% of subjects. In positive subjects median IgG levels in AU/mL were 3.14 if <30 years of age, 4.38 if 30-44 years of age, 7.89 if 45-54 years of age, 9.52 if 55-64 years of age, and 10.64 if ≥65 years of age (P = 2.96 x 10-38). Our data indicate that: 1) combined IL-6 ≥10 pg/mL and CRP ≥10 mg/L identify SARS-CoV-2 positive subjects requiring hospitalization; 2) IgG levels were significantly correlated with neutralizing antibody levels with a wide range of responses; 3) IgG levels have significant utility for case finding in exposed subjects; 4) persistently elevated IgM levels are associated with chronic symptoms; and 5) IgG levels are significantly higher in positive older subjects than their younger counterparts.


Assuntos
COVID-19/sangue , Inflamação/sangue , Adulto , Fatores Etários , Idoso , Envelhecimento , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Proteína C-Reativa/análise , Proteína C-Reativa/imunologia , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/imunologia , Feminino , Ferritinas/sangue , Ferritinas/imunologia , Hospitalização , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Inflamação/diagnóstico , Inflamação/epidemiologia , Inflamação/imunologia , Interleucina-6/sangue , Interleucina-6/imunologia , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação
4.
Fish Shellfish Immunol ; 115: 1-6, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34051346

RESUMO

Iron sequestration through ferritin forms a major part of innate immune response in molluscs and detailed understanding of ferritin gene and its functions can be directly applied in infection and disease management studies. Accordingly, identification and detailed molecular characterization of a ferritin subunit gene from a commercially significant marine mussel Perna viridis was targeted. Molecular screening using degenerate primers in total mantle RNA resulted in the amplification of a novel ferritin gene fragment having <87% identity to the reported ferritin gene sequences. Rapid amplification of cDNA ends-PCR was followed to generate complete cDNA sequence of P.viridis ferritin (PvFer). The complete cDNA was found to be 798 bp, containing an open reading frame of 522 bp, 5' untranslated region (UTR) of 112 bp and 3' UTR of 165 bp. The 5' UTR and 3' UTR were shown to contain an iron response element (IRE) and a polyadenylation signal (767AATAAA772) with poly (A) tail, respectively. Prediction of stem loop structure revealed that, PvFer-IRE can be folded into a typical secondary stem loop structure, having 5-CAGUGA-3' loop, proximal stem of five paired bases followed by a bulged cysteine, and six nucleotide bottom stem, indicating that expression of PvFer is regulated by iron at the translational level. ORF was found to encode 175 amino acid protein with calculated molecular mass of 19.97 kDa and isoelectric point of 4.97. Examination for signal peptide and phylogenetic analysis confirmed that PvFer belonged to cytosolic ferritins of molluscs. Conserved domain analysis showed that PvFer contained both ferroxidase diiron center and ferrihydrite nucleation center, analogous to ferritin M subunit of bony fishes and amphibians. However, amino acid sequence and glycosylation site showed more homology to vertebrate ferritin H subunits. Predicted 3D models of PvFer resembled the typical spatial features of ferritin proteins. The study forms the first comprehensive identification of a ferritin subunit gene in a true/common mussel (Order: Mytilida). Further, the detailed molecular phylogeny conducted through the present study revealed certain thought provoking insights on ferritin genes of the phylum Mollusca.


Assuntos
Ferritinas/genética , Ferritinas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Perna (Organismo)/genética , Perna (Organismo)/imunologia , Animais , Sequência de Bases , DNA Complementar/análise , Ferritinas/química , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA
5.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669238

RESUMO

Protein assemblies provide unique structural features which make them useful as carrier molecules in biomedical and chemical science. Protein assemblies can accommodate a variety of organic, inorganic and biological molecules such as small proteins and peptides and have been used in development of subunit vaccines via display parts of viral pathogens or antigens. Such subunit vaccines are much safer than traditional vaccines based on inactivated pathogens which are more likely to produce side-effects. Therefore, to tackle a pandemic and rapidly produce safer and more effective subunit vaccines based on protein assemblies, it is necessary to understand the basic structural features which drive protein self-assembly and functionalization of portions of pathogens. This review highlights recent developments and future perspectives in production of non-viral protein assemblies with essential structural features of subunit vaccines.


Assuntos
Ferritinas/imunologia , Vacinas de Subunidades/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Animais , Antígenos Virais/imunologia , Bacteriófago T4/imunologia , Humanos , Nanopartículas/química , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta
6.
Sci Transl Med ; 13(583)2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33658355

RESUMO

Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limits their protective efficacy. The H1 and H3 subtypes of influenza A virus cause most of the seasonal epidemics observed in humans and are the major drivers of influenza A virus-associated mortality. The consequences of pandemic spread of COVID-19 underscore the public health importance of prospective vaccine development. Here, we show that headless hemagglutinin (HA) stabilized-stem immunogens presented on ferritin nanoparticles elicit broadly neutralizing antibody (bnAb) responses to diverse H1 and H3 viruses in nonhuman primates (NHPs) when delivered with a squalene-based oil-in-water emulsion adjuvant, AF03. The neutralization potency and breadth of antibodies isolated from NHPs were comparable to human bnAbs and extended to mismatched heterosubtypic influenza viruses. Although NHPs lack the immunoglobulin germline VH1-69 residues associated with the most prevalent human stem-directed bnAbs, other gene families compensated to generate bnAbs. Isolation and structural analyses of vaccine-induced bnAbs revealed extensive interaction with the fusion peptide on the HA stem, which is essential for viral entry. Antibodies elicited by these headless HA stabilized-stem vaccines neutralized diverse H1 and H3 influenza viruses and shared a mode of recognition analogous to human bnAbs, suggesting that these vaccines have the potential to confer broadly protective immunity against diverse viruses responsible for seasonal and pandemic influenza infections in humans.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Primatas/imunologia , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/química , Complexo Antígeno-Anticorpo/química , Anticorpos Amplamente Neutralizantes/biossíntese , Anticorpos Amplamente Neutralizantes/química , COVID-19 , Ferritinas/química , Ferritinas/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/química , Influenza Humana/imunologia , Influenza Humana/virologia , Macaca fascicularis , Modelos Moleculares , Nanopartículas/química , Pandemias , Primatas/virologia , Estrutura Quaternária de Proteína , SARS-CoV-2 , Pesquisa Médica Translacional
7.
J Intern Med ; 289(2): 206-220, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32691471

RESUMO

BACKGROUND: Polymyositis/dermatomyositis (PM/DM) is an autoimmune disease that is sometimes complicated with rapidly progressive interstitial lung disease (RPILD). However, serum and lung biomarkers that can predict RPILD development remain unclear. OBJECTIVES: To determine potential serum and lung biomarkers that can predict RPILD development in patients with PM/DM-ILD. METHODS: In total, 49 patients with PM/DM-ILD were enrolled. We measured the serum levels of 41 cytokines/chemokines, ferritin and anti-MDA5 antibody, compared them between the RPILD (n = 23) and non-RPILD (n = 26) groups, and ranked them by their importance through random forest analysis. To distinguish the two groups, we determined biomarker combinations by logistic regression analysis. We also measured the bronchoalveolar lavage fluid (BALF) levels of 41 cytokines/chemokines. Using immunohistochemistry, we examined IL-15 expression in lung tissues. The IL-15 production was also investigated using A549 and BEAS-2B cells. RESULTS: The RPILD group had significantly higher IL-15, IL-1RA, IL-6, CXCL10, VCAM-1, anti-MDA5 antibody and ferritin serum levels than the non-RPILD group, but it had a significantly low CCL22 level. Meanwhile, anti-MDA5 antibody, IL-15, CXCL8, CCL22, IL-1RA and ferritin were the best combination to distinguish the two groups. IL-15 and CCL22 were also predictive marker for RPILD development in anti-MDA5 antibody-positive patients. Additionally, the RPILD group had significantly high IL-15 levels in BALF. The lung tissues expressed IL-15, which increased after cytokine stimulation in the A549 cells. CONCLUSION: This study identified a combination of biomarkers predicting PM/DM-RPILD progression, and IL-15 is an important cytokine for predicting RPILD development and reflecting ILD severity.


Assuntos
Dermatomiosite/complicações , Interleucina-15/imunologia , Doenças Pulmonares Intersticiais/etiologia , Doenças Pulmonares Intersticiais/imunologia , Biomarcadores , Líquido da Lavagem Broncoalveolar/química , Quimiocinas/imunologia , Citocinas/imunologia , Progressão da Doença , Feminino , Ferritinas/imunologia , Humanos , Japão , Masculino
8.
Blood Rev ; 45: 100707, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32425294

RESUMO

A subset of patients with severe COVID-19 develop profound inflammation and multi-organ dysfunction consistent with a "Cytokine Storm Syndrome" (CSS). In this review we compare the clinical features, diagnosis, and pathogenesis of COVID-CSS with other hematological CSS, namely secondary hemophagocytic lymphohistiocytosis (sHLH), idiopathic multicentric Castleman disease (iMCD), and CAR-T cell therapy associated Cytokine Release Syndrome (CRS). Novel therapeutics targeting cytokines or inhibiting cell signaling pathways have now become the mainstay of treatment in these CSS. We review the evidence for cytokine blockade and attenuation in these known CSS as well as the emerging literature and clinical trials pertaining to COVID-CSS. Established markers of inflammation as well as cytokine levels are compared and contrasted between these four entities in order to establish a foundation for future diagnostic criteria of COVID-CSS.


Assuntos
COVID-19/imunologia , Hiperplasia do Linfonodo Gigante/imunologia , Síndrome da Liberação de Citocina/imunologia , Fatores Imunológicos/uso terapêutico , Linfo-Histiocitose Hemofagocítica/imunologia , SARS-CoV-2/patogenicidade , Corticosteroides/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Biomarcadores/sangue , Proteína C-Reativa/imunologia , Proteína C-Reativa/metabolismo , COVID-19/tratamento farmacológico , COVID-19/patologia , COVID-19/virologia , Hiperplasia do Linfonodo Gigante/tratamento farmacológico , Hiperplasia do Linfonodo Gigante/patologia , Ensaios Clínicos como Assunto , Síndrome da Liberação de Citocina/tratamento farmacológico , Síndrome da Liberação de Citocina/patologia , Síndrome da Liberação de Citocina/virologia , Ferritinas/sangue , Ferritinas/imunologia , Regulação da Expressão Gênica , Humanos , Imunoterapia Adotiva/efeitos adversos , Interleucina-1/antagonistas & inibidores , Interleucina-1/sangue , Interleucina-1/imunologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/sangue , Interleucina-6/imunologia , Linfo-Histiocitose Hemofagocítica/tratamento farmacológico , Linfo-Histiocitose Hemofagocítica/patologia , Transdução de Sinais
9.
JCI Insight ; 6(1)2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33232303

RESUMO

Immune and inflammatory responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contribute to disease severity of coronavirus disease 2019 (COVID-19). However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity and assessed type I IFN-, type II IFN-, and NF-κB-dependent whole-blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and nonhematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients' first samples revealed 12 biomarkers (CCL2, IL-15, soluble ST2 [sST2], NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and 2 additional biomarkers (lactoferrin, CXCL9) that were substantially associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.


Assuntos
COVID-19/imunologia , COVID-19/mortalidade , Corticosteroides/uso terapêutico , Adulto , Idoso , Antibacterianos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Antivirais/uso terapêutico , Azitromicina/uso terapêutico , Biomarcadores , COVID-19/genética , COVID-19/terapia , Calgranulina B/genética , Calgranulina B/imunologia , Estudos de Casos e Controles , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CXCL9/genética , Quimiocina CXCL9/imunologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Ferritinas/genética , Ferritinas/imunologia , Perfilação da Expressão Gênica , Humanos , Hidroxicloroquina/uso terapêutico , Fatores Imunológicos/uso terapêutico , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Interferon gama/genética , Interferon gama/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-15/genética , Interleucina-15/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Lactoferrina/genética , Lactoferrina/imunologia , Lipocalina-2/genética , Lipocalina-2/imunologia , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/imunologia , Pessoa de Meia-Idade , Análise Multivariada , NF-kappa B/genética , NF-kappa B/imunologia
10.
Immunity ; 53(6): 1315-1330.e9, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33275896

RESUMO

Various vaccine strategies have been proposed in response to the global COVID-19 pandemic, each with unique strategies for eliciting immune responses. Here, we developed nanoparticle vaccines by covalently conjugating the self-assembled 24-mer ferritin to the receptor binding domain (RBD) and/or heptad repeat (HR) subunits of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spike (S) protein. Compared to monomer vaccines, nanoparticle vaccines elicited more robust neutralizing antibodies and cellular immune responses. RBD and RBD-HR nanoparticle vaccinated hACE2 transgenic mice vaccinated with RBD and/or RBD-HR nanoparticles exhibited reduced viral load in the lungs after SARS-CoV-2 challenge. RBD-HR nanoparticle vaccines also promoted neutralizing antibodies and cellular immune responses against other coronaviruses. The nanoparticle vaccination of rhesus macaques induced neutralizing antibodies, and T and B cell responses prior to boost immunization; these responses persisted for more than three months. RBD- and HR-based nanoparticles thus present a promising vaccination approach against SARS-CoV-2 and other coronaviruses.


Assuntos
Proteínas de Bactérias/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Ferritinas/imunologia , Helicobacter pylori/metabolismo , Proteínas Recombinantes de Fusão/imunologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Proteínas de Bactérias/química , Vacinas contra COVID-19/química , Ferritinas/química , Humanos , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Pandemias , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/química , Vacinação
11.
Front Immunol ; 11: 606569, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33343580

RESUMO

Cervical cancer remains a global health burden despite the introduction of highly effective vaccines for the prophylaxis of causative human papillomavirus infection (HPV). Current efforts to eradicate cervical cancer focus on the development of broadly protective, cost-effective approaches. HPV minor capsid protein L2 is being recognized as a promising alternative to the major capsid protein L1 because of its ability to induce responses against a wider range of different HPV types. However, a major limitation of L2 as a source of cross-neutralizing epitopes is its lower immunogenicity compared to L1 when assembled into VLPs. Various approaches have been proposed to overcome this limitation, we developed and tested ferritin-based bio-nanoparticles displaying tandemly repeated L2 epitopes from eight different HPV types grafted onto the surface of Pyrococcus furiosus thioredoxin (Pf Trx). Genetic fusion of the Pf Trx-L2(8x) module to P. furiosus ferritin (Pf Fe) did not interfere with ferritin self-assembly into an octahedral structure composed by 24 protomers. In guinea pigs and mice, the ferritin super-scaffolded, L2 antigen induced a broadly neutralizing antibody response covering 14 oncogenic and two non-oncogenic HPV types. Immune-responsiveness lasted for at least one year and the resulting antibodies also conferred protection in a cervico-vaginal mouse model of HPV infection. Given the broad organism distribution of thioredoxin and ferritin, we also verified the lack of cross-reactivity of the antibodies elicited against the scaffolds with human thioredoxin or ferritin. Altogether, the results of this study point to P. furiosus ferritin nanoparticles as a robust platform for the construction of peptide-epitope-based HPV vaccines.


Assuntos
Alphapapillomavirus/efeitos dos fármacos , Anticorpos Antivirais/sangue , Proteínas de Bactérias/farmacologia , Anticorpos Amplamente Neutralizantes/sangue , Proteínas do Capsídeo/farmacologia , Ferritinas/farmacologia , Proteínas Oncogênicas Virais/farmacologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/farmacologia , Alphapapillomavirus/genética , Alphapapillomavirus/imunologia , Animais , Especificidade de Anticorpos , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Epitopos , Feminino , Ferritinas/genética , Ferritinas/imunologia , Cobaias , Imunização , Imunogenicidade da Vacina , Camundongos Endogâmicos BALB C , Nanopartículas , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/sangue , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus/genética , Vacinas contra Papillomavirus/imunologia , Células Sf9 , Spodoptera , Tiorredoxinas/genética , Tiorredoxinas/imunologia , Tiorredoxinas/farmacologia , Fatores de Tempo , Vacinas de DNA/farmacologia
13.
Clin Exp Immunol ; 201(1): 76-84, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32365221

RESUMO

Effective laboratory markers for the estimation of disease severity and predicting the clinical progression of coronavirus disease-2019 (COVID-19) is urgently needed. Laboratory tests, including blood routine, cytokine profiles and infection markers, were collected from 389 confirmed COVID-19 patients. The included patients were classified into mild (n = 168), severe (n = 169) and critical groups (n = 52). The leukocytes, neutrophils, infection biomarkers [such as C-reactive protein (CRP), procalcitonin (PCT) and ferritin] and the concentrations of cytokines [interleukin (IL)-2R, IL-6, IL-8, IL-10 and tumor necrosis factor (TNF)-α] were significantly increased, while lymphocytes were significantly decreased with increased severity of illness. The amount of IL-2R was positively correlated with the other cytokines and negatively correlated with lymphocyte number. The ratio of IL-2R to lymphocytes was found to be remarkably increased in severe and critical patients. IL-2R/lymphocytes were superior compared with other markers for the identification of COVID-19 with critical illness, not only from mild but also from severe illness. Moreover, the cytokine profiles and IL-2R/lymphocytes were significantly decreased in recovered patients, but further increased in disease-deteriorated patients, which might be correlated with the outcome of COVID-19. Lymphopenia and increased levels of cytokines were closely associated with disease severity. The IL-2R/lymphocyte was a prominent biomarker for early identification of severe COVID-19 and predicting the clinical progression of the disease.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Subunidade alfa de Receptor de Interleucina-2/sangue , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Linfócitos T/virologia , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/imunologia , Biomarcadores/sangue , Proteína C-Reativa/imunologia , Proteína C-Reativa/metabolismo , COVID-19 , China/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Progressão da Doença , Feminino , Ferritinas/sangue , Ferritinas/imunologia , Humanos , Interleucina-10/sangue , Interleucina-10/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Interleucina-6/sangue , Interleucina-6/imunologia , Interleucina-8/sangue , Interleucina-8/imunologia , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Neutrófilos/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Pró-Calcitonina/sangue , Pró-Calcitonina/imunologia , Prognóstico , SARS-CoV-2 , Índice de Gravidade de Doença , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/imunologia
15.
Ann Hematol ; 99(3): 421-429, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31984437

RESUMO

ß-thalassemia major is one of the most common hematologic disorders in the world. It causes severe anemia and patients require regular blood transfusions, which causes different complications such as iron overload and alloimmunization. Regulatory T cells (Tregs) have an important role in regulation of immune responses. FoxP3 is the major marker of Tregs and its expression can be influenced by different factors. GDF-15 is another gene that plays a role in iron homeostasis and regulation of immune system in different diseases. The aim of this study was to assess the frequency of Tregs and FoxP3/GDF-15 gene expression in ß-thalassemia major patients with and without alloantibody as well as its correlation with different factors such as serum ferritin and folate levels. This study was conducted on 68 ß-thalassemia major patients with and without alloantibodies in comparison with 20 healthy individuals with matched age and sex as control group. Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and real-time PCR were performed in order to evaluate serum ferritin and folate levels, frequency of Tregs, and the expression of FoxP3 and GDF-15 genes, respectively. The percentage and absolute count of Tregs were increased in patients compared with controls (P = 0.0003), but there was no difference between responders and non-responders (P > 0.05). The Tregs count correlated positively with serum ferritin. No correlation was observed between target genes and serum ferritin and folate, but there was a positive significant correlation between the expression of FoxP3 and GDF-15 genes, which shows the immunosuppressive role of GDF-15.


Assuntos
Ferritinas , Ácido Fólico , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica/imunologia , Fator 15 de Diferenciação de Crescimento , Isoanticorpos , Linfócitos T Reguladores , Talassemia beta , Adolescente , Adulto , Criança , Feminino , Ferritinas/sangue , Ferritinas/imunologia , Ácido Fólico/sangue , Ácido Fólico/imunologia , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/imunologia , Fator 15 de Diferenciação de Crescimento/biossíntese , Fator 15 de Diferenciação de Crescimento/imunologia , Humanos , Isoanticorpos/sangue , Isoanticorpos/imunologia , Masculino , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Talassemia beta/sangue , Talassemia beta/imunologia , Talassemia beta/patologia
16.
Mikrochim Acta ; 186(11): 687, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31595370

RESUMO

A dual-mode assay is described for immunological determination of the anemia biomarker ferritin. It is based on the use of a gold@carbon dot (Au@CD) nanoconjugate as a colorimetric and fluorescent probe. Au@CD is hydrophilic, easily surface modified and stable in aqueous solution. The Au@CD have a red color with blue-green fluorescence and were modified with antibody against ferritin. This allows bi-modal detection of ferritin. Assays can be performed in phosphate buffer and were also analyzed in (Bovine Serum Albumin) BSA and (Fetal Bovine Serum) FBS. Detection is based on antigen-antibody interaction underlying the classical sandwich model. Response to ferritin can be detected by spectrophotometry (at 570 nm) or fluorescence (at excitation/emission maxima of 354/454 nm). Under optimal conditions, the assay has a linear response in the 1 to 120 ngmL-1 ferritin concentration range and detection limits of 20 ng (colorimetrically) and 64 ng (fluorometrically). Graphical abstract Schematic representation of the function of the designed nanoprobe. The Au@CD nanoconjugates are functionalized with ferritin antibody in the initial step which specifically interacts with ferritin molecules leading to aggregation and subsequent changes in the optical and fluorescence signals.


Assuntos
Anticorpos/imunologia , Ferritinas/sangue , Corantes Fluorescentes/química , Imunoensaio/métodos , Nanoconjugados/química , Pontos Quânticos/química , Animais , Biomarcadores/análise , Carbono/química , Bovinos , Colorimetria/métodos , Ferritinas/imunologia , Ouro/química , Limite de Detecção , Espectrometria de Fluorescência/métodos
17.
Neotrop Entomol ; 48(4): 706-716, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30941675

RESUMO

Aedes aegypti (Linnaeus) (Diptera: Culicidae) is the main vector of viruses causing dengue, chikungunya, Zika, and yellow fever, worldwide. This report focuses on immuno-blocking four critical proteins in the female mosquito when fed on blood containing antibodies against ferritin, transferrin, one amino acid transporter (NAAT1), and acetylcholinesterase (AchE). Peptides from these proteins were selected, synthetized, conjugated to carrier proteins, and used as antigens to immunize New Zealand rabbits. After rabbits were immunized, a minimum of 20 female mosquitos were fed on each rabbit, per replicate. No effect in their viability was observed after blood-feeding; however, the number of infertile females was 20% higher than the control when fed on AchE-immunized rabbits. The oviposition period was significantly longer in females fed on immunized rabbits than those fed on control (non-immunized) rabbits. Fecundity (eggs/female) of treated mosquitoes was significantly reduced (about 50%) in all four treatments, as compared with the control. Fertility (hatched larvae) was also significantly reduced in all four treatments, as compared with the control, being the effect on AchE and transferrin the highest, by reducing hatching between 70 and 80%. Survival to the adult stage of the hatched larvae showed no significant effect, as more than 95% survival was observed in all treatments, including the control. In conclusion, immuno-blocking of these four proteins caused detrimental effects on the mosquito reproduction, being the effect on AchE the most significant.


Assuntos
Acetilcolinesterase/imunologia , Aedes/fisiologia , Anticorpos/imunologia , Imunização/veterinária , Proteínas de Insetos/imunologia , Sistemas de Transporte de Aminoácidos/imunologia , Animais , Feminino , Ferritinas/imunologia , Fertilidade , Hemolinfa , Imunoglobulina G/imunologia , Mosquitos Vetores/fisiologia , Oviposição , Coelhos , Reprodução , Transferrina/imunologia
18.
Fish Shellfish Immunol ; 89: 701-709, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31004801

RESUMO

Based on the transcriptome database, we screened out four ferritin subunit genes (MnFer2-5) from the oriental river prawn Macrobrachium nipponense, which encode two non-secretory and two secretory peptides. MnFer2 and 4 possess a strictly conserved ferroxidase site, and MnFer3 has a non-typical ferroxidase site. MnFer5 seems to be a number of ferritin families, which has a distinct dinuclear metal binding motif, but lacks an iron ion channel, a ferroxidase site and a nucleation site. Diverse tissue-specific transcriptions of the four genes indicate their functional diversity in the prawn. Among them, MnFer2 is mainly expressed in hepatopancreas and intestines, MnFer3 and 4 are predominantly expressed in gills, and MnFer5 is widely expressed in various tissues with high presence in intestines, hepatopancreas and haemocytes. The transcription of all the four MnFer genes can be strongly induced by doxorubicin, indicating the involvement of these ferritin subunits in protection from oxidative stress. Upon Aeromonas hydrophila infection, only MnFer5 is persistently up-regulated, while other subunits including MnFer2-4 are down-regulated during the early stage, followed by recovery and even a slight increase at 48 h post bacterial challenge. Moreover, the iron binding capacity of recombinant MnFer2 is also demonstrated in vitro. The E. coli expressing MnFer2 displays increased resistance to hydrogen peroxidase cytotoxicity. These results suggest a protective role of ferritins from M. nipponense in iron homeostasis, redox biology and antibacterial immunity and shed light on the molecule evolution of crustacean ferritin subunits.


Assuntos
Ferritinas/genética , Ferritinas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Estresse Oxidativo/imunologia , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Ferritinas/química , Perfilação da Expressão Gênica , Homeostase/imunologia , Oxirredução , Palaemonidae , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Distribuição Aleatória , Alinhamento de Sequência
19.
Fish Shellfish Immunol ; 87: 679-687, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30731213

RESUMO

Ferritins play vital roles in maintenance of iron homeostasis as iron storage proteins. Recently, the immune function of ferritins have attracted increasing attention, especially their roles in defense against pathogenic infections. However, the immune regulatory mechanism of fish ferritins are not well known. In the present study, comparative digital gene expression (DGE) profiling was performed to explore the regulatory effects of the Megalobrama amblycephala ferritins (MamFers) using MamFers overexpressed and control L8824 cells (Ctenopharyngodon idella hepatic cell line). Clean reads were aligned to the C. idella genome and differential expression analysis was conducted with representative differentially expressed genes pointed out. On that basis, further studies were performed to verify two pivotal regulated pathways in L8824 and EPC (Epithelioma Papulosum Cyprini cell line) cells, respectively. The results showed that NLRC5 (NOD-like Receptor Family CARD Domain Containing 5) mediated the regulation of MamFers on expression of MHC I (Major Histocompatibility Complex Class I) and its chaperone ß2M (Beta-2-Microglobulin) in L8824 cells. Then, ß2M further mediated the regulation of MamFers on hepcidin expression, indicating that MamFers regulated the expression of hepcidin via NLRC5/MHC I/ß2M axis. In addition, MamFers regulated the adhesion of Aeromonas hydrophila to EPC cells by regulating the expression of two extracellular matrix proteins Intgß1 (integrin ß1) and FN (fibronectin). In a word, the present study provided novel insights into the immune regulatory functions of fish ferritins.


Assuntos
Cyprinidae/genética , Cyprinidae/imunologia , Ferritinas/genética , Ferritinas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Aeromonas hydrophila/fisiologia , Animais , Aderência Bacteriana , Carpas , Linhagem Celular , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária
20.
Fish Shellfish Immunol ; 86: 882-891, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30553892

RESUMO

Ferritin is a protein related to the storage of iron and widely distributed in animals. It participates in many biological process, including antioxidation, cell activation, angiogenesis, regulation of iron metabolic balance and immune defense. In the present study, a novel ferritin gene was identified from red swamp crayfish Procambarus clarkii, with a cDNA sequence encoding a predicted 221 amino-acid residues. The ferritin protein contains a 19-residue signal peptide and 145-residue classic ferritin domain. The NJ phylogenetic analysis showed PcFer clustered with other crustacean peptides. The recombinant PcFer protein was produced and purified in E. coli, and the anti-rabbit polyclonal antibody was obtained. The rPcFer exhibited iron binding activity at a dose-dependent effect. The qPCR and western blot analysis revealed that PcFer was highly expressed in hemocytes, hepatopancreas, and gills. After challenged with WSSV and Aeromonas hydrophila, the mRNA and protein expression patterns of PcFer were significantly up-regulated in hemocytes and hepatopancreas. dsRNA interfering technique was utilized to silence the expression of PcFer gene. The WSSV copy number in PcFer silenced shrimp was much higher than that in the control group. The present study indicated that PcFer was involved in the immune defense against WSSV and Aeromonas hydrophila, and might inhibit WSSV replication in P. clarkii. These results will provide important data support for further study of the functional role of the ferritin gene.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Ferritinas/genética , Ferritinas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Ferritinas/química , Perfilação da Expressão Gênica , Brânquias/metabolismo , Hemócitos/metabolismo , Hepatopâncreas/metabolismo , Filogenia , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/fisiologia
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