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Neutrophils mediate the early innate immune response through extracellular traps comprising intracellular protein and DNA. These traps play a pivotal role in both immunity against invading pathogens and the development of immunopathological reactions through the production of reactive oxygen species (ROS). Proteins serve as the main target for ROS, resulting in the formation of protein adducts. Herein, we report that the superoxide anion radical (O2Ë-) plays a vital role in neutrophil function through sequential events involving 5-lipoxygenase (5-LOX) and NADPH oxidase (NOX). More specifically, differences in NOX homologs expression were observed post-stimulation with PMA and LPS. Differentiation conditions and O2Ë- generation were confirmed using flow cytometry. Immunoblotting analysis confirmed the time-dependent expression of NOX underlying its requirement and 5-LOX-mediated lipid peroxidation events in neutrophil function. Protein-malondialdehyde (MDA) adducts formed were detected using immunoblotting, and quercetin was evaluated for its ability to scavenge free radicals through electron paramagnetic resonance (EPR) spin-trapping spectroscopy and results were confirmed with blotting analysis. Free radical-mediated protein oxidation events influence neutrophil function and protein adducts formed serve as markers of neutrophil activation upon infection and inflammation. The study warrants further corroboration and the study of specific proteins involved in neutrophil activation and their role in inflammation.
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BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target. METHODS: An albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking. RESULTS: We demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells. CONCLUSION: We report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.
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Movimiento Celular , Proliferación Celular , Receptores de Interleucina-6 , Humanos , Proliferación Celular/efectos de los fármacos , Receptores de Interleucina-6/metabolismo , Movimiento Celular/efectos de los fármacos , Células HEK293 , Línea Celular Tumoral , Unión Proteica/efectos de los fármacosRESUMEN
Introduction: Immunoglobulin A1 (IgA1) with galactose-deficient O-glycans (Gd-IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). Mucosal-tissue infections increase IL-6 production and, in patients with IgAN, are often associated with macroscopic hematuria. IgA1-secreting cell lines derived from the circulation of patients with IgAN, compared to those of healthy controls (HCs), produce more IgA1 that has O-glycans with terminal or sialylated N-acetylgalactosamine (GalNAc). GalNAc residues are added to IgA1 hinge region by some of the 20 GalNAc transferases, the O-glycosylation-initiating enzymes. Expression of GALNT2, encoding GalNAc-T2, the main enzyme initiating IgA1 O-glycosylation, is similar in cells derived from patients with IgAN and HCs. In this report, we extend our observations of GALNT14 overexpression in IgA1-producing cell lines from patients with IgAN. Methods: GALNT14 expression was analyzed in peripheral blood mononuclear cells (PBMCs) from patients with IgAN and from HCs. Moreover, the effect of GALNT14 overexpression or knock-down on Gd-IgA1 production in Dakiki cells was assessed. Results: GALNT14 was overexpressed in PBMCs from patients with IgAN. IL-6 increased GALNT14 expression in PBMCs from patients with IgAN and HCs. We used IgA1-producing cell line Dakiki, a previously reported model of Gd-IgA1-producing cells, and showed that overexpression of GalNAc-T14 enhanced galactose deficiency of IgA1, whereas siRNA-mediated GalNAc-T14 knock-down reduced it. GalNAc-T14 was localized in trans-Golgi network, as expected. Conclusions: Overexpression of GALNT14 due to inflammatory signals during mucosal infections may contribute to overproduction of Gd-IgA1 in patients with IgAN.
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BACKGROUND: IgA nephropathy (IgAN) primary glomerulonephritis is characterized by the deposition of circulating immune complexes composed of polymeric IgA1 molecules with altered O-glycans (Gd-IgA1) and anti-glycan antibodies in the kidney mesangium. The mesangial IgA deposits and serum IgA1 contain predominantly λ light (L) chains, but the nature and origin of such IgA remains enigmatic. METHODS: We analyzed λ L chain expression in peripheral blood B cells of 30 IgAN patients, 30 healthy controls (HCs), and 18 membranous nephropathy patients selected as disease controls (non-IgAN). RESULTS: In comparison to HCs and non-IgAN patients, peripheral blood surface/membrane bound (mb)-Gd-IgA1+ cells from IgAN patients express predominantly λ L chains. In contrast, total mb-IgA+, mb-IgG+, and mb-IgM+ cells were preferentially positive for kappa (κ) L chains, in all analyzed groups. Although minor in comparison to κ L chains, λ L chain subsets of mb-IgG+, mb-IgM+, and mb-IgA+ cells were significantly enriched in IgAN patients in comparison to non-IgAN patients and/or HCs. In contrast to HCs, the peripheral blood of IgAN patients was enriched with λ+ mb-Gd-IgA1+, CCR10+, and CCR9+ cells, which preferentially home to the upper respiratory and digestive tracts. Furthermore, we observed that mb-Gd-IgA1+ cell populations comprise more CD138+ cells and plasmablasts (CD38+) in comparison to total mb-IgA+ cells. CONCLUSIONS: Peripheral blood of IgAN patients is enriched with migratory λ+ mb-Gd-IgA1+ B cells, with the potential to home to mucosal sites where Gd-IgA1 could be produced during local respiratory or digestive tract infections.
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Glomerulonefritis por IGA , Femenino , Galactosa , Humanos , Inmunoglobulina A/metabolismo , Inmunoglobulina G , Inmunoglobulina M , MasculinoRESUMEN
IgA nephropathy (IgAN) is the dominant type of primary glomerulonephritis worldwide. However, IgAN rarely affects African Blacks and is uncommon in African Americans. Polymeric IgA1 with galactose-deficient hinge-region glycans is recognized as auto-antigen by glycan-specific antibodies, leading to formation of circulating immune complexes with nephritogenic consequences. Because human B cells infected in vitro with Epstein-Barr virus (EBV) secrete galactose-deficient IgA1, we examined peripheral blood B cells from adult IgAN patients, and relevant controls, for the presence of EBV and their phenotypic markers. We found that IgAN patients had more lymphoblasts/plasmablasts that were surface-positive for IgA, infected with EBV, and displayed increased expression of homing receptors for targeting the upper respiratory tract. Upon polyclonal stimulation, these cells produced more galactose-deficient IgA1 than did cells from healthy controls. Unexpectedly, in healthy African Americans, EBV was detected preferentially in surface IgM- and IgD-positive cells. Importantly, most African Blacks and African Americans acquire EBV within 2 years of birth. At that time, the IgA system is naturally deficient, manifested as low serum IgA levels and few IgA-producing cells. Consequently, EBV infects cells secreting immunoglobulins other than IgA. Our novel data implicate Epstein-Barr virus infected IgA+ cells as the source of galactose-deficient IgA1 and basis for expression of relevant homing receptors. Moreover, the temporal sequence of racial-specific differences in Epstein-Barr virus infection as related to the naturally delayed maturation of the IgA system explains the racial disparity in the prevalence of IgAN.
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Linfocitos B/inmunología , Infecciones por Virus de Epstein-Barr/epidemiología , Glomerulonefritis por IGA/virología , Herpesvirus Humano 4/fisiología , Grupos Raciales , República Checa/epidemiología , Galactosa , Glomerulonefritis por IGA/epidemiología , Humanos , Inmunoglobulina A/metabolismo , Lactante , PrevalenciaRESUMEN
AIMS: Epstein-Barr virus (EBV) targets predominantly B cells and these cells could acquire new phenotype characteristics. Here we analyzed whether EBV-infected and -uninfected B cells from healthy subjects differ in proportion of dominant phenotypes, maturation stage, and homing receptors expression. METHODS: EBV-infected and -uninfected cells were identified by flow cytometry using fluorophore-labeled EBV RNA-specific DNA probes combined with fluorophore-labeled antibody to surface lineage markers, integrins, chemokine receptors, and immunoglobulin isotypes, including intracellular ones. RESULTS: Our results show that the trafficking characteristics of EBERpos B cells are distinct from EBERneg B cells with most dominant differences detected for α4ß1 and α4ß7 and CCR5 and CCR7. EBV-positive cells are predominantly memory IgM+ B cells or plasmablasts/plasma cells (PB/PC) positive for IgA or less for IgM. In comparison to uninfected B cells, less EBV-positive B cells express α4ß7 and almost no cells express α4ß1. EBV-positive B cells contained significantly higher proportion of CCR5+ and CCR7+ cells in comparison to EBV-negative cells. In vitro exposure of blood mononuclear cells to pro-inflammatory cytokine IL-6 reduces population of EBV-positive B cell. CONCLUSION: Although EBV-infected B cells represent only a minor subpopulation, their atypical functions could contribute in predisposed person to development abnormities such as some autoimmune diseases or tumors. Using multi-parameter flow cytometry we characterized differences in migration of EBV-positive and -negative B cells of various maturation stage and isotype of produced antibodies particularly different targeting to mucosal tissues of gastrointestinal and respiratory tracts.
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Linfocitos B/inmunología , Sangre/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/fisiopatología , Proteínas de Transporte Vesicular/inmunología , Proteínas de Transporte Vesicular/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Citometría de Flujo , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana EdadRESUMEN
New synthetic aminooxy lipid was designed and synthesized as a building block for the formulation of functionalised nanoliposomes (presenting onto the outer surface of aminooxy groups) by microfluidic mixing. Orthogonal binding of cellular mannan (Candida glabrata (CCY 26-20-1) onto the outer surface of functionalised nanoliposomes was modified by orthogonal binding of reducing termini of mannans to oxime lipids via a click chemistry reaction based on aminooxy coupling (oxime ligation). The aminooxy lipid was proved as a suitable active component for preparation of functionalised nanoliposomes by the microfluidic mixing method performed with the instrument NanoAssemblr™. This "on-chip technology" can be easily scaled-up. The structure of mannan-liposomes was visualized by transmission and scanning electron microscopy, including immunogold staining of recombinant mannan receptor bound onto mannosylated-liposomes. The observed structures are in a good correlation with data obtained by DLS, NTA, and TPRS methods. In vitro experiments on human and mouse dendritic cells demonstrate selective internalisation of fluorochrome-labelled mannan-liposomes and their ability to stimulate DC comparable to lipopolysaccharide. We describe a potentially new drug delivery platform for mannan receptor-targeted antimicrobial drugs as well as for immunotherapeutics. Furthermore, the platform based on mannans bound orthogonally onto the surface of nanoliposomes represents a self-adjuvanted carrier for construction of liposome-based recombinant vaccines for both systemic and mucosal routes of administration.
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Células Dendríticas/inmunología , Lectinas Tipo C/inmunología , Liposomas/inmunología , Mananos/inmunología , Lectinas de Unión a Manosa/inmunología , Nanopartículas/química , Receptores de Superficie Celular/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Antígenos de Superficie/metabolismo , Candida glabrata/química , Química Clic , Humanos , Hidroxilaminas/síntesis química , Hidroxilaminas/química , Lípidos/síntesis química , Lípidos/química , Liposomas/química , Liposomas/farmacología , Mananos/química , Mananos/farmacología , Receptor de Manosa , Ratones Endogámicos BALB C , Microfluídica/métodos , Tamaño de la PartículaRESUMEN
OBJECTIVES: The Czech Republic ranks among countries with the highest alcohol consumption per capita. Several older studies discuss Czech media portrayal of health effects of alcohol, but we found no recent analysis of media portrayal of harms caused by alcohol consumption. Our analysis aims to fill this gap in. METHODS: The dataset of texts (n = 903) consisting of articles from press, radio, television and the internet published within a 30-day interval in 2017 (Newton Media computerized database) was coded and analyzed using mixed quantitative and qualitative approach to content analysis. The frequency of references to acute and long-term alcohol harms of various types were counted, and the results were compared to the classification of (alcohol) harms by the Independent Scientific Committee of Drugs (ISCD). RESULTS: The short-term intoxication effects in the areas of crime and road safety, in particular reports on traffic accidents, are over-represented, while topics describing the impact of alcohol use on health, family and society, as well as economic costs or environmental issues seem to be marginal. That corresponds to the fact that police and courts were the information source in more than half of the articles, while information sourced from physicians, sociologists and drug field professionals was rather scarce. CONCLUSIONS: Media portrayal of the harms caused by alcohol use does not match up to real harm effects on the society. In terms of public health, it is imperative to strengthen media presentation of the impact of alcohol use on health and social issues.
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Consumo de Bebidas Alcohólicas/efectos adversos , Medios de Comunicación de Masas/estadística & datos numéricos , Accidentes de Tránsito , Crimen , República Checa , HumanosRESUMEN
Vaccines currently in the clinical use contain adjuvants stimulating preferably Th2 type of immune response associated with the production of specific antibodies, mostly of neutralizing isotypes. This kind of immune response is effective only against some types of pathogens and has limited effect against tumors and many viruses where parallel activation of antigen-specific humoral and cell-mediated immunity is required. One of the main objectives of the current vaccine research is the development of approaches leading to the induction of antigen-specific CD8(+) T cell response including cytotoxic T lymphocyte (CTL). Induction of antigen-specific CD8(+) T cell response to exogenously delivered antigen requires their cross-presentation by antigen presenting cells, especially dendritic cells. The cross-presentation principles seem to be crucial for effective activation of CTL. In this paper, we discuss some approaches to employing heat shock proteins for induction of antigen-specific CD8(+) T cells in the context of cross-presentation and cross-priming principles.
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Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer , Reactividad Cruzada , Células Dendríticas/inmunología , Proteínas de Choque Térmico/inmunología , Neoplasias/terapia , Vacunas Virales , Virosis/terapia , Animales , Citotoxicidad Inmunológica , Células Dendríticas/trasplante , Humanos , Activación de Linfocitos , Neoplasias/inmunología , Virosis/inmunologíaRESUMEN
BACKGROUND: HIV-1 entry into host cells is mediated by interactions between the virus envelope glycoprotein (gp120/gp41) and host-cell receptors. N-glycans represent approximately 50% of the molecular mass of gp120 and serve as potential antigenic determinants and/or as a shield against immune recognition. We previously reported that N-glycosylation of recombinant gp120 varied, depending on the producer cells, and the glycosylation variability affected gp120 recognition by serum antibodies from persons infected with HIV-1 subtype B. However, the impact of gp120 differential glycosylation on recognition by broadly neutralizing monoclonal antibodies or by polyclonal antibodies of individuals infected with other HIV-1 subtypes is unknown. METHODS: Recombinant multimerizing gp120 antigens were expressed in different cells, HEK 293T, T-cell, rhabdomyosarcoma, hepatocellular carcinoma, and Chinese hamster ovary cell lines. Binding of broadly neutralizing monoclonal antibodies and polyclonal antibodies from sera of subtype A/C HIV-1-infected subjects with individual gp120 glycoforms was assessed by ELISA. In addition, immunodetection was performed using Western and dot blot assays. Recombinant gp120 glycoforms were tested for inhibition of infection of reporter cells by SF162 and YU.2 Env-pseudotyped R5 viruses. RESULTS: We demonstrated, using ELISA, that gp120 glycans sterically adjacent to the V3 loop only moderately contribute to differential recognition of a short apex motif GPGRA and GPGR by monoclonal antibodies F425 B4e8 and 447-52D, respectively. The binding of antibodies recognizing longer peptide motifs overlapping with GPGR epitope (268 D4, 257 D4, 19b) was significantly altered. Recognition of gp120 glycoforms by monoclonal antibodies specific for other than V3-loop epitopes was significantly affected by cell types used for gp120 expression. These epitopes included CD4-binding site (VRC03, VRC01, b12), discontinuous epitope involving V1/V2 loop with the associated glycans (PG9, PG16), and an epitope including V3-base-, N332 oligomannose-, and surrounding glycans-containing epitope (PGT 121). Moreover, the different gp120 glycoforms variably inhibited HIV-1 infection of reporter cells. CONCLUSION: Our data support the hypothesis that the glycosylation machinery of different cells shapes gp120 glycosylation and, consequently, impacts envelope recognition by specific antibodies as well as the interaction of HIV-1 gp120 with cellular receptors. These findings underscore the importance of selection of appropriately glycosylated HIV-1 envelope as a vaccine antigen.
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Lyme disease caused by spirochete Borrelia burgdorferi sensu lato, is a tick-born illness. If the infection is not eliminated by the host immune system and/or antibiotics, it may further disseminate and cause severe chronic complications. The immune response to Borrelia is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies associated with Th1 cell activation. A new experimental vaccine was constructed using non-lipidized form of recombinant B. burgdorferi s.s. OspC protein was anchored by metallochelating bond onto the surface of nanoliposomes containing novel nonpyrogenic lipophilized norAbuMDP analogues denoted MT05 and MT06. After i.d. immunization, the experimental vaccines surpassed Alum with respect to OspC-specific titers of IgG2a, IgG2b isotypes when MT06 was used and IgG3, IgM isotypes when MT05 was used. Both adjuvants exerted a high adjuvant effect comparable or better than MDP and proved themselves as nonpyrogenic.
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Acetilmuramil-Alanil-Isoglutamina/química , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Borrelia burgdorferi/inmunología , Quelantes/química , Portadores de Fármacos/química , Vacunas contra Enfermedad de Lyme/inmunología , Nanopartículas/química , Acetilmuramil-Alanil-Isoglutamina/toxicidad , Animales , Rastreo Diferencial de Calorimetría , Quelantes/toxicidad , Portadores de Fármacos/toxicidad , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Luz , Liposomas , Vacunas contra Enfermedad de Lyme/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Nanopartículas/toxicidad , Dispersión de Radiación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Borrelia burgdorferi sensu lato (Spirochaetes) is a group of at least 12 closely related species, some of which are responsible for chronic zoonotic infection that may cause Lyme disease. The only experimentally confirmed vector transmitting Borrelia to mammals is the Ixodes ticks. Borrelia is a highly adapted pathogen that can survive in the host organism in spite of the intense immune responses. Some patients have chronic long-lasting complications despite antibiotic therapy, probably due to adverse effects of the immune responses. A preventive vaccine against this bacterium has not been available due to the relatively broad spectrum and antigenic variability of Borrelia-surface lipoproteins and the different epitope recognition by experimental animals and humans. Although a human vaccine was marketed in the USA, it has been already pulled off the market. In addition, this vaccine was effective only in the USA, where the only pathogenic species is B. burgdorferi sensu stricto. Recent data indicate that a broadly effective vaccine will to be composed of a mixture of several antigens or multiple epitopes.
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Antígenos Bacterianos/inmunología , Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Borrelia burgdorferi , Lipoproteínas/inmunología , Enfermedad de Lyme , Animales , Anticuerpos Antibacterianos/inmunología , Variación Antigénica , Antígenos Bacterianos/metabolismo , Enfermedades Autoinmunes/etiología , Proteínas Bacterianas/metabolismo , Borrelia burgdorferi/inmunología , Borrelia burgdorferi/metabolismo , Humanos , Ixodes/inmunología , Ixodes/metabolismo , Ixodes/microbiología , Lipoproteínas/metabolismo , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/microbiología , Enfermedad de Lyme/patología , Enfermedad de Lyme/prevención & control , Ratones , Datos de Secuencia Molecular , Estados Unidos , Vacunación/efectos adversosRESUMEN
Hsp90-CA is present in cell wall of Candida pseudohyphae or hyphae-typical pathogenic morphotype for both systemic and mucosal Candida infections. Heat shock protein from Candida albicans (hsp90-CA) is an important target for protective antibodies during disseminated candidiasis of experimental mice and human. His-tagged protein rHsp90 was prepared and used as the antigen for preparation of experimental recombinant liposomal vaccine. Nickel-chelating liposomes (the size around 100nm, PDI≤0.1) were prepared from the mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (molar ratio 95:5%) by hydration of lipid film and extrusion methods. New non-pyrogenic hydrophobised derivative of MDP (C18-O-6-norAbuMDP) was incorporated into liposomes as adjuvans. rHsp90 was attached onto the surface of metallochelating liposomes by metallochelating bond and the structure of these proteoliposomes was studied by dynamic light scattering, AF microscopy, TEM and GPC. The liposomes with surface-exposed C18-O-6-norAbuMDP were well recognised and phagocyted by human dendritic cells in vitro. In vivo the immune response towards this experimental vaccine applied in mice (i.d.) demonstrated both TH1 and TH2 response comparable to FCA, but without any side effects. Metallochelating liposomes with lipophilic derivatives of muramyl dipeptide represent a new biocompatible platform for construction of experimental recombinant vaccines and drug-targeting systems.
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Antígenos Fúngicos/inmunología , Quelantes/metabolismo , Proteínas HSP90 de Choque Térmico/inmunología , Inmunidad Celular , Níquel/metabolismo , Animales , Antígenos Fúngicos/metabolismo , Candida/inmunología , Células Cultivadas , Quelantes/química , Materiales Biocompatibles Revestidos/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Liposomas , Ratones , Ratones Endogámicos BALB C , Níquel/inmunología , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/metabolismoRESUMEN
Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the interaction between a variably glycosylated envelope glycoprotein (gp120) and host-cell receptors. Approximately half of the molecular mass of gp120 is contributed by N-glycans, which serve as potential epitopes and may shield gp120 from immune recognition. The role of gp120 glycans in the host immune response to HIV-1 has not been comprehensively studied at the molecular level. We developed a new approach to characterize cell-specific gp120 glycosylation, the regulation of glycosylation, and the effect of variable glycosylation on antibody reactivity. A model oligomeric gp120 was expressed in different cell types, including cell lines that represent host-infected cells or cells used to produce gp120 for vaccination purposes. N-Glycosylation of gp120 varied, depending on the cell type used for its expression and the metabolic manipulation during expression. The resultant glycosylation included changes in the ratio of high-mannose to complex N-glycans, terminal decoration, and branching. Differential glycosylation of gp120 affected envelope recognition by polyclonal antibodies from the sera of HIV-1-infected subjects. These results indicate that gp120 glycans contribute to antibody reactivity and should be considered in HIV-1 vaccine design.
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Anticuerpos/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , Síndrome de Inmunodeficiencia Adquirida/inmunología , Especificidad de Anticuerpos , Línea Celular , Línea Celular Tumoral , ADN Complementario/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Glicósido Hidrolasas/metabolismo , Glicosilación , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/aislamiento & purificación , Seropositividad para VIH/inmunología , Seropositividad para VIH/metabolismo , VIH-1/inmunología , VIH-1/metabolismo , Células Hep G2/metabolismo , Humanos , Células Jurkat/metabolismo , Manosa/metabolismo , Lectina de Unión a Manosa/genética , Espectrometría de Masas , Oligosacáridos/química , Oligosacáridos/aislamiento & purificación , Plásmidos , Polisacáridos/química , Polisacáridos/aislamiento & purificaciónRESUMEN
Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-D-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite, N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other His.