RESUMO
Antigen-binding fragments (Fabs) are preferred alternatives to antibodies for medical application, whereas their short half-lives limit therapeutic effectiveness. Albumin binding domain (ABD) with high affinity for albumin possesses a great potential in enhancing in vivo performance of biotherapeutics. In this study, to mitigate the poor pharmacokinetics of adalimumab Fab targeting tumor necrosis factor-α (TNFα), an ABD fusion strategy was applied innovatively using GA3, ABD035, ABD094 and ABDCon with high affinities for albumin. The prokaryotic expression, bioactivities and half-lives of those novel Fab-ABD fusions were investigated in vitro and in vivo. All Fab-ABD fusions were successfully purified, and they retained similar TNFα-binding activities with the unmodified Fab control, also presented high affinities for human/mouse serum albumin (HSA/MSA). Additionally, the simultaneous binding of the difunctional Fab-ABD fusions to TNFα and albumin was verified, and ABD fused to Fab neither interfered with Fab-TNFα binding nor impaired the association between Fc fragment of IgG receptor and transporter (FcRn) and albumin. Based on the highest binding affinity for HSA and maximal yield, Fab-ABDCon was selected for further evaluation. Fab-ABDCon showed similar thermostability with the Fab control and robust stability in human and mouse plasma. Most notably, the pharmacokinetics of Fab-ABDCon in mice was significantly improved with a 22-fold longer plasma half-life (28.2 h) compared with that of Fab control (1.31 h), which have contributed to its satisfactory therapeutic efficacy in murine TNFα-induced hepatonecrosis model. Thus, Fab-ABDCon could be a promising long-acting candidate suitable for drug development targeting TNFα-mediated inflammatory disease.
Assuntos
Adalimumab/biossíntese , Adalimumab/farmacologia , Albuminas/metabolismo , Anti-Inflamatórios/farmacologia , Fragmentos Fab das Imunoglobulinas/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia , Albuminas/imunologia , Animais , Anti-Inflamatórios/imunologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Desenho de Fármacos , Feminino , Galactosamina/administração & dosagem , Galactosamina/toxicidade , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Fragmentos Fab das Imunoglobulinas/farmacologia , Injeções Intraperitoneais , Camundongos Endogâmicos BALB C , Necrose/induzido quimicamente , Necrose/prevenção & controle , Ligação Proteica/genética , Domínios Proteicos/genética , Receptores Fc/imunologia , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Albumina Sérica Humana/imunologia , Albumina Sérica Humana/metabolismo , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
Tissue-resident memory T cells (TRMs) can profoundly enhance mucosal immunity, but parameters governing TRM induction by vaccination remain poorly understood. Here, we describe an approach exploiting natural albumin transport across the airway epithelium to enhance mucosal TRM generation by vaccination. Pulmonary immunization with albumin-binding amphiphile conjugates of peptide antigens and CpG adjuvant (amph-vaccines) increased vaccine accumulation in the lung and mediastinal lymph nodes (MLNs). Amph-vaccines prolonged antigen presentation in MLNs over 2 weeks, leading to 25-fold increased lung-resident T cell responses over traditional immunization and enhanced protection from viral or tumor challenge. Mimicking such prolonged exposure through repeated administration of soluble vaccine revealed that persistence of both antigen and adjuvant was critical for optimal TRM induction, mediated through T cell priming in MLNs after prime, and directly in the lung tissue after boost. Thus, vaccine persistence strongly promotes TRM induction, and amph-conjugates may provide a practical approach to achieve such kinetics in mucosal vaccines.
Assuntos
Adjuvantes Imunológicos , Albuminas/imunologia , Imunidade nas Mucosas , Memória Imunológica , Pulmão/imunologia , Células T de Memória/imunologia , Animais , Biomarcadores , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunização , Imunofenotipagem , Pulmão/metabolismo , Ativação Linfocitária/imunologia , Células T de Memória/metabolismo , Camundongos , Camundongos Knockout , Especificidade de Órgãos/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Vacinas/imunologiaAssuntos
Albuminas/imunologia , Alérgenos/imunologia , Hipersensibilidade Alimentar , Gliadina/imunologia , Período Pós-Parto , Triticum/imunologia , Adulto , Feminino , Hipersensibilidade Alimentar/sangue , Hipersensibilidade Alimentar/diagnóstico , Hipersensibilidade Alimentar/imunologia , Humanos , Imunoglobulina E/sangue , Reino UnidoRESUMO
The formulations of peptide-based antitumour vaccines being tested in clinical studies are generally associated with weak potency. Here, we show that pharmacokinetically tuning the responses of peptide vaccines by fusing the peptide epitopes to carrier proteins optimizes vaccine immunogenicity in mice. In particular, we show in immunized mice that the carrier protein transthyretin simultaneously optimizes three factors: efficient antigen uptake in draining lymphatics from the site of injection, protection of antigen payloads from proteolytic degradation and reduction of antigen presentation in uninflamed distal lymphoid organs. Optimizing these factors increases vaccine immunogenicity by up to 90-fold and maximizes the responses to viral antigens, tumour-associated antigens, oncofetal antigens and shared neoantigens. Protein-peptide epitope fusions represent a facile and generalizable strategy for enhancing the T-cell responses elicited by subunit vaccines.
Assuntos
Vacinas Anticâncer/imunologia , Vacinas Anticâncer/farmacologia , Imunogenicidade da Vacina/imunologia , Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/farmacocinética , Albuminas/imunologia , Animais , Antígenos de Neoplasias , Fatores de Transcrição de Zíper de Leucina Básica , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Epitopos , Imunidade Celular , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Repressoras/genéticaRESUMO
Aliphatic di- and polyisocyanates are crucial chemical ingredients in many industrial processes and are a well-recognized cause of occupational asthma. Serologic detection of "chemical epitopes" in biological samples could serve as an exposure surveillance approach toward disease prevention, and thus we sought to generate aliphatic isocyanate-specific monoclonal antibodies (mAbs). Three hybridomas were generated from Balb/c mice immunized with a commercial product containing a combination of uretdione, homopolymer, and monomeric forms of hexamethylene diisocyanate (HDI). Three stable hybridomas were subcloned by limiting dilution, two secreting IgG1κ and one secreting IgMκ mAb that bind aliphatic di- and polyisocyanates (conjugated to albumin), but not aromatic toluene or methylene diphenyl diisocyanate (TDI or MDI). Each mAb demonstrates slight differences in epitope specificity, for example, recognition of hydrogenated MDI (HMDI) or different carrier proteins (transferrin, actin) reacted with vapor phase HDI, and is encoded by unique recombination of different germline antibody genes, with distinct complementary determining regions. By western blot, all three mAbs detect a molecule with characteristics of an albumin adduct uniquely in urine from mice skin exposed to a mixture of aliphatic di- and polyisocyanate. Together, the data define molecular determinants of humoral immune recognition of aliphatic di- and polyisocyanates through new mAbs, which will serve as useful research reagents and may be applicable to future exposure surveillance efforts.
Assuntos
Actinas/imunologia , Anticorpos Monoclonais/farmacologia , Epitopos/imunologia , Transferrina/imunologia , Actinas/isolamento & purificação , Albuminas/imunologia , Albuminas/isolamento & purificação , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Epitopos/química , Humanos , Hibridomas/imunologia , Isocianatos/química , Isocianatos/imunologia , Camundongos , Poliuretanos/química , Ligação Proteica/imunologia , Tolueno 2,4-Di-Isocianato/química , Tolueno 2,4-Di-Isocianato/imunologiaRESUMO
Although pizza is one of the most popular foods in the world, allergic responses after ingesting pizza are relatively uncommon. However, precisely identifying the allergens responsible for these allergic reactions is challenging because of the high and diverse number of ingredients used in pizza preparation. In this report, we aim to identify the allergens responsible for systemic allergic reactions following ingestion of pizza in two patients. Using a skin prick by prick test (SPPT) and in vitro techniques, with natural and recombinant purified allergens from tomato and mustard seeds, we identified 2S albumin and non-specific lipid transfer proteins (nsLTP) as the proteins involved. However, IgE responses to the four nsLTPs differed before and after denaturation and reduction, thus suggesting additional complexity around nsLTP in food processing.
Assuntos
Albuminas/imunologia , Anafilaxia/imunologia , Antígenos de Plantas/imunologia , Proteínas de Transporte/imunologia , Hipersensibilidade Alimentar/imunologia , Imunoglobulina E/imunologia , Anafilaxia/etiologia , Pré-Escolar , Hipersensibilidade Alimentar/complicações , Humanos , Solanum lycopersicum/imunologia , Masculino , Mostardeira/imunologia , Sementes/imunologia , Testes Cutâneos , Adulto JovemRESUMO
Green pea (Pisum sativum) is a component of European cuisine; however, an estimated 0.8% of Europeans suffer from allergies to pea proteins. We examined the immunoreactive potential of pea albumins (PA) in BALB/c and C57BL/6 mice. Mice were orally gavaged with PA or glycated pea albumins (G-PA) for 10 consecutive days, in combination with an adjuvant. Both PA and G-PA increased PA-specific serum antibody titers to about 212 for anti-PA IgG, â¼27 for anti-PA IgA, and â¼27.8 for anti-PA IgA in fecal extracts (p < 0.001). On day 42 postexposure, the antibodies titers decreased and were greater in BALB/c compared to C57BL/6 mice (p < 0.05). Distribution of CD4+ and CD8+ T cells in lymphoid tissues presented strain-specific differences. PA was found to induce lymphocyte proliferation; however, G-PA did not. Both PA and G-PA changed CD4+ and CD8+ T cells percentages in some lymphoid tissues; however, this did not impact cytokines production by splenocyte cultures evidenced by the stimulation of Th1, Th2, and Th17 cells. The observed immunomodulatory properties of PA and G-PA and lack of a sign of allergic reaction render them suitable for supplements in personalized diets, but further research is needed to precisely understand this activity.
Assuntos
Albuminas/imunologia , Hipersensibilidade Alimentar/imunologia , Pisum sativum/imunologia , Proteínas de Plantas/imunologia , Animais , Anticorpos/imunologia , Linfócitos T CD8-Positivos , Feminino , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Th1/imunologia , Células Th2/imunologiaRESUMO
STUDY DESIGN: Case report. OBJECTIVES: To describe intraoperative administration of albumin as a cause of immunoglobulin E (IgE)-mediated anaphylaxis and cardiac arrest in an adolescent with adolescent idiopathic scoliosis. BACKGROUND: Albumin is considered the reference intraoperative colloidal solution, and is used commonly as a volume expander for treating hypovolemia. Albumin rarely causes an anaphylactic reaction, with a documented rate of only 0.099%. METHOD: An adolescent with scoliosis experienced acute, intraoperative hypotension during exposure for planned T5-L4 posterior spinal fusion shortly after infusion of albumin. She was treated rapidly and successfully with CPR and epinephrine. RESULTS: Intraoperative transesophageal echocardiogram, chest radiograph, and serum histamine, serum tryptase, and urine N-methyl-histamine laboratory tests confirmed albumin anaphylaxis to be the etiology of the intraoperative event. Further postoperative complications were avoided as a result of the rapid diagnosis and treatment. CONCLUSIONS: Although rare, IgE-mediated anaphylaxis to albumin, if administered, must be considered a possible cause of acute, intraoperative hypotension. Rapid management of anaphylaxis with communication between the surgeon, anesthesia team, and operative staff are essential if additional complications are to be avoided.
Assuntos
Albuminas/administração & dosagem , Albuminas/efeitos adversos , Anafilaxia/diagnóstico , Anafilaxia/etiologia , Cuidados Intraoperatórios/efeitos adversos , Complicações Intraoperatórias/diagnóstico , Complicações Intraoperatórias/etiologia , Escoliose/cirurgia , Fusão Vertebral/métodos , Doença Aguda , Adolescente , Albuminas/imunologia , Anafilaxia/imunologia , Anafilaxia/terapia , Reanimação Cardiopulmonar , Diagnóstico Precoce , Epinefrina/uso terapêutico , Feminino , Humanos , Hipotensão/etiologia , Imunoglobulina E , Complicações Intraoperatórias/terapia , Período Intraoperatório , Resultado do TratamentoRESUMO
From whole tissues to single-cell lysate, heterogeneous immunoassays are widely utilized for analysis of protein targets in complex biospecimens. Recently, benzophenone-functionalized hydrogel scaffolds have been used to immobilize target protein for immunoassay detection with fluorescent antibody probes. In benzophenone-functionalized hydrogels, multiplex target detection occurs via serial rounds of chemical stripping (incubation with sodium-dodecyl-sulfate (SDS) and ß-mercaptoethanol at 50-60 °C for ≥1 h), followed by reprobing (interrogation with additional antibody probes). Although benzophenone facilitates covalent immobilization of proteins to the hydrogel, we observe 50% immunoassay signal loss of immobilized protein targets during stripping rounds. Here, we identify and characterize signal loss mechanisms during stripping and reprobing. We posit that loss of immobilized target is responsible for ≥50% of immunoassay signal loss, and that target loss is attributable to disruption of protein immobilization by denaturing detergents (SDS) and incubation at elevated temperatures. Furthermore, our study suggests that protein losses under non-denaturing conditions are more sensitive to protein structure (i.e., hydrodynamic radius), than to molecular mass (size). We formulate design guidance for multiplexed in-gel immunoassays, including that low-abundance proteins be immunoprobed first, even when targets are covalently immobilized to the gel. We also recommend careful scrutiny of the order of proteins targets detected via multiple immunoprobing cycles, based on the protein immobilization buffer composition.
Assuntos
Benzofenonas/química , Hidrogéis/química , Proteínas Imobilizadas/química , Microfluídica/métodos , Albuminas/química , Albuminas/imunologia , Animais , Bovinos , Galinhas , Proteínas Imobilizadas/imunologia , Imunoensaio/métodos , Mercaptoetanol/química , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Ribonuclease Pancreático/química , Ribonuclease Pancreático/imunologia , Dodecilsulfato de Sódio/química , Inibidores da Tripsina/química , Inibidores da Tripsina/imunologiaRESUMO
Adjuvants are substances that enhance adaptive immune response to antigen. Development of a safe and effective immunostimulant adjuvant is essential for the efficacy of a vaccine to protect against infectious pathogens. Purple non-sulfur photosynthetic bacteria exhibited nontoxic natural lipid A variants that are distinct in their chemical structures from that of the Escherichia coli-type lipid A. In this study, the adjuvant efficacy of attenuated lipid A variants and their corresponding lipopolysaccharides (LPSs), derived from purple photosynthetic bacteria (Rhodocyclus tenuis and Rhodobacter sphaeroides) were evaluated. LPS was extracted using modified phenol-chloroform-petroleum ether method and lipid A was separated by mild acid hydrolysis. Trinitrophenol (TNP) was conjugated to hen egg albumin (TNP-HEA) and used as haptenic antigen. The LPS and lipid A adjuvant candidates were formulated in oil-in-water emulsion (OIWE) and evaluated to elicit anti-TNP IgG against TNP-HEA conjugate in BALB/c female mice. The anti-TNP IgG titers were measured using ELISA. The intact LPS-based adjuvants present in OIWE formulation showed significantly higher efficacy to elicit anti-TNP IgG titers against TNP-HEA conjugate compared to their corresponding lipid A-based adjuvants. As expected, the OIWE formulations of all LPS- and lipid A-based adjuvant candidates showed higher activities compared to the aqueous formulations. Slow reduction in the levels of anti-TNP IgG antibodies in the serum was observed over 4â¯months after immunization using the LPS- and lipid A-based adjuvant candidates which may provide a long protection against pathogens. The attenuated LPSs and lipid A's from the photosynthetic bacteria showed promising results to develop novel safe and effective adjuvants that can evoke the immune response. The most promising adjuvant candidate was the LPS-based adjuvant from R. tenuis.
Assuntos
Lipídeo A/imunologia , Lipopolissacarídeos/imunologia , Rhodobacter sphaeroides/imunologia , Rhodocyclaceae/imunologia , Adjuvantes Imunológicos/administração & dosagem , Albuminas/imunologia , Animais , Anticorpos/imunologia , Galinhas/imunologia , Feminino , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Atenuadas/imunologiaRESUMO
Depression is the common and early symptoms associated with early onset of SLE, 16α-hydroxyestrone (16α-OHE1) levels were found to be significantly higher in serum and urine in patients with SLE. This study was carried out in order to know whether depression and its related parameters in the SLE patients enhanced the production of autoantibodies against 16α-OHE1-albumin (A) complexes. The autoantibodies in the serum of 100 SLE [including 65 depressed SLE (DSLE)] patients and 37 control subjects were detected by using direct binding, inhibition ELISA and quantitative precipitin titration. Autoantibodies from DSLE patients (and also the patients who were taken anti-depressant and with neurological symptoms) showed high binding to 16α-OHE1-A in contrast to SLE (pâ¯<â¯0.05) and control subjects (pâ¯<â¯0.001). Although, SLE sera showed high recognition to 16α-OHE1-A in comparison to A (pâ¯<â¯0.05) or 16α-OHE1 (pâ¯<â¯0.001). The affinity of autoantibodies for 16α-OHE1-A was found to be high for DSLE (1.16â¯×â¯10-7â¯M) and SLE (1.24â¯×â¯10-7â¯M) patients as detected by Langmuir plot. The concentration of 16α-OHE1 (pâ¯<â¯0.05) and inflammatory cytokines (IL-6, pâ¯<â¯0.05 and IL-17, pâ¯<â¯0.001) in the serum of SLE patients was found to be significantly higher than controls. Depression and its related parameters in SLE enhanced the production of autoantibodies against 16α-OHE1-A through the generation of inflammatory conditions. Depression in SLE patients increased the release of pro-inflammatory cytokine (IL-6 and IL-17) that in turn generating more autoantibodies and showed strong recognition to 16α-OHE1-A.
Assuntos
Albuminas/imunologia , Autoanticorpos/sangue , Depressão/imunologia , Hidroxitestosteronas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Adulto , Albuminas/química , Estudos de Casos e Controles , Feminino , Humanos , Hidroxitestosteronas/química , Mediadores da Inflamação/sangue , Interleucina-17/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Regulação para CimaRESUMO
The protozoan parasite, Leishmania donovani, undergoes several molecular adaptations and secretes many effector molecules for host cell manipulation and successful parasitism. The current study identifies an albumin-like secretory protein, expressed in its extracellular promastigote forms. A leishmanial complementary DNA sequence of a partial gene has been cloned, and the encoded peptide (14 kD) is used for the production of polyclonal antibody. This targeted antibody identifies a large native protein (66.421 kD), expressed stage-specifically in promastigotes. Through electron microscopic studies, the native protein is found to be localized in the flagellar pocket and flagella and at the surface of the promastigotes. This native protein is purified with the same customized antibody for future characterization and sequencing. The sequence analysis reveals its homology with the mammalian serum albumin. It is evidenced from in silico studies that this albumin-like protein remains associated with long-chain fatty acids while in vitro studies indicate its close association with membrane cholesterol. Since antibody-mediated blocking compromises the parasite infectivity, these leishmanial albumin-like molecules are hereby proposed to play an instrumental role in the infectivity of L. donovani to peripheral blood monocyte cells. Thus, identification and characterization of an albumin-like protein in L. donovani promastigotes may be interpreted as a molecular adaptation candidate. It may be hypothesized that the parasite mimics the mammalian system for importing fatty acids into the intracellular amastigotes, facilitating its host cell infectivity.
Assuntos
Albuminas/análise , Flagelos/metabolismo , Leishmania donovani/genética , Leishmania donovani/metabolismo , Leishmaniose Visceral/parasitologia , Proteínas de Protozoários/genética , Albuminas/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Flagelos/imunologia , Leishmania donovani/crescimento & desenvolvimento , Proteínas de Protozoários/imunologiaAssuntos
Albuminas/imunologia , Asma/imunologia , Menstruação/imunologia , Adulto , Alérgenos/imunologia , Feminino , HumanosAssuntos
Albuminas/imunologia , Alérgenos/imunologia , Pelo Animal/imunologia , Asma Ocupacional/diagnóstico , Lipocalinas/imunologia , Técnicos em Manejo de Animais , Animais , Asma Ocupacional/imunologia , Gatos , Reações Cruzadas , Cães , Humanos , Camundongos , Análise em Microsséries , Exposição Ocupacional/efeitos adversos , Animais de Estimação , Ratos , Risco , Médicos VeterináriosRESUMO
Wheat allergy specifically refers to the adverse reaction involving IgE antibody to one or more protein fraction of wheat such as albumin, globulin, gliadin and glutenin (gluten). The majority of IgE-mediated reactions to wheat involve albumin and globulin fraction while gluten (gliadin & glutenin) also cause allergy (Celiac disease). Allergic reactions to wheat may be caused by ingestion of wheat containing foods or inhalation of flour (Bakers asthma). The present study was an effort to explore the antibody response of different proteins present in wheat. ELISA results revealed that the antibody response for albumin varied from 0.92-1.78, whereas, for globulin ranged from 1.39-1.60. Antibody response against glutenin and gliadin ranged from 0.57-1.05 and 0.98-1.95 respectively, among the different varieties of wheat. All the tested wheat varieties showed the significant difference antibody response against the different fractions of protein.
Assuntos
Albuminas/imunologia , Globulinas/imunologia , Glutens/imunologia , Imunoglobulina E/sangue , Triticum/imunologia , Albuminas/isolamento & purificação , Animais , Ensaio de Imunoadsorção Enzimática , Gliadina/imunologia , Gliadina/isolamento & purificação , Globulinas/isolamento & purificação , Glutens/isolamento & purificação , Imunoglobulina E/imunologia , Coelhos , Triticum/metabolismoRESUMO
BACKGROUND: Most food allergens sensitizing via the gastrointestinal tract are stable proteins that are resistant to pepsin digestion, in particular major peanut allergens, Ara h 2 and Ara h 6. Survival of their large fragments is essential for sensitizing capacity. However, the immunoreactive proteins/peptides to which the immune system of the gastrointestinal tract is exposed during digestion of peanut proteins are unknown. Particularly, the IgE reactivity of short digestion-resistant peptides (SDRPs; <10 kDa) released by gastric digestion under standardized and physiologically relevant in vitro conditions has not been investigated. OBJECTIVE: The aim of this study was to investigate and identify digestion products of major peanut allergens and in particular to examine IgE reactivity of SDRPs released by pepsin digestion of whole peanut grains. METHODS: Two-dimensional gel-based proteomics and shotgun peptidomics, immunoblotting with allergen-specific antibodies from peanut-sensitized patients, enzyme-linked immunosorbent inhibition assay and ImmunoCAP tests, including far ultraviolet-circular dichroism spectroscopy were used to identify and characterize peanut digesta. RESULTS: Ara h 2 and Ara h 6 remained mostly intact, and SDRPs from Ara h 2 were more potent in inhibiting IgE binding than Ara h 1 and Ara 3. Ara h 1 and Ara h 3 exhibited sequential digestion into a series of digestion-resistant peptides with preserved allergenic capacity. A high number of identified SDRPs from Ara h 1, Ara h 2 and Ara h 3 were part of short continuous epitope sequences and possessed substantial allergenic potential. CONCLUSION AND CLINICAL RELEVANCE: Peanut grain digestion by oral and gastric phase enzymes generates mixture of products, where the major peanut allergens remain intact and their digested peptides have preserved allergenic capacity highlighting their important roles in allergic reactions to peanut.
Assuntos
Alérgenos/imunologia , Arachis/efeitos adversos , Hipersensibilidade a Amendoim/diagnóstico , Hipersensibilidade a Amendoim/imunologia , Albuminas/química , Albuminas/imunologia , Alérgenos/química , Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Estudos de Coortes , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/química , Glicoproteínas/imunologia , Humanos , Imunoglobulina E/imunologia , Proteínas de Membrana , Modelos Moleculares , Peptídeos/química , Peptídeos/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Conformação Proteica , Proteoma , Proteômica/métodos , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/imunologia , Relação Estrutura-AtividadeRESUMO
Oxidation-associated malondialdehyde (MDA) modification of proteins can generate immunogenic neo-epitopes that are recognized by autoantibodies. In health, IgM antibodies to MDA-adducts are part of the natural antibody pool, while elevated levels of IgG anti-MDA antibodies are associated with inflammatory and autoimmune conditions. Yet, in human autoimmune disease IgG anti-MDA responses have not been well characterized and their potential contribution to disease pathogenesis is not known. Here, we investigate MDA-modifications and anti-MDA-modified protein autoreactivity in rheumatoid arthritis (RA). While RA is primarily associated with autoreactivity to citrullinated antigens, we also observed increases in serum IgG anti-MDA in RA patients compared to controls. IgG anti-MDA levels significantly correlated with disease activity by DAS28-ESR and serum TNF-alpha, IL-6, and CRP. Mass spectrometry analysis of RA synovial tissue identified MDA-modified proteins and revealed shared peptides between MDA-modified and citrullinated actin and vimentin. Furthermore, anti-MDA autoreactivity among synovial B cells was discovered when investigating recombinant monoclonal antibodies (mAbs) cloned from single B cells, and 3.5% of memory B cells and 2.3% of plasma cells were found to be anti-MDA positive. Several clones were highly specific for MDA-modification with no cross-reactivity to other antigen modifications such as citrullination, carbamylation or 4-HNE-carbonylation. The mAbs recognized MDA-adducts in a variety of proteins including albumin, histone 2B, fibrinogen and vimentin. Interestingly, the most reactive clone, originated from an IgG1-bearing memory B cell, was encoded by near germline variable genes, and showed similarity to previously reported natural IgM. Other anti-MDA clones display somatic hypermutations and lower reactivity. Importantly, these anti-MDA antibodies had significant in vitro functional properties and induced enhanced osteoclastogenesis, while the natural antibody related high-reactivity clone did not. We postulate that these may represent distinctly different facets of anti-MDA autoreactive responses.
Assuntos
Anticorpos Monoclonais/metabolismo , Artrite Reumatoide/imunologia , Autoantígenos/imunologia , Linfócitos B/imunologia , Epitopos Imunodominantes/imunologia , Malondialdeído/imunologia , Oxirredução , Membrana Sinovial/imunologia , Actinas/imunologia , Albuminas/imunologia , Anticorpos Monoclonais/genética , Autoanticorpos/sangue , Autoantígenos/metabolismo , Autoimunidade , Células Cultivadas , Progressão da Doença , Humanos , Epitopos Imunodominantes/metabolismo , Imunoglobulina G/sangue , Peroxidação de Lipídeos , Malondialdeído/metabolismo , Osteogênese , Hipermutação Somática de Imunoglobulina , Vimentina/imunologiaRESUMO
Objectives: Anti-carbamylated protein (anti-CarP) antibodies are detected in RA patients. Fetal calf serum is used as an antigen source in anti-CarP ELISA, and the precise target antigens have not been found. We aimed to identify the target antigens of anti-CarP antibodies. Methods: Western blotting of anti-CarP antibodies was conducted. Anti-carbamylated human albumin (CarALB) antibody was detected by in-house ELISA for 493 RA patients and 144 healthy controls (HCs). An inhibition ELISA of anti-CarP antibodies by CarALB and citrullinated albumin (citALB) was performed using eight RA patients' sera. Serum CarALB was detected by liquid chromatography-tandem mass spectroscopy (LC/MS/MS), and the serum MPO concentration was measured by ELISA. Results: We focused on carbamylated albumin because it corresponded to the size of the thickest band detected by western blotting of anti-CarP antibodies. Anti-CarALB antibody was detected in 31.4% of RA patients, and the correlation of the titres between anti-CarALB and anti-CarP was much closer than that between anti-citALB and anti-CCP antibodies (ρ = 0.59 and ρ = 0.16, respectively). The inhibition ELISA showed that anti-CarP antibodies were inhibited by CarALB, but not by citALB. CarALB was detected in sera from RA patients by LC/MS/MS. The serum MPO concentration was correlated with disease activity and was higher in RA patients with anti-CarALB antibody than in those without. Conclusion: We found that carbamylated albumin is a novel target antigen of anti-CarP antibodies, and it is the first reported target antigen that has not been reported as the target of ACPA.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Autoantígenos/sangue , Carbamatos/imunologia , Peptídeos Cíclicos/sangue , Adulto , Albuminas/imunologia , Albuminas/metabolismo , Artrite Reumatoide/fisiopatologia , Biomarcadores/sangue , Western Blotting , Cromatografia Líquida , Bases de Dados Factuais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Peptídeos Cíclicos/imunologia , Estudos Retrospectivos , Fator Reumatoide/sangue , Estatísticas não Paramétricas , Espectrometria de Massas em TandemRESUMO
Summary: It has been shown that allergen immunotherapy (AIT) is effective in reducing symptoms of allergic asthma and rhinitis. Data on the efficacy are less convincing with regard to AIT for allergens of common pets (cats/dogs). We describe a case of dog allergy in which we explored if dog AIT (DAI) could reduce a concomitant allergic sensitization to other allergens of furry animals. Our case demonstrates the efficacy of sublingual DAI on SPTs, symptom score, and spirometric responses despite persistent exposure to dog allergens at home in a patient sensitized, but not exposed, to several other furry animals. Moreover, this is the first report suggesting that DAI is able to reduce SPTs responses not only to dog, but also to other furry animals such as rabbit, horse, mouse, rat, hamster, cow. We recommend an accurate anamnesis and diagnosis of dog allergy before prescribing DAI. In particular, the use of ImmunoCAP ISAC is essential to verify the presence of IgE to lipocalins / albumins belonging to other furry animals. Obviously further studies carried out by using different DAI schedules, allergen amount and time of re-evaluation, laboratory procedure should be performed to confirm our findings.
Assuntos
Alérgenos/administração & dosagem , Asma/terapia , Cães/imunologia , Cabelo/imunologia , Imunoterapia Sublingual/métodos , Administração Sublingual , Adulto , Albuminas/imunologia , Alérgenos/imunologia , Animais , Asma/diagnóstico , Asma/imunologia , Biomarcadores/sangue , Humanos , Imunoglobulina E/sangue , Testes Intradérmicos , Lipocalinas/imunologia , Masculino , Especificidade da Espécie , Espirometria , Resultado do TratamentoRESUMO
Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.