ABSTRACT
Selective removal of excess or damaged mitochondria is an evolutionarily conserved process that contributes to mitochondrial quality and quantity control. This catabolic event relies on autophagy, a membrane trafficking system that sequesters cytoplasmic constituents into double membrane-bound autophagosomes and delivers them to lysosomes (vacuoles in yeast) for hydrolytic degradation and is thus termed mitophagy. Dysregulation of mitophagy is associated with various diseases, highlighting its physiological relevance. In budding yeast, the pro-mitophagic single-pass membrane protein Atg32 is upregulated under prolonged respiration or nutrient starvation, anchored on the surface of mitochondria, and activated to recruit the autophagy machinery for the formation of autophagosomes surrounding mitochondria. In this chapter, we provide protocols to assess Atg32-mediated mitophagy using fluorescence microscopy and immunoblotting.
Subject(s)
Microscopy, Fluorescence , Mitochondria , Mitophagy , Saccharomycetales , Microscopy, Fluorescence/methods , Saccharomycetales/metabolism , Mitochondria/metabolism , Immunoblotting/methods , Autophagy-Related Proteins/metabolism , Autophagy-Related Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Autophagy/physiology , Autophagosomes/metabolism , Receptors, Cytoplasmic and NuclearABSTRACT
Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.
Subject(s)
Antibodies , Electrophoresis, Polyacrylamide Gel , Peptides , Electrophoresis, Polyacrylamide Gel/methods , Peptides/chemistry , Peptides/immunology , Antibodies/chemistry , Antibodies/immunology , Blotting, Western/methods , Humans , Calreticulin/chemistry , Calreticulin/immunology , Calreticulin/metabolism , Immunoblotting/methods , Antibody Specificity , AnimalsABSTRACT
Background: Alpha-synuclein (aSyn) is a key player in neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies, or multiple system atrophy. aSyn is expressed throughout the brain, and can also be detected in various peripheral tissues. In fact, initial symptoms of PD are non-motoric and include autonomic dysfunction, suggesting that the periphery might play an important role in early development of the disease. aSyn is expressed at relatively low levels in non-central tissues, which brings challenges for its detection and quantification in different tissues. Objective: Our goal was to assess the sensitivity of aSyn detection in central and peripheral mouse tissues through capillary electrophoresis (CE) immunoblot, considering the traditional SDS-PAGE immunoblot as the current standard. Methods: Tissues from central and non-central origin from wild type mice were extracted, and included midbrain, inner ear, and esophagus/stomach. aSyn detection was assessed through immunoblotting using Simple Western size-based CE and SDS-PAGE. Results: CE immunoblots show a consistent detection of aSyn in central and peripheral tissues. Through SDS-PAGE, immunoblots revealed a reliable signal corresponding to aSyn, particularly following membrane fixation. Conclusion: Our results suggest a reliable detection of aSyn in central and peripheral tissues using the CE Simple Western immunoblot system. These observations can serve as preliminary datasets when aiming to formally compare CE with SDS-PAGE, as well as for further characterization of aSyn using this technique.
Subject(s)
Electrophoresis, Capillary , alpha-Synuclein , Animals , alpha-Synuclein/metabolism , alpha-Synuclein/analysis , Mice , Electrophoresis, Capillary/methods , Mice, Inbred C57BL , Immunoblotting/methods , Esophagus/metabolism , Mesencephalon/metabolismABSTRACT
We compared the performance of a new modified two-tier testing (MTTT) platform, the Diasorin Liaison chemiluminescent immunoassay (CLIA), to the Zeus enzyme-linked immunoassay (ELISA) MTTT and to Zeus ELISA/Viramed immunoblot standard two-tier testing (STTT) algorithm. Of 537 samples included in this study, 91 (16.9%) were positive or equivocal by one or more screening tests. Among these 91 samples, only 57 samples were concordant positive by first-tier screening tests, and only 19 of 57 were concordant by the three second-tier methods. For IgM results, positive percent agreement (PPA) was 68.1% for Diasorin versus 89.4% for Zeus compared to immunoblot. By contrast, the PPA for IgG for both Diasorin and Zeus was 100%. Using a 2-out-of-3 consensus reference standard, the PPAs for IgM were 75.6%, 97.8%, and 95.6% for Diasorin, Zeus, and immunoblot, respectively. The difference between Zeus MTTT and Diasorin MTTT for IgM detection was significant (P = 0.0094). PPA for both Diasorin and Zeus MTTT IgG assays was 100% but only 65.9% for immunoblot STTT (P = 0.0005). In total, second-tier positive IgM and/or IgG results were reported for 57 samples by Diasorin MTTT, 63 by Zeus MTTT, and 54 by Viramed STTT. While Diasorin CLIA MTTT had a much more rapid, automated, and efficient workflow, Diasorin MTTT was less sensitive for the detection of IgM than Zeus MTTT and STTT including in 5 early Lyme cases that were IgM negative but IgG positive. IMPORTANCE: The laboratory diagnosis of Lyme disease relies upon the detection of antibodies to Borrelia species. Standard two tier testing (STTT) methods rely upon immunoblots which have clinical and technical limitations. Modified two-tier testing (MTTT) methods have recently become available and are being widely adopted. There are limited independent data available assessing the performance of MTTT and STTT methods.
Subject(s)
Algorithms , Antibodies, Bacterial , Immunoglobulin G , Immunoglobulin M , Lyme Disease , Sensitivity and Specificity , Serologic Tests , Humans , Lyme Disease/diagnosis , Lyme Disease/immunology , Lyme Disease/blood , Immunoglobulin M/blood , Immunoglobulin G/blood , Serologic Tests/methods , Serologic Tests/standards , Antibodies, Bacterial/blood , Luminescent Measurements/methods , Immunoblotting/methodsABSTRACT
INTRODUCTION: For the diagnosis of liver diseases, clinical criteria, biochemical, immunological and histological parameters are included. The autoimmune panel is an immunoblot that contemplates the detection of antibodies against 9 different hepatic antigens, which could guide the diagnosis of these pathologies. OBJECTIVE: To describe the usefulness of the autoimmune panel in the diagnosis of liver diseases. Methods: Observational, descriptive study. All autoimmune panels performed between January 2020 and August 2021 (n = 279) were reviewed, and the ones with positive result selected (n = 101). Clinical records were reviewed, including: clinical, biochemical, immunological and histological characteristics. Diagnosis was determined by clinical suspicion (clinical, biochemical and immunological parameters), only through autoimmune panel, and according to liver biopsy in available cases. RESULTS: 45 patients with complete clinical history were included in the analysis; 82% women, median age 58 years (16-79). Clinical suspicions included autoimmune hepatitis (AIH) in 12 patients (27%), primary biliary cholangitis (PBC) in 10 patients (22%), overlap syndrome (AIH/PBC) in 17 (38%), and others in 6 (13%). The diagnosis of PBC was confirmed by autoimmune panel in 9/10 and 11/17 patients with clinical suspicion of PBC and HAI/PBC, respectively. Of the 27 patients with initial clinical suspicion of PBC, 14 had negative AMA and AMA-M2 (6 had Sp100 and 5 gp210 as the only markers and 3 had positive Sp100 and PML). In 10/14 patients, the diagnosis was confirmed by panel and/or compatible liver biopsy. CONCLUSION: The autoimmune panel turns out to be a useful diagnostic tool for liver diseases, especially PBC in isolation or in overlap syndrome.
Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Aged , Young Adult , Autoantibodies/blood , Immunoblotting/methods , Hepatitis, Autoimmune/diagnosis , Hepatitis, Autoimmune/immunology , Hepatitis, Autoimmune/blood , Liver Diseases/diagnosis , Liver Diseases/immunology , Autoimmune Diseases/diagnosis , Autoimmune Diseases/immunology , Autoimmune Diseases/blood , Liver Cirrhosis, Biliary/diagnosis , Liver Cirrhosis, Biliary/immunology , Liver Cirrhosis, Biliary/bloodABSTRACT
INTRODUCTION: For the diagnosis of liver diseases, clinical criteria, biochemical, immunological and histological parameters are included. The autoimmune panel is an immunoblot that contemplates the detection of antibodies against 9 different hepatic antigens, which could guide the diagnosis of these pathologies. OBJECTIVE: To describe the usefulness of the autoimmune panel in the diagnosis of liver diseases. METHODS: Observational, descriptive study. All autoimmune panels performed between January 2020 and August 2021 (n = 279) were reviewed, and the ones with positive result selected (n = 101). Clinical records were reviewed, including: clinical, biochemical, immunological and histological characteristics. Diagnosis was determined by clinical suspicion (clinical, biochemical and immunological parameters), only through autoimmune panel, and according to liver biopsy in available cases. RESULTS: 45 patients with complete clinical history were included in the analysis; 82% women, median age 58 years (16-79). Clinical suspicions included autoimmune hepatitis (AIH) in 12 patients (27%), primary biliary cholangitis (PBC) in 10 patients (22%), overlap syndrome (AIH/PBC) in 17 (38%), and others in 6 (13%). The diagnosis of PBC was confirmed by autoimmune panel in 9/10 and 11/17 patients with clinical suspicion of PBC and HAI/PBC, respectively. Of the 27 patients with initial clinical suspicion of PBC, 14 had negative AMA and AMA-M2 (6 had Sp100 and 5 gp210 as the only markers and 3 had positive Sp100 and PML). In 10/14 patients, the diagnosis was confirmed by panel and/or compatible liver biopsy. CONCLUSION: The autoimmune panel turns out to be a useful diagnostic tool for liver diseases, especially PBC in isolation or in overlap syndrome.
Subject(s)
Autoantibodies , Hepatitis, Autoimmune , Immunoblotting , Liver Diseases , Humans , Female , Autoantibodies/blood , Male , Middle Aged , Adult , Aged , Adolescent , Young Adult , Immunoblotting/methods , Hepatitis, Autoimmune/immunology , Hepatitis, Autoimmune/diagnosis , Hepatitis, Autoimmune/blood , Liver Diseases/immunology , Liver Diseases/diagnosis , Liver Cirrhosis, Biliary/immunology , Liver Cirrhosis, Biliary/diagnosis , Liver Cirrhosis, Biliary/blood , Autoimmune Diseases/immunology , Autoimmune Diseases/diagnosis , Autoimmune Diseases/bloodABSTRACT
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Subject(s)
Humans , Male , Adult , Anaphylaxis/immunology , Food Hypersensitivity/immunology , Egg Hypersensitivity/immunology , Salmon , Immunoblotting/methods , Asthma/complications , Rhinitis, Allergic/complications , Fish Products/adverse effectsABSTRACT
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Subject(s)
Humans , Female , Adult , Egg Hypersensitivity/diagnosis , Food Hypersensitivity/diagnosis , Food Hypersensitivity/immunology , Ducks , Chickens , Immunoblotting/methods , Egg Hypersensitivity/immunologyABSTRACT
Background. Current methods for the production of Histoplasma capsulatum antigens are problematic in terms of standardization, specificity, stability, repeatability and reproducibility. Aims. In this study, we sought to optimize the methodology for producing H. capsulatum antigens, and to evaluate its applicability. Methods. Antigenic preparations obtained from 12 H. capsulatum isolates were evaluated by double immunodiffusion and immunoblotting assays against homologous and heterologous sera. Results. The evaluated and optimized protocol allowed a more stable production, as well as repeatable, reproducible, with shorter culture time and less costly. By double immunodiffusion and immunoblotting assays, the best pattern of reactivity was observed for antigens obtained with 33 days of culture from the isolates 200 and 406 against the M antigen and for the isolate 200 with 15 days against H antigen. The SDS-PAGE presented antigenic components of molecular masses between 17 and 119kDa. The immunoblotting sensitivity was 95.5% and 100% with histoplasmosis sera from ill patients and sera from H. capsulatum infected but otherwise healthy patients, respectively, to the antigen derived from isolates 200 and 406. Conclusions. We suggest the employment of the antigen from isolate 200, with 15 or 30 days of culture, in the double immunodiffusion and immunoblotting assays due to its good ability to discriminate both sera from patients with histoplasmosis illness and histoplasmosis infection, in addition to its high specificity against heterologous sera (AU)
Antecedentes. Los métodos actuales para la producción de antígenos de Histoplasma capsulatum son problemáticos en términos de estandarización, especificidad, estabilidad, repetitividad y reproducibilidad. Objetivos. En este estudio se buscó optimizar la metodología para la producción de antígenos y evaluar su aplicabilidad. Métodos. Las preparaciones antigénicas obtenidas de 12 cepas de H. capsulatum se evaluaron por doble inmunodifusión en gel de agar e inmunotransferencia frente a sueros homólogos y heterólogos. Resultados. El protocolo evaluado y optimizado permitió mayor sensibilidad en la producción, más estabilidad, repetitividad y reproducibilidad con menos tiempo de cultivo y menor coste. Los mejores patrones de reactividad por inmunodifusión en gel de agar e inmunotransferencia se observaron en el antígeno M obtenido tras 33 días de cultivo de las cepas 200 y 406, y en el antígeno H de la cepa 200 tras 15 días de cultivo. Con la técnica SDS-PAGE se separaron componentes antigénicos de masas moleculares entre 17 y 119kDa. La sensibilidad de la inmunotransferencia fue del 95,5% y del 100% con sueros obtenidos de pacientes con enfermedad y con sueros de pacientes infectados, pero sanos, respectivamente, con antígenos derivados de las cepas 200 y 406. Conclusiones. Sugerimos el empleo del antígeno de la muestra 200, con 15 o 30 días de cultivo, en los ensayos de doble inmunodifusión en gel e inmunotransferencia debido a su buena capacidad para discriminar los sueros de pacientes enfermos de histoplasmosis y los de pacientes portadores sanos, además de su elevada especificidad frente a sueros heterólogos (AU)
Subject(s)
Humans , Histoplasmosis/immunology , Histoplasma/isolation & purification , Antigens, Differentiation/immunology , Immunologic Tests/methods , Immunoblotting/methods , Immunodiffusion/methodsABSTRACT
Background: There are no studies on cross-reactivity between Salsola kali and Salsola imbricata pollens. The main goals of the present study were to compare the degree of the cross-reactivity between S kali and S imbricata and to compare the various allergenic components shared by S kali and S imbricata. Methods: Serum samples were obtained from rhinitis patients with or without asthma living in Kuwait and presenting with a positive skin test result to S kali. SDS-PAGE/IgE Western blot and ELISA inhibition assay were performed. Results: The study population comprised 37 patients. The most frequent IgE proteins against S imbricata weighed around 12, 15, 18, 37, and 50+55 kDa. 2D electrophoresis revealed a correlation between S kali and S imbricata at 40, 60, and 75 kDa, with similar isoelectric points. ELISA inhibition revealed an Ag50 value of 1.7 μg/mL for S kali and 500.5 μg/mL for S imbricata when the solid phase was S kali and an Ag50 value of 1.4 μg/mL for S kali and 3.0 μg/mL for S imbricata when the solid phase was S imbricata. Conclusions: ELISA inhibition revealed strong cross-reactivity between S kali and S imbricata. This finding might be clinically relevant for the efficacy of allergen-specific immunotherapy. We report, for the first time, the allergenic profile of S imbricata and potentially allergenic proteins for S kali and S imbricata (AU)
Introducción: No existen estudios sobre la reactividad cruzada entre pólenes de Salsola kali y Salsola imbricata. El objetivo principal de este estudio es comparar el grado de reactividad cruzada entre Salsola kali y Salsola imbricata, y el objetivo secundario es comparar los diversos componentes alergénicos entre Salsola kali y Salsola imbricata. Métodos: Se obtuvo suero de pacientes con rinitis con o sin asma, que vivieran en Kuwait y tuvieran un test positivo a Salsola kali. Se realizaron SDS PAGE/IgE Western blot, ELISA inhibición, a los sueros de pacientes kuwaitíes. Resultados: Se incluyeron 37 pacientes kuwaitíes. Las proteínas que reaccionaron con más frecuencia contra Salsola imbricata se encontraron alrededor de 12, 15, 18, 37 y 50+55 kDa. La electroforesis 2D mostró una correlación entre Salsola kali y Salsola imbricata a 40, 60, 75 KDa con puntos isoeléctricos similares. El estudio de ELISA inhibición mostró un Ag50 de 1,7 μg/mL para Salsola kali y 500,5 μg/mL para Salsola imbricata cuando la fase sólida era Salsola kali, y un Ag50 de 1,4 μg/mL para Salsola kali y 3,0 μg/mL para Salsola imbricata cuando la fase sólida era Salsola imbricata. Conclusión: Salsola kali y Salsola imbricata presentan una fuerte reactividad cruzada como se observa en el ELISA inhibición y esto podría ser clínicamente relevante para la eficacia de la inmunoterapia específica contra alérgenos. Hemos descrito por primera vez el perfil alergénico para Salsola imbricata y los posibles alérgenos comunes entre Salsola kali y Salsola imbricata (AU)
Subject(s)
Humans , Male , Female , Adolescent , Young Adult , Adult , Middle Aged , Cross-Priming/immunology , Salsola/adverse effects , Respiratory Hypersensitivity/epidemiology , Rhinitis, Allergic, Seasonal/epidemiology , Kuwait/epidemiology , Asthma/epidemiology , Desensitization, Immunologic , Immunoblotting/methods , Enzyme-Linked Immunosorbent Assay/methodsABSTRACT
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Subject(s)
Humans , Pimenta/adverse effects , Capsicum/adverse effects , Food Hypersensitivity/diagnosis , Anaphylaxis/diagnosis , Eosinophilic Esophagitis/diagnosis , Cross Reactions/immunology , Immunoblotting/methods , Cellulase/adverse effects , Peroxidase/adverse effects , Allergens/isolation & purificationABSTRACT
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Subject(s)
Humans , Female , Adult , Milk Hypersensitivity/diagnosis , Anaphylaxis/diagnosis , Respiratory Hypersensitivity/complications , Food Hypersensitivity/diagnosis , Basophil Degranulation Test/methods , Immunoblotting/methods , Spectrum Analysis/methodsABSTRACT
Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.(AU)
Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Phospholipase D/isolation & purification , Spider Venoms/toxicity , Antibodies, Heterophile/blood , Antivenins/therapeutic use , Enzyme-Linked Immunosorbent Assay/methods , Immunoblotting/methodsABSTRACT
Meningococcus serogroup B (MenB), clonal complex 32 (cc 32), was the Brazilian epidemic strain of meningococcal disease (MD) in the 1990’s. Currently, meningococcus serogroup C (MenC), cc 103, is responsible for most of the cases of the disease in Brazil. The aim of this study was to investigate the seroprevalence of bactericidal antibody (SBA) against representative epidemic strains of MenC, (N753/00 strain, C:23:P1.22,14-6, cc103) and MenB, (Cu385/83 strain, B:4,7:P1.15,19, cc32) in students and employees of a university hospital in the State of Rio Grande do Sul (RS, Brazil). A second MenC strain (N79/96, C:2b:P1.5-2,10, cc 8) was used as a prototype strain of Rio de Janeiro’s outbreak that occurred in the 1990’s. Our previous study showed a 9% rate of asymptomatic carriers in these same individuals. A second goal was to compare the SBA prevalence in meningococcal carriers and non-carriers. Fifty-nine percent of the studied population showed protective levels of SBA titers (log2≥2) against at least one of the three strains. About 40% of the individuals had protective levels of SBA against N753/00 and Cu385/83 strains. Nonetheless, only 22% of the individuals showed protective levels against N79/96 strain. Significantly higher antibody levels were seen in carriers compared to non-carriers (P≤0.009). This study showed that, similar to other States in Brazil, a MenC (23:P1.22,14-6, cc103) strain with epidemic potential is circulating in this hospital. Close control by the Epidemiological Surveillance Agency of RS of the number of cases of MD caused by MenC strains in the State is recommended to prevent a new disease outbreak.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Young Adult , Antibodies, Bacterial/blood , Neisseria meningitidis, Serogroup B/immunology , Neisseria meningitidis, Serogroup C/immunology , Brazil , Hospitals, University , Immunoblotting/methods , Meningococcal Infections/immunology , Neisseria meningitidis, Serogroup B/isolation & purification , Neisseria meningitidis, Serogroup C/isolation & purification , Seroepidemiologic Studies , Serogroup , Serum Bactericidal Antibody Assay , Statistics, NonparametricABSTRACT
BACKGROUND: Storage mites of the genus Acarus can be responsible for allergic sensitisation in domestic environments.Acarus gracilis is a frequent species in some geographical regions of the Iberian Peninsula. Since the allergenicity of this mite has not been described before, the objectives of this study were to characterise it immunologically, and to compare it with the closely related and more extensively studied speciesAcarus siro. METHODS: Extracts from A. gracilis and A. siro cultures were characterised by Lowry, 1D and 2D-SDS and IEF. Zymogram, and determination of different enzymatic activities were performed. Skin prick solution of A. gracilis was tested in consecutive patients attending the Hospital of Mérida (Extremadura, Spain). Serum samples from eight individuals with positive skin prick test were collected. IgE determination, immunoblot and immunoblot-inhibition studies were performed. RESULTS: Extracts of both species showed a very similar protein and allergenic profile. Allergens at 14 and 17 kDa were clearly recognised in both extracts by serum samples. Immunoblot-inhibition studies demonstrated that both extracts were totally inhibited by the opposite one. Enzymatic activity was similar in both cases with the most important differences being in kallikrein, serine protease and collagenase activities. CONCLUSION: The storage mite A. gracilis has a similar protein and allergen profile to A. siro and can induce allergic sensitisation. Due to the higher prevalence of this species respect to A. siro in some regions, more studies are needed to determine the clinical significance of sensitisation to this storage mite species
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Subject(s)
Humans , Pyroglyphidae/pathogenicity , Mite Infestations/epidemiology , Mites/pathogenicity , Respiratory Hypersensitivity/immunology , Cross-Priming/immunology , Immunoblotting/methods , Cysteine Proteases/immunology , Immunoglobulin E/immunology , Enzyme Activation/immunologyABSTRACT
No disponible
Subject(s)
Adult , Female , Humans , Lipocalin 1/analysis , Serum Albumin/administration & dosage , Serum Albumin/analysis , Allergens/isolation & purification , Molecular Weight , Serum Albumin/isolation & purification , Hypersensitivity/complications , Hypersensitivity/diagnosis , Hedgehogs/immunology , Allergy and Immunology/standards , Allergens/immunology , Hypersensitivity, Immediate/complications , Hypersensitivity, Immediate/diagnosis , Immunoglobulin E/immunology , Immunoglobulin E/isolation & purification , Respiratory Tract Diseases/immunology , Immunoblotting/methods , ImmunoblottingABSTRACT
Los serogrupos A, B, C, W, Y y X de Neisseria meningitidis (Nm) son los principales responsables de la enfermedad meningocóccica y contra ellos va dirigido actualmente el desarrollo de vacunas para la prevención de la enfermedad. El Instituto Finlay se encuentra desarrollando vacunas antimeningocóccicas polisacarídicas contra los serogrupos A, C, W, Y y X. En el desarrollo de estas vacunas, el ensayo de identidad constituye un requisito obligatorio para la liberación final del producto. Este ensayo se realiza mediante la técnica Dot Blot, utilizando anticuerpos policlonales (AcP) comerciales; sin embargo, el Instituto Finlay cuenta en este momento con una plataforma de Anticuerpos Monoclonales (AcM) contra los cinco polisacáridos (PsC) de Nm. El objetivo del trabajo fue evaluar la sensibilidad y especificidad de estos AcM para su uso en los ensayos de identidad de las vacunas antimeningocócicas polisacarídicas, con vista a sustituir los AcP comerciales empleados hasta el momento. La sensibilidad de los AcM se evaluó frente al PsC homólogo y la especificidad frente al resto de los PsC de Nm. Se realizó el ensayo de identidad por Dot Blot a cuatro vacunas polisacarídicas multivalentes, usando los AcM comparativamente con los AcP. Todos los AcM evaluados mostraron una alta sensibilidad y especificidad, no encontrándose reactividad cruzada y siendo capaces de identificar en las formulaciones vacunales multivalentes la presencia del PsC homólogo. Concluimos que los AcM obtenidos en nuestro instituto pueden ser usados para los ensayos de identidad de las vacunas(AU)
Neisseria meningitidis serogroups A, B, C, W, Y and X are the main responsible of meningococcal disease. Vaccines are necessary to prevent this disease. The Finlay Institute is developing polysaccharide vaccines against serogroups A, C, W, Y and X. In the development of vaccines, the identity assay is a mandatory requirement for final product release. This test is performed by Dot Blot technique using commercial polyclonal antibodies (PAb). However, the Finlay Institute has monoclonal antibodies (mAbs) against capsular polysaccharides from N. meningitidis (CPs). The objective of this work was to use these mAbs in identity tests to replace commercial PAb. The specificity of the mAbs, the cross-reactivity with other CPs and the identity of the CPs present in four multivalent polysaccharide vaccines, were evaluated. All tested mAbs showed high specificity, and no cross-reactivity was found. Also all mAbs were able to identify the presence of homologous PsC in multivalent vaccine formulations. We conclude that the mAbs obtained in our institution can be used for identity assays of vaccines(AU)
Subject(s)
Meningococcal Infections/complications , Immunoblotting/methods , Antibodies, Monoclonal , Meningitis, Meningococcal/prevention & controlABSTRACT
INTRODUÇÃO O tratamento da Síndrome da Imunodeficiência Adquirida vem sofrendo importantes avanços desde a introdução da terapia antirretroviral altamente ativa, conhecida como HAART (high active antirretroviral therapy). Este tratamento levou à eliminação do vírus na corrente sanguínea e ao aumento na sobrevida, entretanto alterações metabólicas e estruturais tornaram-se evidentes. Uma dessas alterações é a redistribuição de gordura corpórea, também denominada lipodistrofia. Com uma das maiores casuísticas mundiais, o objetivo deste trabalho é demonstrar algumas das alternativas cirúrgicas, bem como os resultados obtidos na tentativa de minimizar o impacto da lipodistrofia. MÉTODO: No período de julho de 2005 a julho de 2013, 510 pacientes portadores de lipodistrofia secundária ao uso de HAART foram operados pela Clínica de Cirurgia Plástica do Hospital Heliópolis. Todos esses pacientes foram submetidos à prévia avaliação clínica e imunológica, sob auxílio da equipe de Infectologia. O presente trabalho foi aprovado pelo Comitê de Ética em Pesquisa da Fundação do ABC. RESULTADO: Dentre os 510 pacientes, 335 eram do sexo feminino e 175 do sexo masculino, com idades variando entre 16 e 74 anos. Quanto aos procedimentos, destacou-se lipoaspiração da giba e dorso, com 199 casos. Quanto à resposta estimulada através de questionário subjetivo, observou-se elevado grau de satisfação, aumento significativo da autoestima e maior adesão ao tratamento antirretroviral. CONCLUSÃO: A correção cirúrgica da lipodistrofia corporal comprovadamente melhora o aspecto estético do paciente que faz uso da HAART; porém, o efeito psicológico e social é ainda mais importante, elevando a autoestima, com diminuição dos estigmas, e proporcionando uma maior adesão ao tratamento antirretroviral.
INTRODUCTION The treatment of acquired immunodeficiency syndrome has undergone important advances since the introduction of highly active antiretroviral therapy (HAART). This treatment led to the elimination of the virus in the bloodstream and increased survival; however, metabolic and structural changes became evident. One of these changes is lipodystrophy, the redistribution of body fat. With one of the largest samples worldwide, the aim of this work was to present some of the various surgical alternatives as well as the results obtained for minimizing the impact of lipodystrophy. METHOD: From July 2005 to July 2013, 510 patients with HAART-associated lipodystrophy underwent surgery in the Clinic of Plastic Surgery, Heliópolis Hospital. All patients submitted to prior clinical and immunological assessments made with the aid of the infectious diseases team. The present study was approved by the Research Ethics Committee of the ABC Foundation. RESULTS: The 510 patients included 335 women and 175 men with an age range of 16-74 years. Liposuction of the cervicodorsal fat pad (buffalo hump) was predominant (199 cases). With regard to the response stimulated through a subjective questionnaire, a high degree of satisfaction was observed with a significant increase in self-esteem and greater adherence to antiretroviral treatment. CONCLUSION: The surgical correction of body lipodystrophy demonstrably improves the aesthetics of patients using HAART; however, its psychological and social effects are even more important since self-esteem increases and stigma decreases, which leads to better adherence to antiretroviral treatment.