ABSTRACT
Histoplasma capsulatum causes systemic mycosis that depends on host susceptibility, fungal virulence, and factors associated with the infectious process. We evaluated the possible interference of the phenotype of 12 samples of H. capsulatum isolated from HIV-positive and negative patients in obtaining antigens, aiming at the serological diagnosis through the gender-specific recognition of the H and M fractions. The antigens were evaluated by double immunodiffusion against H. capsulatum anti-antigen polyclonal antibody and serum samples from patients with histoplasmosis.The phenotypic evaluation revealed differences in the identification of the fungal agent and in the expression of H and M antigens, considered serological markers of the disease, associated with pigmentation and the production of conidia. It was found that antigenic preparations obtained from H. capsulatum isolated from HIV-positive patients may have satisfactory antigenic capacity. The patient's immune status does not seem to interfere with the expression of antigenic proteins secreted by H. capsulatum. However, we suggest that prolonged use of antiretrovirals drugs or steroids can cause important phenotypic alterations. We showed that some fungal samples from patients with a long history of immunosuppressive drugs produced atypical cellular elements and low reactivity against the H and M fractions. (AU)
Histoplasma capsulatum causa micose sistêmica endêmica que depende da suscetibilidade do hospedeiro, da virulência fúngica e de fatores associados ao processo infeccioso. Avaliamos a possível interferência do fenótipo de 12 amostras de H. capsulatumisolados de pacientes HIV positivos e negativos na obtenção de antígenos, visando o diagnóstico sorológico por meio do reconhecimento gênero-específico das frações H e M. Os antígenos foram avaliados por imunodifusão dupla, frente a anticorpo policlonal anti-antígeno de H. capsulatum e frente a amostras de soro de pacientes com histoplasmose. A avaliação fenotípica revelou diferenças, não só na identificação do agente fúngico, mas também na expressão dos antígenos H e M, considerados marcadores sorológicos da doença, associados à pigmentação e produção de conídios. Verificou-se que preparações antigênicas obtidas de H. capsulatum isoladas de pacientes HIV positivos podem ter capacidade antigênica satisfatória. O estado imunológico do paciente parece não interferir na expressão de proteínas antigênicas secretadas por H. capsulatum. No entanto, sugerimos que o uso prolongado de antirretrovirais e/ou esteróides pode causar alterações fenotípicas importantes. Verificou-se que algumas amostras fúngicas isoladas de pacientes com longo histórico de uso de imunossupressores produziram elementos celulares atípicos e baixa reatividade sorológica contra as frações H e M de H. capsulatum. (AU)
Subject(s)
Antiretroviral Therapy, Highly Active , Biological Variation, Population , Histoplasma , Histoplasmosis , AntigensABSTRACT
Introduction: The most important genetic association in rheumatoid arthritis (RA) is presented with some alleles from the HLA-DRB1 gene that encode the shared epitope (SE). Objectives: To apply the SE classification methods of Gregersen, de Vries, Raychaudhuri, Mattey, and Tezenas du Montcel in a group of Colombian patients with RA and determine the most common HLA-DRB1 alleles in the population. Methods: RA diagnosis, genetic study of the HLA-DRB1 region using Luminex technology in 50 RA and 50 healthy subjects. For the classification analysis, Fisher's exact test and chi-squared test were applied. Tables were created to count the RA-related alleles. We used odds ratio to determine the risk between the presence of the shared epitope (SE) and anti-cyclic citrullinated peptides (Anti-CCP). Results: Gregersen and de Vries methods were suitable for the characterization of RA in this population (p = .006). The most prevalent HLA-DRB1 alleles in the RA group were 14:02,04:04, 08:02,04:05, and 10:01. High frequencies of the 07:01, 03:01,13:02,01:02, and 12:01 HLA-DRB1 alleles were found in the healthy population. HLA-DRB1 alleles with similar distribution in both populations were 04:07, 15:01, 11:01, 16:02, and 01:01. A high frequency of SE + was observed in Anti-CCP + individuals (63.15%); however, this was not statistically significant [OR2.4 (.63-9.01); p = .19]. Conclusion: The SE classification methods of Gregersen and de Vries were adequate in characterizing RA in a Colombian population group. An equivalence of 100% was verified between the susceptibility alleles defined by de Vries and the alleles assigned as SE according to Gregersen.
Introducción: La asociación genética más importante en artritis reumatoide (AR) se presenta con algunos alelos del gen HLA DRB1 que codifican el epítope compartido (EC). Objetivos: Aplicar los métodos de clasificación de EC de Gregersen et al., de Vries et al., Raychaudhuri et al., Mattey et al., y Tezenas du Montcel et al., en un grupo de pacientes colombianos con AR, y determinar los alelos HLA DRB1 más frecuentes en esta población. Métodos: Diagnóstico para AR, estudio genético de la región HLA DRB1 por tecnología Luminex® de 50 sujetos AR y 50 sanos. Para análisis comparativos de clasificaciones EC, se aplicaron las pruebas test exacto de Fisher y Chi-cuadrado y se realizaron tablas de conteos para los alelos relacionados con AR. Se estimó la razón de odds para determinar el riesgo entre la presencia de EC y los anticuerpos antipéptidos cíclicos citrulinados (anti-PCC). Resultados: Los métodos de Gregersen et al. y de Vries et al. fueron adecuados para la caracterización de AR en esta población (p = 0,006). Los alelos HLA DRB1 más prevalentes en el grupo AR fueron 14:02, 04:04, 08:02, 04:05 y 10:01. Se encontraron altas frecuencias de los alelos HLA DRB1 07:01, 03:01,13:02, 01:02 y 12:01 en población sana. Alelos HLA DRB1 con distribución similar en ambas poblaciones fueron: 04:07, 15:01, 11:01, 16:02 y 01:01. Se observó alta frecuencia de individuos EC+ en el grupo AR anti-PCC+ (63,15%); no obstante, sin asociación estadística (OR: 2,4 [0,63-9,01]; p = 0,19). Conclusión: Los métodos de clasificación para EC de Gregersen et al. y de Vries et al. fueron adecuados caracterizando AR en un grupo de población colombiana. Se corroboró equivalencia del 100% entre los alelos de susceptibilidad definidos por de Vries y los alelos asignados como EC según Gregersen et al.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Arthritis, Rheumatoid , Biological Factors , Musculoskeletal Diseases , Joint Diseases , Epitopes , AntigensABSTRACT
Abstract Improving vaccine immunity and reducing antigen usage are major challenges in the clinical application of vaccines. Microneedles have been proven to be painless, minimally invasive, highly efficient, and have good patient compliance. Compared with traditional transdermal drug delivery, it can effectively deliver a large-molecular-weight drug into the skin, resulting in a corresponding immune response. However, few studies have examined the relationship between microneedle loading dose and immune effects. In this study, the hyaluronic acid (HA) conical and pyramidal dissolving microneedles were prepared by the two-step vacuum drying method, respectively. The model drug ovalbumin (OVA) was added to HA to prepare dissolving microneedles with different loading amounts. The mass ratios of HA to OVA were 5:1, 5:3, and 5:5. The mechanical properties of the dissolving microneedles were characterized using nanoindentation and in vitro puncture studies. The immune effects of the matrix and drug content were studied in Sprague-Dawley (SD) rats. Finally, the diffusion behavior of OVA and the binding mode of HA and OVA in the microneedles were simulated using Materials Studio and Autodocking software. The experimental results showed that the conical microneedles exhibited better mechanical properties. When the mass ratio of HA to OVA was 5:3, the immune effect can be improved by 37.01% compared to subcutaneous injection, and achieved a better immune effect with relatively fewer drugs. This conclusion is consistent with molecular simulations. This study provides theoretical and experimental support for the drug loading and efficacy of microneedles with different drug loadings
Subject(s)
Injections, Subcutaneous/adverse effects , Pharmaceutical Preparations/analysis , Vaccines/analysis , Immunization/classification , Mechanical Tests/instrumentation , Hyaluronic Acid/agonists , Antigens/adverse effectsABSTRACT
Introdução: Com a emergência do SARS-CoV-2 foi disponibilizado uma grande quantidade de ferramentas de diagnóstico. Neste contexto, a falta de vacina, de tratamento e o grande número de casos graves e morte, possibilitou a aprovação emergencial de diversos testes, que ainda necessitam de estudos populacionais para seu registro definitivo. Objetivo: Realizar uma revisão de literatura para avaliar as metodologias de diagnóstico disponíveis no Brasil, de acordo com a realidade local de saúde, explorando o momento epidemiológico a complexidade do teste e a finalidade da sua aplicação. Metodologia: Trata-se de um estudo bibliográfico, descritivo do tipo revisão de literatura. Foram utilizadas as seguintes bases de dados científicos para buscas: PUBMED, MEDLINE, LILACS E COCHRANE LIBRARY, através de descritores selecionados na plataforma DECS. Resultados: O cenário de diversos ensaios, baseados em diferentes metodologias, como os testes baseados em RNA viral, em detecção de antígenos virais ou de anticorpos, associados ao conhecimento da história natural do vírus, possibilita uma análise crítica do melhor diagnóstico de acordo com a clínica do paciente, os epidemiológicos, o objetivo do diagnóstico e a acurácia do ensaio. Atualmente, há mudança no padrão imunológico da população e a descrição de tipos e subtipos de SARS-CoV-2 com mudanças gênicas, que podem levar a mudanças na acurácia diagnóstica ou a re-emergência em surtos de doença grave. Conclusão: Ainda é incerto o caminho evolutivo da história natural da Covid-19 e os ensaios diagnósticos estão em diferentes estágios de desenvolvimento, validação e produção e cada tipo de teste tem suas próprias vantagens e desvantagens distintas inerentes a plataforma tecnológica de origem e uma combinação de tipos de testes usados em momentos diferentes pode ser útil para a condução clínica dos pacientes e no controle da pandemia por SARS-CoV-2.
Introduction: With the emergence of SARS-CoV-2, a large number of diagnostic tools were made available. In this context, the lack of vaccine, treatment and the large number of severe cases and death, allowed the emergency approval of several tests, which still require population studies for their definitive registration. Objective: To carry out a literature review to evaluate the diagnostic methodologies available in Brazil, according to the local health reality, exploring the epidemiological moment, the complexity of the test and the purpose of its application. Methodology: This is a bibliographic, descriptive study of the literature review type. The following scientific databases were used for searches: PUBMED, MEDLINE, LILACS AND COCHRANE LIBRARY, through selected descriptors on the DECS platform. Results: The scenario of several tests, based on different methodologies, such as tests based on viral RNA, on detection of viral antigens or antibodies, associated with knowledge of the natural history of the virus, allows a critical analysis of the best diagnosis according to the patient's clinical, epidemiological, diagnostic objective and assay accuracy. Currently, there is a change in the immune pattern of the population and the description of types and subtypes of SARS-CoV-2 with genetic changes, which can lead to changes in diagnostic accuracy or the re-emergence in outbreaks of severe disease. Conclusion: The evolutionary path of the natural history of Covid-19 is still uncertain and diagnostic assays are at different stages of development, validation and production and each type of test has its own distinct advantages and disadvantages inherent in the technology platform of origin and a combination of types of tests used at different times can be useful for the clinical management of patients and in the control of the SARS-CoV-2 pandemic.
Introducción: Con la aparición del SARS-CoV-2, se dispuso de un gran número de herramientas diagnósticas. En este contexto, la falta de vacuna, tratamiento y el gran número de casos graves y muerte, permitieron la aprobación de urgencia de varias pruebas, que aún requieren estudios poblacionales para su registro definitivo. Objetivo: Realizar una revisión bibliográfica para evaluar las metodologías diagnósticas disponibles en Brasil, de acuerdo con la realidad sanitaria local, explorando el momento epidemiológico, la complejidad de la prueba y la finalidad de su aplicación. Metodología: Se trata de un estudio bibliográfico, descriptivo, del tipo revisión de literatura. Para las búsquedas se utilizaron las siguientes bases de datos científicas PUBMED, MEDLINE, LILACS Y COCHRANE LIBRARY, a través de descriptores seleccionados en la plataforma DECS. Resultados: El escenario de varias pruebas, basadas en diferentes metodologías, como pruebas basadas en el ARN viral, en la detección de antígenos virales o anticuerpos, asociado al conocimiento de la historia natural del virus, permite un análisis crítico del mejor diagnóstico de acuerdo con la clínica del paciente, epidemiológica, objetivo diagnóstico y precisión de la prueba. Actualmente, hay un cambio en el patrón inmunológico de la población y la descripción de tipos y subtipos de SARS-CoV-2 con cambios genéticos, que pueden conducir a cambios en la precisión diagnóstica o la reaparición en brotes de enfermedad grave. Conclusiones: El camino evolutivo de la historia natural del Covid-19 es aún incierto y los ensayos de diagnóstico se encuentran en diferentes etapas de desarrollo, validación y producción y cada tipo de prueba tiene sus propias ventajas y desventajas distintas inherentes a la plataforma tecnológica de origen y una combinación de tipos de pruebas utilizadas en diferentes momentos puede ser útil para el manejo clínico de los pacientes y en el control de la pandemia de SARS- CoV-2.
Subject(s)
Systematic Reviews as Topic , COVID-19 Serological Testing/methods , COVID-19 Testing/methods , COVID-19 Nucleic Acid Testing/methods , Health Services Research , Antibodies/analysis , Antigens/analysisABSTRACT
La enfermedad de von Willebrand (EVW) es el trastorno hemorrágico hereditario más común, y se caracteriza por presentar disminución de la capacidad del factor von Willebrand (FVW) de unirse a las plaquetas y al colágeno de la matriz extracelular durante la hemostasia primaria, debido a defectos cuantitativos o cualitativos. La EVW se clasifica en tres fenotipos principales: el 1 y el 3 que son trastornos cuantitativos, y el 2 que se subclasifica en 2A, 2B, 2M y 2N, y refleja los trastornos cualitativos. Para su diagnóstico son necesarios varios pasos: 1) la evaluación del historial de sangrado personal y familiar del paciente, 2) detección inicial de trastornos hemorrágicos, 3) pruebas para la detección de la EVW, 4) pruebas para la tipificación de la EVW, y 5) el análisis molecular. Tanto la subclasificación de la EVW como su diagnóstico continúan planteando desafíos importantes, motivo por el cual se realiza esta revisión, de manera que los profesionales de la salud tengan una guía que los oriente al momento de tener pacientes con algún trastorno hemorrágico que amerite descartar una EVW e implementar un tratamiento adecuado
von Willebrand disease (VWD) is the most common hereditary bleeding disorder, and is characterized by a decreased ability of the von Willebrand factor (VWF) to bind to platelets and extracellular matrix collagen during primary hemostasis, due to quantitative or qualitative defects. VWD is classified into three main phenotypes: 1 and 3, which are quantitative disorders, and 2 (2A, 2B, 2M and 2N) that reflects qualitative disorders. Several steps are necessary for its diagnosis: 1) evaluation of the patient's personal and family bleeding history, 2) initial screening tests for bleeding disorders, 3) tests for the detection of VWD, 4) tests for the classification of VWD, and 5) molecular analysis. Both the subclassification of VWD and its diagnosis continue to represent important challenges, which we aimed to describe in this review, so that health professionals have a guide to assist them when they have patients with a bleeding disorder that requires exclusion of VWD, and implementation of an appropriate treatment.
Subject(s)
Humans , von Willebrand Diseases , von Willebrand Factor , Ristocetin , Platelet Aggregation , Genetics , Hemorrhage , Hemostasis , AntigensABSTRACT
Utilidad clínica de la prueba El factor von Willebrand (FVW) es una glicoproteína compuesta por multímeros con pesos moleculares que pueden variar desde 500 KDa hasta 20.000 kDa, que se sintetiza en las células endoteliales y en los megacariocitos, y se almacena en los cuerpos de Weibel-Palade y en los gránulos alfa de las plaquetas [1]. El papel del FVW en la hemostasia primaria es mediar la adhesión de las plaquetas a los componentes de la matriz extracelular, a través de los complejos glucoproteicos plaquetarios GPIbα y αIIb3ß; en la hemostasia secundaria, se asocia con el factor VIII para prevenir su degradación y favorecer la generación de trombina para la formación del trombo final
Subject(s)
Humans , von Willebrand Factor , von Willebrand Diseases , Platelet Membrane Glycoproteins , Hemostasis , AntigensABSTRACT
Cerebral small vessel disease (CSVD) is one of the most prevalent pathologic processes affecting 5% of people over 50 years of age and contributing to 45% of dementia cases. Increasing evidence has demonstrated the pathological roles of chronic hypoperfusion, impaired cerebral vascular reactivity, and leakage of the blood-brain barrier in CSVD. However, the pathogenesis of CSVD remains elusive thus far, and no radical treatment has been developed. NG2 glia, also known as oligodendrocyte precursor cells, are the fourth type of glial cell in addition to astrocytes, microglia, and oligodendrocytes in the mammalian central nervous system. Many novel functions for NG2 glia in physiological and pathological states have recently been revealed. In this review, we discuss the role of NG2 glia in CSVD and the underlying mechanisms.
Subject(s)
Animals , Neuroglia/metabolism , Central Nervous System/metabolism , Astrocytes/metabolism , Oligodendroglia/metabolism , Cerebral Small Vessel Diseases/metabolism , Antigens/metabolism , Mammals/metabolismABSTRACT
In order to increase the ability of oil-emulsion adjuvant to stimulate cellular immunity, chitosan hydrochloride with positive charge was selected to stabilize oil-in-water emulsion (CHE). In this paper, model antigen ovalbumin was selected to prepare vaccines with emulsion adjuvant, commercial adjuvant or no adjuvant. The emulsion was characterized by measuring the particle size, electric potential and antigen adsorption rate. BALB/c mice were immunized by intramuscular injection. Serum antibody levels, the numbers of IL-4-secreting cells in splenocytes, cytotoxic T lymphocyte (CTL) response, and the expression of central memory T cells were measured to evaluate the immunostimulatory effect. The results showed that chitosan hydrochloride can effectively stabilize the emulsion. The emulsion size is about 600 nm, and the antigen adsorption rate is more than 90%. After immunization, CHE could increase serum antibodies levels and increase IL-4 secretion. Expression of CTL surface activation molecules was also increased to stimulate CTL response further and to increase the CD44+CD62L+ in T cells proportion. CHE as adjuvant can stimulate humoral and cellular immunity more efficiently, and is expected to extend the duration of protection.
Subject(s)
Animals , Mice , Chitosan , Interleukin-4 , Emulsions , Immunization , Adjuvants, Immunologic/pharmacology , Antigens , Mice, Inbred BALB CABSTRACT
Tecnologia: Detecção do antígeno galactomanana no soro. Contexto: A aspergilose pulmonar invasiva (API) é uma infecção fúngica oportunista de grande risco para pacientes imunocomprometidos. A detecção do antígeno galactomanana no soro por meio de um imunoensaio (ELISA) pode ser um teste não invasivo que auxilie no diagnóstico precoce da doença nestes pacientes. Objetivo: Avaliar a acurácia da detecção do antígeno galactomana no soro para o diagnóstico precoce de aspergilose pulmonar invasiva. Métodos: Revisão rápida sistematizada sobre acurácia de diagnóstico. As bases de dados utilizadas na pesquisa foram: PUBMED, EMBASE, SCOPUS, BVS e Cochrane Library. A avaliação da qualidade metodológica dos estudos incluídos foi realizada por meio da ferramenta AMSTAR-2. Resultados: Foram selecionadas três revisões sistemáticas que atendiam aos critérios de elegibilidade com as quais foi realizada uma análise descritiva dos dados encontrados. A avaliação da qualidade metodológica demonstrou que duas das revisões sistemáticas (RS) apresentaram qualidade criticamente baixa e uma das RS apresentou qualidade alta. Conclusão: A detecção da galactomanana sérica por ELISA pode ser um teste auxiliar no diagnóstico de API, entretanto, possui várias limitações e deve ser utilizado juntamente com outros critérios diagnósticos do consenso do EORTC/MSG. Novas pesquisas devem ser fomentadas para avaliar a utilização do teste no tempo do diagnóstico e no monitoramento da API
Technology: Detection of galactomannan antigen in serum. Background: Invasive pulmonary aspergillosis (IPA) is an opportunistic fungal infection of serious risk for immunocompromised patients. Detection of galactomannan antigen in serum by immunoassay (ELISA) could be a noninvasive test that contributes to the early diagnosis of the disease in this group of patients. Objective: To evaluate the accuracy of serum galactomannan antigen detection for the early diagnosis of invasive pulmonary aspergillosis. Methods: Rapid review of diagnostic accuracy. Databases used in the search were: PUBMED, EMBASE, SCOPUS, BVS, and Cochrane Library. The methodological quality of the included studies was assessed using the AMSTAR-2 tool. Results: Three systematic reviews that satisfied the eligibility criteria were selected, and a descriptive analysis of the data found was performed. The methodological quality assessment showed that two of the systematic reviews (SR) presented critically low quality, and one of the SR presented high quality. Conclusion: Detection of serum galactomannan by ELISA may be a valuable test for diagnosing IPA; however, it has a series of limitations and should be used in conjunction with other diagnostic criteria of the EORTC/MSG consensus. Further research should be encouraged to evaluate the use of this assay, considering the time to diagnosis and IPA monitoring
Subject(s)
Humans , Male , Female , Invasive Pulmonary Aspergillosis/diagnosis , Antigens , Dimensional Measurement Accuracy , Invasive Fungal Infections/diagnosisABSTRACT
A nivel mundial, 300 millones de personas están infectadas por el virus de la hepatitis B (VHB). A pesar de que existe una vacuna que previene la infección y se dispone de tratamiento antiviral que suprime la replicación del virus, no hay cura aún. El principal problema que evita la recuperación total del paciente, incluso para aquel que recibe tratamiento, es la persistencia de dos formas del genoma viral en los hepatocitos: el ADN circular covalentemente cerrado (ADNccc), el cual se encuentra en forma de episoma y tiene la capacidad de replicarse, y las secuencias lineales subge-nómicas que se integran en el genoma humano, con potencial oncogénico. Hasta el momento se dispone de unos pocos biomarcadores para monitorear o predecir la progresión de la enfermedad y la respuesta al tratamiento. Estos biomarcadores se detectan durante la infección, y son la base para la monitorización de la enfermedad y hacer un diagnóstico de la fase clínica de la infección. Recientemente han surgido nuevos biomarcadores como el antígeno relacionado con el core del virus de la hepatitis B (HBcrAg) y la detección del ARN del VHB, que parecen correlacionarse con los niveles transcripcionales del ADNccc, además, durante el tratamiento parecen ayudar a predecir la respuesta y podrían identificar aquellos a quienes se les puede suspender la terapia sin riesgo de recaída. En esta revisión, se describe la utilidad de los principales biomarcadores convencionales en hepatitis B, y se abordan los dos biomarcadores emergentes más estudiados que prometen evaluar el curso de la infección, al igual que determinar la progresión de la enfermedad y la respuesta al tratamiento.
Globally, 300 million people are infected with hepatitis B virus (HBV). Although there is a vaccine that prevents infection and antiviral treatment that suppresses the replication of the virus, there is still no cure. The main problem that prevents the total recovery of the patient, even for those who recei-ve treatment, is the persistence of two forms of the viral genome in hepatocytes: covalently close circular DNA (cccDNA), which is in the form of an episome that has the ability to replicate, and linear subgenomic sequences that are integrated into the human genome, with oncogenic potential. Few biomarkers are currently available to monitor or predict disease progression and response to treatment. These biomarkers are detected during infection and are the basis for monitoring the di-sease and making a diagnosis of the clinical phase of the infection. New biomarkers have recently emerged, such as hepatitis B core-related antigen (HBcrAg) and HBV RNA detection, which seem to correlate with cccDNA transcriptional levels while during treatment seem to help predict response, and could identify those for whom therapy can be discontinued without risk of relapse. In this review, the usefulness of the main conventional biomarkers in hepatitis B is described, and the two most studied emerging biomarkers are mentioned, which promise to evaluate the course of the infection, as well as to determine disease progression and treatment response.
Subject(s)
Humans , Biomarkers , Hepatitis B virus , Hepatitis , Hepatitis B , DNA, Circular , RNA , Risk , Genome , Diagnosis , AntigensABSTRACT
Introduction. The existing methods for Paracoccidioides spp. antigen production are problematic in terms of standardization, specificity, stability, repeatability, and reproducibility. Objective. To optimize the methodology for Paracoccidioides spp. antigen production and evaluate its applicability in paracoccidioidomycosis immunodiagnosis. Materials and methods. The antigens were obtained from Paracoccidioides lutzii isolates (01, 66, and 8334), Paracoccidioides brasiliensis sensu stricto (113), and Paracoccidioides restripiensis (B-339). These fungi were grown at 36 °C ± 1 °C, on modified Fava-Netto agar, according to Freitas et al. (2018). Paracoccidioides lutzii antigens were obtained after 5, 10, and 20 days of culture, whereas P. brasiliensis and P. restripiensis antigens were obtained after 10 days. Antigens were evaluated in natura, 10 and 20 times concentrated. Antigenic capacity was evaluated using a double immunodiffusion assay against serum samples from patients with paracoccidioidomycosis, histoplasmosis, and aspergillosis, and random blood donors. Results. Cross-reactivity between Paracoccidioides spp. antigens was observed when P. brasiliensis, P. restrepiensis antigens, and P. lutzii antigens were evaluated with the polyclonal antibodies against P. lutzii and P. brasiliensis, respectively. No cross-reactivity was obtained for polyclonal antibodies against Histoplasma capsulatum, Aspergillus fumigatus, and random blood donors. The proposed protocol allowed stable, repeatable, and reproducible genus-specific antigen production at a low cost and in a short cultivation time. Conclusion. The proposed protocol allowed us to obtain genus-specific antigens that can be developed and reproduced in all laboratories in Brazil and South America, where paracoccidioidomycosis is a neglected disease, contributing to an early diagnosis, especially in endemic regions, regardless of the species.
Introducción. Los métodos existentes para la producción de los antígenos de Paracoccidioides spp. son problemáticos en su estandarización, especificidad, estabilidad, repetibilidad y reproducibilidad. Objetivo. Optimizar la metodología para la producción de antígenos de Paracoccidioides spp. y evaluar su aplicabilidad en el inmunodiagnóstico de la paracoccidioidomicosis. Materiales y métodos. Los antígenos se obtuvieron de aislamientos de P. lutzii (01, 66 y 8334), P. brasiliensis sensu stricto (113) y P. restripiensis (B-339). Estos hongos se cultivaron a 36 °C ± 1 °C en agar Fava-Netto modificado, según Freitas et al. (2018). Los antígenos de P. lutzii se obtuvieron a los 5, 10 y 20 días de cultivo y los antígenos de P. brasiliensis y P. restripiensis se obtuvieron a los 10 días. Los antígenos se evaluaron in natura, concentrados 10 y 20 veces. La capacidad antigénica se evaluó mediante un ensayo de inmunodifusión doble con muestras de suero de pacientes con paracoccidioidomicosis, histoplasmosis, aspergilosis y donantes de sangre aleatorios. Resultados. Se observó reacción cruzada con Paracoccidioides spp. cuando se evaluaron los antígenos de P. brasiliensis, P. restrepiensis y P. lutzii frente a los anticuerpos policlonales contra P. lutzii y P. brasiliensis, respectivamente. No hubo reactividad cruzada con los anticuerpos policlonales contra Histoplasma capsulatum y Aspergillus fumigatus, ni contra los donantes de sangre aleatorios. El protocolo propuesto permitió la producción estable, repetible y reproducible de antígenos dirigidos de un género específico (Paracoccidiodes) en un tiempo corto de cultivo y a un menor costo. Conclusión. El protocolo propuesto permitió obtener antígenos específicos de un género, que pueden ser desarrollados y reproducidos en todos los laboratorios de Antígenos de Paracoccidioides spp.: protocolo rápido Brasil y Surámerica donde la paracoccidioidomicosis es una enfermedad endémica y desatendida. Estos antígenos pueden contribuir al diagnóstico precoz de la infección, independientemente de la especie.
Subject(s)
Paracoccidioides , Antigens , Paracoccidioidomycosis , Immunologic TestsABSTRACT
Antecedentes: El recuento de unidades formadoras de colonia (UFC) de Cryptococcus en el líquido cefalorraquídeo (LCR) sería un marcador fiable para el pronóstico del paciente y una herramienta simple y económica. Objetivo: Evaluar la utilidad del recuento de UFC de Cryptococcus spp. y compararlo con las variaciones de antígeno capsular de Cryptococcus (AgCr) en LCR.Materiales y métodos: Se realizó la revisión de historias clínicas de pacientes con meningoencefalitis por Cryptococcus asociada con el sida en nuestro centro, entre febrero de 2016 y julio de 2020. Se evaluaron los valores de UFC y AgCr en LCR durante la evolución de la micosis. Resultados y discusión: Se analizaron datos de 94 episodios clínicos de 85 pacientes, con un total de 297 observaciones de muestras de LCR. Se evidenció el valor del recuento de UFC por ser un marcador de viabilidad y de carga fúngica. El recuento de UFC bajo no necesariamente coexistió con un nivel bajo de AgCr. Con respecto a la evolución en el tiempo, la mayoría de los pacientes fueron diagnosticados con una alta carga fúngica y su descenso ocurrió más rápido que el del AgCr, por lo que reflejaría la mejora del paciente, permitiendo tomar conductas al respecto.Palabras clave: Criptococosis, carga fúngica, ufc/mL.
Background. The Cryptococcus' colony-forming unit (CFU) count in cerebrospinal fluid (CSF) would be a reliable marker for patient prognosis and a simple and inexpensive tool. Objectives: To evaluate the usefulness of the CFU count of Cryptococcus spp. And to compare it with the variations of Cryptococcus' capsular antigen (CrAg) in CSF.Materials and methods. Clinical records of patients with aids-related meningoencephalitis caused by Cryptococcusassisted in our center between February 2016 and July 2020 were reviewed. CFU count and CrAg values in CSF were evaluated during the evolution of the mycosis.Results and Discussion. Data from 94 clinical episodes of 85 patients with a total of 297 observations of CSF samples were analyzed.The importance of using the CFU count was evidenced as it is a viability and fungal load marker.Low CFU count did not necessarily coexist with low CrAg.Regarding the evolution over time, most of the patients were diagnosed with a high fungal load and its decrease occurred faster than that the one of AgCr. This would reflect the improvement of the patient, allowing behaviors to be taken in this regard
Subject(s)
Humans , Male , Female , Colony Count, Microbial , Cerebrospinal Fluid/immunology , Acquired Immunodeficiency Syndrome/immunology , Cryptococcosis/immunology , AntigensABSTRACT
ABSTRACT Objectives: Investigate the prevalence of Rh and the K antigens and their phenotypes in the red blood cells of blood donors in Riyadh, Saudi Arabia. Methods: This is a retrospective study. The five principal Rh antigens (D, C, c, E, e) and the Kell antigen from the Kell blood group were tested in 4,675 random samples collected from four blood bank centers in Riyadh. Data were collected for seven weeks (from January 4, 2019 to February 28, 2019). Antigens were tested using the TANGO Optimo system. Results: We found that approximately 86% of the donors had the D antigen, 66% had C, 78% had c, 26% had E, 97% had e and 14% had K. The most common Rh phenotypes were R1r (31%) and R1R1 (22%). Conclusion: The differences in the results between the study population and other populations, such as Caucasian, Indian and African populations indicate the importance of establishing a population-specific database.
Subject(s)
Blood Donors , Phenotype , AntigensABSTRACT
Objetivos. Determinar el rendimiento diagnóstico de la prueba rápida SD dengue DUO (Inyecta) para la detección de NS1, IgM e IgG en comparación con la prueba de ELISA. Materiales y métodos. Es una evaluación de prueba diagnóstica que incluyó 286 muestras de suero de pacientes con sintomatología atribuible a dengue de zonas endémicas del Perú. Las muestras se analizaron por ELISA y la prueba rápida SD dengue DUO (Inyecta) para IgM, NS1 e IgG en el Instituto de Investigación Nutricional en Lima. Resultados. La sensibilidad de la prueba rápida fue de 68% para NS1 e IgM, y 86% para IgG, mejorando este parámetro a 75% y 81% para NS1 e IgM, respectivamente, en los tres primeros días. La especificidad para los tres analitos fue mayor a 87%. La concordancia de los resultados obtenidos medidos por el coeficiente Kappa para los tres analitos fue buena y no se encontró reacción cruzada con otros arbovirus. Conclusiones. La prueba rápida SD Dengue DUO permite detectar con una adecuada sensibilidad y especificidad NS1, IgM e IgG. La sensibilidad para IgM y NS1 aumenta cuando se detecta en los tres primeros días de síntomas, por lo que se recomienda su implementación en los centros de primer nivel de atención para un diagnóstico temprano y oportuno.
Objectives . To assess the diagnostic performance of the SD dengue DUO rapid test (Inyecta) for the detection of NS1, IgM and IgG in comparison to the ELISA test. Materials and methods . This is a diagnostic test evaluation that included 286 serum samples from patients with symptomatology attributable to dengue from endemic areas of Peru. The samples were analyzed by ELISA and the SD dengue DUO rapid test (Inyecta) for IgM, NS1 and IgG at the Instituto de Investigación Nutricional in Lima. Results . The sensitivity of the rapid test was 68.0% for NS1 and IgM, and 86.0% for IgG, improving to 75.0% and 81.0% for NS1 and IgM, respectively, during the first three days. The specificity for all three analytes was greater than 87.0%. The concordance of the results, measured by the Kappa coefficient for the three analytes, was good and no cross-reaction with other arboviruses was found. Conclusions . The SD dengue DUO rapid test allows detection of NS1, IgM and IgG with adequate sensitivity and specificity. Sensitivity for IgM and NS1 increases when detected during the first three days of symptoms. Therefore, we recommend its implementation in primary care centers for early and timely diagnosis.
Subject(s)
Humans , Male , Female , Immunoglobulin M , Dengue , Dengue Virus , Antigens , Signs and Symptoms , Immunoglobulin G , Sensitivity and SpecificityABSTRACT
ABSTRACT Introduction: Spondyloarthritis is a group of chronic inflammatory diseases. Several factors of the disease remain unknown, including clinical and radiological behavior, the demographic characteristics and burden of disease in Colombian patients. Objective: To characterize the demographic aspects, the clinical and paraclinical behaviour, and the therapeutic requirements of a cohort of patients with spondyloarthritis followed-up in the Hospital Pablo Tobón Uribe from January 1, 2005 to December 31, 2017. Methodology: Cohort study. The population was characteriszed using descriptive statistics, qualitative variables using simple and relative frequencies, and quantitative variables using means and standard deviation or medians with their interquartile ranges. Results: The cohort consisted of 181 patients, 100 men (54.9%) and 81 women (44.5%). Just under one half (45.1%) had ankylosing spondylitis, 18.1% undifferentiated spondyloarthritis, 17.1% psoriatic arthropathy, 14.8% reactive arthritis, and 4.4% inflammatory bowel disease. More than two-thirds (69.8%) of the patients had peripheral manifestations, and 67% had axial. A positive HLAB27 was observed in 55.6% of patients. The MRI showed acute and chronic changes in the sacroiliac in 69% and 37%, respectively, with radiological sacroiliitis being observed in 59.5% of cases. The large majority (91.1%) of the patients were treated with PII of original article: S0121-8123(21)00018-9 NSAIDs, 60.1% with sulfasalazine, 43.4% with COX2 inhibitors, and 33.7% with methotrexate. TNFa inhibitors were required by 56.6% of the subjects 3 years after the onset of symptoms. The most commonly used biological drugs were Adalimumab (31.1%), etanercept (21.7%), infliximab (13.1%), golimumab 6.1%, and certolizumab 0.5%. Conclusions: Ourpopulation was characterized by a high activity and functional compromise demonstrated by the high scores of BASDAI and BASFI, and because 56.6% of the patients required anti-TNFa agents.
RESUMEN Introducción: Las espondiloartritis son un grupo de enfermedades inflamatorias crónicas. Se desconoce su comportamiento en nuestro medio, al igual que el comportamiento clínico y radiológico, las características demográficas y la carga de enfermedad en los pacientes colombianos. Objetivos: Caracterizar los aspectos demográficos, el comportamiento clínico y paraclínico y los requerimientos terapéuticos de la cohorte de pacientes con espondiloartritis seguidos en el Hospital Pablo Tobón Uribe desde el 1.° de enero del 2005 hasta el día 31 de diciembre del 2017. Metodología: Estudio de cohorte. La población se caracterizó mediante estadística descrip tiva, las variables cualitativas mediante frecuencias simples y relativas, en tanto que para las cuantitativas se emplearon medias y desviación estándar o medianas con sus rangos intercuartílicos. Resultados: La cohorte está constituida por 181 pacientes, 100 hombres (54,9%) y 81 mujeres (44,5%). El 45,1% tenía espondilitis anquilosante, el 18,1% espondiloartritis indiferenciada, el 17,1% artropatía psoriásica, el 14,8% artritis reactiva y el 4,4% enfermedad inflamatoria intestinal. El 69,8% de los pacientes tenía manifestaciones periféricas y el 67% axiales. El 55,6% de los pacientes tuvo HLAB27 positivo. La RMN mostró cambios agudos y crónicos en las sacroilíacas en el 69% y 37%, respectivamente; en el 59,5% de los casos se observó sacroileítis radiológica. el 91,1% de los pacientes se trató con AINE, el 60,1% con sulfasa lazina, el 43,4% con inhibidores COX2 y el 33,7% con metotrexato. El 56,6% de los sujetos requirió inhibidores-TNFa 3 arios después del inicio de los síntomas. Los biológicos más uti lizados fueron adalimumab (31,1%), etanercept (21,7%), infliximab (13,1%), golumimab (6,1%) y certolizumab (0,5%). Conclusiones: Nuestra población se caracterizó por una alta actividad y gran compromiso funcional, lo que se refleja en altos puntajes de Basdai y Basfi y en que el 56,6% de los pacientes requirió agentes anti-TNFa.
Subject(s)
Humans , Male , Female , Bone Diseases , Biological Factors , Musculoskeletal Diseases , Spondylarthritis , AntigensABSTRACT
As células CAR-T são linfócitos geneticamente modificados para reconhecerem um espectro amplo de antígenos de superfície celulares. Além disso, atacam células tumorais malignas, que expressam esses antígenos, por meio da ativação da coestimulação citoplasmática, secreção de citocinas, citólise de células tumorais e proliferação de células T. O objetivo desse estudo é abordar a imunoterapia com células CAR-T, a fim de explicar seu conceito, processo de fabricação e papel no tratamento de neoplasias hematológicas e tumores sólidos. Foi realizada uma revisão através do portal PubMed, utilizando como descritores: "car-t cell therapy" e "neoplasms", determinados com base nos "Descritores em Ciências da Saúde". Foram obtidos, inicialmente, 10 artigos, os quais foram lidos integralmente para a confecção dessa revisão. Além disso, foram adicionados 3 ensaios clínicos atualizados sobre o tema. Na terapia com células CAR-T, as células T são coletadas do paciente, geneticamente modificadas para incluir receptores de antígeno específicos e, posteriormente, expandidas em laboratórios e transfundidas de volta para o paciente. Assim, esses receptores podem reconhecer células tumorais que expressam um antígeno associado a um tumor. A terapia com células CAR-T é mais conhecida por seu papel no tratamento de malignidades hematológicas de células B, sendo a proteína CD19 o alvo antigênico mais bem estudado até o momento. Entretanto, estudos estão sendo feitos para verificar a eficácia desse tratamento, também, em tumores sólidos. Portanto, apesar de inicialmente ser indicada apenas para um grupo seleto de pessoas, essa terapia tem demonstrado grande potencial para atuar em um espectro maior de pacientes.
The CAR-T cells are lymphocytes genetically modified to recognize a broader spectrum of cell surface antigens. In addition, they attack malignant tumor cells, which express these antigens, by activating cytoplasmic co-stimulation, cytokine secretion, tumor cell cytolysis and T cell proliferation. The aim of this study is to address immunotherapy with CAR-T cells, in order to explain its concept, manufacturing process and role in the treatment of hematological neoplasms and solid tumors. This is a literature review conducted through the PubMed portal, that uses the terms "car-t cell therapy" and "neoplasms" as descriptors, determined based on the DeCS (Descritores em Ciências da Saúde). To prepare this review, initially 10 articles were found and read in full. In addition, 3 updated clinical trials on the subject were added. For CAR-T cell therapy, T cells are collected from the patient, genetically modified to include specific antigen receptors, and later expanded in laboratories and transfused back to the patient. Thus, these receptors can recognize tumor cells that express a tumor-associated antigen. CAR-T cell therapy is best known for its role in the treatment of B cell hematological malignancies, with the CD19 protein being the most studied antigenic target to date. However, studies are being conducted to verify the effectiveness of this treatment, also, in solid tumors. Therefore, despite being formulated only for a selected group of patients, this therapy has great potential to act on a broader spectrum of patients.
Subject(s)
Humans , Immunotherapy, Adoptive , Hematologic Neoplasms , Cellular Reprogramming , Cell- and Tissue-Based Therapy , Receptors, Antigen , Inducible T-Cell Co-Stimulator Ligand , Epithelial Cell Adhesion Molecule/therapeutic use , Immunotherapy/methods , Antigens/immunology , NeoplasmsABSTRACT
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the disease coronavirus 2019 (COVID-19) in humans. SARS-CoV-2 has been identified in cats with or without clinical signs. Case presentation: We describe the pathological and molecular findings in a six-month-old asymptomatic cat with SARS-CoV-2 infection from Brazil, belonging to a human family with COVID-19 cases. The pool of nasopharynx and oropharynx swabs at day zero tested positive by RT-qPCR for SARS-CoV-2. No amplification resulted from molecular testing performed on days 7 and 14. The cat was hit by a car and died 43 days after the molecular diagnosis. Immunohistochemistry at post-mortem examination demonstrated nucleocapsid protein in samples from the lungs, kidneys, nasal conchae, trachea, intestine, brain and spleen. Conclusion: The present study has highlighted the possibility that viral antigens can be detected by immunohistochemistry in multiple organs six weeks after infection, although the same tissues tested negative by RT-PCR.(AU)
Subject(s)
Animals , Cats , Immunohistochemistry , SARS-CoV-2/immunology , COVID-19/diagnosis , Antigens/analysis , Oropharynx , NasopharynxABSTRACT
Stem cells are undifferentiated cells that can be distinguished from others by their ability to self-renew and to differentiate into new specific cell types. Mesenchymal stem cells (MSC) are adult stem cells that can be obtained from different sources, such as adipose tissue, bone marrow, dental pulp, and umbilical cord. They can either replicate, originating new identical cells, or differentiate into cells of mesodermal origin and from other germ layers. MSC have been studied as new tools for regenerative therapy. Although encouraging results have been demonstrated, MSC-based therapies still face a great barrier: the difficulty of isolating these cells from heterogeneous environments. MSC are currently characterized by immunolabelling through a set of multiple surface membrane markers, including CD29, CD73, CD90 and CD105, which are also expressed by other cell types. Hence, the present work aimed to identify new specific biomarkers for the characterization of human MSC using DNA aptamers produced by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique. Our results showed that MSC from different origins bound to DNA candidate aptamers, that is, DNA or RNA oligonucleotides selected from random libraries that bind specifically to biological targets. Aptamer-bound MSC could be isolated by fluorescenceactivated cell sorting (FACS) procedures, enhancing the induction of differentiation into specific phenotypes (chondrocytes, osteocytes and adipocytes) when compared to the whole MSC population. Flow cytometry analyses revealed that candidate aptamers bound to 50% of the MSC population from dental pulp and did not present significant binding rates to human fibroblasts or lymphocytes, both used as negative control. Moreover, immunofluorescence images and confocal analyses revealed staining of MSC by aptamers localized in the surfacemembrane of these cells. The results also showed internal staining of human monocytes by our investigated aptamers. A non-specific control aptamer (CNTR APT) obtained from the random pool was then utilized to compare the specificity of the aptamers bound to the analyzed non-apoptotic cells, showing no staining for MSC. However, 40% of the monocytes bound to the CNTR APT. Normalized data based on the cells bound to candidate aptamers compared to those bound to the CNTR APT, revealed a 10 to 16-fold higher binding rate for MSC against 2-fold for monocytes. Despite its low specificity, monocyte-aptamer binding occurs probably due to the expression of shared markers with MSC, since monocytes are derived from hematopoietic stem cells and are important for the immune system ability to internalize/phagocyte external molecules. Given that, we performed a pull-down assay followed by mass spectrometry analysis to detect which MSC-specific protein or other target epitope not coexpressed by monocytes or the CNTR APT would bind to the candidate aptamer. Distinguishing between MSC and monocyte epitopes is important, as both cells are involved in immunomodulatory effects after MSC transplantations. ADAM17 was found to be a target of the APT10, emerging as a possible biomarker of MSC, since its involvement in the inhibition of the TGF signaling cascade, which is responsible for the differentiation of MSC. Thus, MSC with a higher stemness profile should overexpress the protein ADAM17, which presents a catalytic site with affinity to APT10. Another target of Apt 10 is VAMP3, belonging to a transmembrane protein complex that is involved in endocytosis and exocytosis processes during immune and inflammatory responses. Overall, proteins identified as targets of APT10 may be cell surface MSC biomarkers, with importance for MSC-based cell and immune therapies
Células tronco são células indiferenciadas que podem ser distinguidas de outros tipos celulares por meio da habilidade de se auto renovarem e de se diferenciarem em novos tipos celulares. Células tronco mesenquimais (MSC) são células tronco adultas encontradas em diferentes tecidos como tecido adiposo, polpa de dente e cordão umbilical. Estas células podem se autodividir em células idênticas ou se diferenciarem em células de origem mesodermal. Estas células têm sido estudadas em novas aplicações que envolvem terapia regenerativas. Embora resultados encorajadores tenham sido demonstrados, terapias que utilizam MSC ainda encontram uma grande barreira: a dificuldade no isolamento destas células a partir de um ambiente heterogêneo. MSC são caracterizadas por populações positivas em ensaios de imunomarcação para os epítopos membranares CD29, CD73, CD90 e CD105, presentes também em outros tipos celulares. Assim, o presente trabalho tem o objetivo de identificar novos biomarcadores de MSC de origem humana, utilizando aptâmeros de DNA produzidos pela técnica SELEX (Systematic Evolution of Ligands by EXponential Enrichment) como ferramenta. Nossos resultados mostraram que MSC de diferentes origens ligam-se a aptâmeros (oligonucleotídeos de DNA ou RNA que atuam como ligantes específicos de alvos moleculares) de DNA candidatos que atuam no isolamento de MSC por meio da técnica FACS de separação celular, promovendo uma maior indução de diferenciação em células específicas (condrócitos, osteócitos e adipócitos) comparada com a população total de MSC. Análises de citometria de fluxo mostraram que os aptâmeros candidatos se ligam a 50% das MSC de polpa de dente e não apresentam taxa de ligação significante para fibroblastos e linfócitos de origem humana - utilizados como controles negativo. Além domais, imagens de imunofluorescência e confocal mostraram ligação na superfície da membrana de MSC e a marcação interna de monócitos a estes aptâmeros. Portanto, um aptâmero controle (CNTR APT) foi utilizado para comparar a especificidade dos aptâmeros ligados a células viáveis, mostrando a não ligação deste aptâmero a MSC. Porém, 40% da população de monócitos ligou-se ao CNTR APT. Uma normalização baseada na comparação entre as taxas de ligação entre células ligadas com aptâmeros candidatos e o aptâmero controle gerou uma taxa de especificidade entre 10-16 vezes maior para MSC contra 2,5 vezes para os monócitos. Deste modo, embora os resultados tenham mostrado uma taxa de ligação entre monócitos e aptâmeros, as MSC ligadas aos aptâmeros candidatos possuem uma maior taxa de especificidade devido a uma maior presença de antígenos que são expressos em ambas as células. Um ensaio de Pull Down seguido de espectrometria de massas foi utilizado para a identificação de biomarcadores que se ligariam aos aptâmeros candidatos, e que não seriam co-expressos por monócitos e por antígenos ligados ao aptâmero controle. Deste modo, a proteína ADAM17 foi identificada nas amostras de APT10 ligadas às MSC. Tal proteína está relacionada à inibição de uma cascata de sinalização da família de proteínas TGF, responsável pela diferenciação de MSC. Assim, MSC com maior potencial tronco deveriam expressar ADAM17 em maior quantidade. Tal proteína apresenta um sítio catalítico que demonstra interagir com o APT10, de acordo com predição Docking entre proteína e DNA. Foi identificada também, a proteína VAMP3, que pertence a um complexo proteico transmembranar responsável pelos processos de endocitose e exocitose, e que podem ter um papel importante na liberação de citocinas e outras moléculas relacionadas às respostas imune e inflamatórias. Deste modo, o APT10 identificou proteínas importantes que devem estar relacionas com a melhora de imunoterapias que utilizam MSC
Subject(s)
Stem Cells , Biomarkers/analysis , SELEX Aptamer Technique/instrumentation , Mesenchymal Stem Cells/classification , ADAM17 Protein/pharmacology , Patient Isolation , Mass Spectrometry/methods , Staining and Labeling/methods , Transplantation/adverse effects , Umbilical Cord , DNA/agonists , Transforming Growth Factors/agonists , Cell Separation/instrumentation , Cytokines/adverse effects , Adipocytes/metabolism , Chondrocytes/classification , Scientists for Health and Research for Development , Adult Stem Cells/classification , Fibroblasts/chemistry , Flow Cytometry/instrumentation , Germ Layers , Antigens/adverse effectsABSTRACT
Recombinant HLA-Ⅰ molecules/antigenic peptide complexes (pHLA complexes) are applied in the research of human T cell-specific immune responses. The preparation of pHLA complex is based on genetic engineering and protein in vitro dilution and folding-refolding technology. In an in vitro refolding system, recombinant HLA-Ⅰ molecules correctly fold and bind with antigenic peptides to form complexes. In this study, ultrafiltration-high performance liquid chromatography (ultrafiltration-HPLC) was used for quantitative determination of the antigenic peptides in recombinant pHLA complexes, especially for those in a small amount of prepared products. By adding the recombinant HLA-Ⅰ molecules and antigenic peptides into the refolding buffer, the heavy chain (HC) and light chain (β2m) of recombinant HLA-Ⅰ molecules were refolded and bond with the VYF antigenic peptide containing anchor residues to form a pHLA complex. The unbound free antigenic peptide VYF was removed by ultrafiltration to retain the complex. Finally, the pHLA complex was treated by acid to destroy its interaction, thus releasing the antigenic peptide. The results showed that the prepared recombinant pHLA complex was recognized by HLA-Ⅰ molecule specific antibody W6/32, which indicated that the recombinant HLA-Ⅰ class molecule had correct folding and was identified as pHLA complex. The antigen peptide VYF contained in the pHLA complex was also detected by ultrafiltration-HPLC, so it is feasible to apply ultrafiltration-HPLC for determination of pHLA complex. Compared with Western blotting, the concentration of antigenic peptides detected by ultrafiltration-HPLC was 0-9 μg/mL. The binding conditions can be optimized according to the amount of antigenic peptides bound in the complex in order to improve the folding efficiency of HLA-Ⅰ molecules and promote the binding of HLA-Ⅰ molecules to antigenic peptides. The production rate of pHLA complexes in the refolding system can also be calculated according to the content of antigenic peptides bound by pHLA complexes. Therefore, ultrafiltration-HPLC in this study can be used for the quality control of the preparation process of pHLA complexes, and may facilitate the research of T cell-specific immunity, artificial antigen-presenting cells, and development of specific tetramer probe applications.