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1.
J Clin Apher ; 32(3): 163-169, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27258774

RESUMO

Transplanting immunized patients requires immunological monitoring in the pretransplant phase to follow reduction of donor specific HLA antibodies (DSA) after Staphylococcus aureus protein A (SPA) immunoadsorption (IA) or therapeutic plasma exchange followed by IVIG and Rituximab administration. Pretreatment aims to significantly reduce DSA strength. The Tissue Typing Lab at Aarhus University Hospital performs immunological monitoring of approximately 150 kidney transplantation patients per year from two transplant centers. From 2012 to 2013, we experienced seven patients desensitized using SPA IA, initially presenting negative cytotoxic complement dependent (CDC) T-cell crossmatches but positive B and T cell flowcytometric crossmatch, who despite significant DSA reduction developed weakly positive CDC T-cell crossmatch shortly prior to transplantation. We hypothesised that leached SPA during IA could be the cause, as the complication was not observed in patients who received plasma exchanges. We found that the positive CDC was not donor specific and SPA column material incubated with control serum reproduced a positive CDC T-cell crossmatch. Finally, we detected leached SPA in one of the patient samples using a highly sensitive time-resolved fluorescent assay. In conclusion, the results emphasize the importance of carefully considering CDC crossmatch results subsequent to IA, before a planned transplantation is either postponed or cancelled. J. Clin. Apheresis 32:163-169, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Técnicas de Imunoadsorção/efeitos adversos , Proteína Estafilocócica A/imunologia , Feminino , Antígenos HLA/sangue , Antígenos HLA/isolamento & purificação , Teste de Histocompatibilidade/normas , Humanos , Transplante de Rim , Masculino
2.
Reumatol. clín. (Barc.) ; 12(2): 85-90, mar.-abr. 2016. tab, ilus
Artigo em Espanhol | IBECS | ID: ibc-150874

RESUMO

La enfermedad de Behçet es una vasculitis caracterizada por úlceras bucales y genitales. La afectación neurológica o neuro-Behçet es una manifestación infrecuente, de predominio en el género masculino y que aparece de 2 a 4 años después de la primera manifestación clínica. El neuro-Behçet cursa ocasionalmente lesiones cerebrales pseudotumorales. Presentamos 2 casos de pacientes diagnosticados de neuro-Behçet tras la detección de lesiones cerebrales pseudotumorales y se realiza una revisión de la literatura (AU)


Behçet‘s disease is a systemic vasculitis characterized by the presence of oral and genital ulcers. Neurological involvement or neuro-Behçet is an uncommon manifestation. It manifestation has predominance in the male gender appearing 2 to 4 years after the first clinical manifestation. However, neuro-Behçet disease sometimes occurs with pseudotumoral brain lesions. Herein, we present the cases of two patients diagnosed with neuro-Behçet after detection of pseudotumoral brain lesions. A review of the literature is performed (AU)


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Síndrome de Behçet/patologia , Síndrome de Behçet , Pseudotumor Cerebral/patologia , Pseudotumor Cerebral , Metilprednisolona/uso terapêutico , Prednisona/uso terapêutico , Colchicina/uso terapêutico , Azatioprina/uso terapêutico , Ciclofosfamida/uso terapêutico , Agressão/fisiologia , Anamnese/métodos , Foliculite/complicações , Foliculite/patologia , Foliculite , Esclerose/complicações , Esclerose , Antígenos HLA/isolamento & purificação , Glucocorticoides/uso terapêutico , Infliximab/uso terapêutico
3.
Inmunología (1987) ; 33(3): 81-86, jul.-sept. 2014. tab
Artigo em Espanhol | IBECS | ID: ibc-125468

RESUMO

Hoy conocemos algo más de la participación de la región HLA-DP en la patogénesis de las leucemias gracias a varios estudios demostrando la injerencia temprana de estas moléculas en la modulación de la respuesta inmunitaria frente a patógenos. Para la evaluación de alelos y haplotipos HLA-DPA1*/DPB1* (28 alelos del locus HLA-DPA1* y 123 alelos HLA-DPB1*) se utilizó PCR SSPTM Olerup (Genovision) en 48 pacientes con LLA, 48 con LMC y 48 controles mestizos venezolanos. Para la asociación HLA/leucemias se tomó el riesgo relativo (RR) > 3 como positivo y negativo RR < 3, con una p corregida < 0,05. Pacientes LLA confirmaron asociaciones positivas con el alelo DPA1*0105 y negativa con el alelo DPA1*010301-010302. Además, resultaron asociados positivamente DPA1*0106 y *0107; DPA1*020101-020106 se asoció negativamente con la LLA. DPA1*0105, *0108 y *0109 resultaron asociados negativamente con la LMC. Las frecuencias observadas de los alelos HLA-DPB1*01:01, 02:01, 03:01, 04:01 y 04:02 en los controles mestizos venezolanos ubicadas entre 7 y 16% fueron superiores a las de pacientes leucémicos. Se confinaron asociaciones negativas de los alelos DPB1*02:01 y DPB1*03:01 con las LLA. No se observaron asociaciones positivas con las LLA. DPB1*99:01 se asoció negativamente con las LLA. La región DPB1* sola no parece estar asociada a las leucemias en esta población venezolana. La fuerte asociación LMC con varios haplotipos DPA/DPB indica importantes diferencias entre las patogénesis de ambas enfermedades


More is now known of the involvement of HLA-DP region in the pathogenesis of the leukemias through several previousstudies showing interference of these molecules in modulating the immune response to pathogens. For evaluation of HLA alleles and haplotypes DPA1*/DPB1* (28 alleles HLA DPA1* and 123 of HLA- DPB1*) Olerup SSP ™PCR (Genovision) was used in 48 patients with ALL, 48 CML, and 48 Venezuelan twins as controls. For HLA/leukemias, a relative risk (RR) > 3 was considered to be a positive association and negative with an RR<3, with a p corrected P<.05. ALL patients confirmed positive associations with DPA1*0105 allele, and negative with DPA1*010301-010302. In addition, they were positively associated with DPA1*0106 and *0107, with DPA1*020101-020106 being negatively associated with ALL. DPA1*0105, *0108 and *0109 were negatively associated with CML. The observed frequencies of HLA-DPB1* 01:01, 02:01, 03:01, 04:01 and 4:02 alleles in Venezuelan, which twins were between 7 and 16%, were higher than those of leukemic patients. Negative associations of DPB1*2:01, *3:01 and LLA were confined. No positive associations were observed with ALL. Non-confirmed positive associations were observed between DPB1*99:01 and CML. Haplotypes HLA-DPA1*01:03-DPB1*4:01, *2:01, *99:01 were strongly positively associated with CML. DPA1*1:09-DPB1*2:01, *4:01 were negatively associated with the CML. DPA1*1:03-DPB1*4:02; DPA1*01:09-DPB1*2:01, *4:01 and DPA1*02:01-DPB1*04:02 were negatively associated with ALL. The DPB1* single region does not appear to be associated with leukemia in the Venezuelan population. The strong association with several haplotypes DPA*1/DPB1* and LMC suggests massive differences between the pathogenesis of both diseases


Assuntos
Humanos , Antígenos HLA-DP/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Antígenos HLA/isolamento & purificação , Substâncias Protetoras/análise , Alelos , Fenótipo
4.
Methods Mol Biol ; 1034: 197-219, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23775738

RESUMO

Next-generation sequencing (NGS) of HLA class I and II loci (HLA-A, HLA-B, HLA-C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPB1) is described here in detail using the 454 Life Sciences GS FLX System and Titanium chemistry. An overview of the protocol with our experience on sequence performance efficiencies, read depth and ambiguity analyses using the GS FLX System are also presented. A total of 14 HLA primer pairs with multiplex identifiers (MIDs) are used in clonal, amplicon-based pyrosequencing of up to 44 samples per plate using the GS FLX. Genotype assignment and ambiguity reduction -analysis is performed using Conexio Assign ATF 454 software. Clonal NGS gives a significant reduction in genotyping ambiguity during analysis of the highly complex HLA system.


Assuntos
Antígenos HLA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade/métodos , Biologia Molecular/métodos , Alelos , Frequência do Gene , Genótipo , Antígenos HLA/genética , Antígenos HLA/isolamento & purificação , Antígenos HLA-B/genética , Antígenos HLA-B/isolamento & purificação , Antígenos HLA-C/genética , Antígenos HLA-C/isolamento & purificação , Haplótipos , Humanos , Polimorfismo Genético
5.
Rev. esp. pediatr. (Ed. impr.) ; 66(5): 282-293, sept.-oct. 2010. ilus, tab
Artigo em Espanhol | IBECS | ID: ibc-91723

RESUMO

La diabetes mellitus de tipo 1 es una enfermedad crónica autoinmune caracterizada por la destrucción selectiva de las células beta de los islotes de Langerhans. Este proceso, probablmente causado por uno o más factores ambientales, tiene lugar en individuos genéticamente susceptible portadores de un HLA determinado, y puede ocurrir durante meses o años antes de que la enfermedad se ponga de manifiesto. La autoinmunidad está en relación con células T autorreactivas, es decir, células que desencadenan una reacción autoinmune contra antígenos propios. La fase preclínica de la diabetes puede ser detectada por la presencia de anticuerpos anticélulas beta (ICA), anticuerpos antiinsulina (IAA), y anticuerpos frente a la decarboxilasa del ácido glutámico (GAD). Dadas sus características específicas inmunológicas, genéticas y metabóicas, el riesgo para desarrollar una diabetes de tipo 1ª es bastante predecible. Aunque hoy día aún no disponemos de una estrategia terapétuica adecuada, es importante identificar a estas personas puesto que podrían beneficiarse de actuaciones terapéuticas para prevenir la enfermedad. La diabetes mellitus tipo 1 se asocia con frecuencia a otras enfermedades de etiología o patogenia autoinmune: enfermedad tiroidea autoinmune, en enfermedad celíaca y, en menor grado, a la enfermedad gástrica autoinmune y a la enfermedad de Addison. La determinación de los autoanticuerpos órgano-específicos proporcionan una manera sencilla de cribado de la autoinmunidad en una población susceptible, como es la de los enfermos diabéticos tipo 1, con la posibilidad de prevenir morbilidad y mortalidad (AU)


Type 1 diabetes mellitus (DM1) is a chronic immune mediated disease characterized by the selective destruction of insulin-producing beta cells in the islets of Langerhans. This process, probably caused by one or more environmental factors, takes place in HLA-genetically susceptible individuals and may be present for months or years before the disease manifests itself. Diabetes autoimmunity is related to autoreactive T cells, this is, T cells that trigger an immune reaction against self antigens. The preclinical period of the DM1 can be detected by the presence of islet cell antibodies (ICA), insulin autoantibodies (IAA), and antibodies to glutamic acid decarboxylase (GAD). Because of its specific immunological, genetic and metabolic character, the risk of developing DM1 is very predictable. Even if we still do not have an adequate therapeutic response/strategy it is important to identify those susceptible subjects in order to help them prevent the disease. DM1 is commonly related to other autoimmune diseases: thyroiditis and celiac disease and much less to pernicious anemia and Addison´s disease. The detection of organ specific autoantibodies in an easy way to determine autoimmunity among a susceptible population such as DM1 patients, and thus helping to prevent morbidity and mortality (AU)


Assuntos
Humanos , Antígenos HLA/isolamento & purificação , Diabetes Mellitus Tipo 1/imunologia , Autoimunidade/imunologia , Estado Pré-Diabético/imunologia , Biomarcadores/análise , Células Secretoras de Insulina/imunologia
6.
Curr Pharm Des ; 15(28): 3325-35, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19860682

RESUMO

In addition to a variety of other techniques used in T-cell epitope identification, mass spectrometery coupled to liquid chromatography have now become an important and sensitive tool in separation, detection, and sequence analysis of highly complex natural major histocompatibility complex (MHC) ligand mixtures. In this article, we present current strategies for the identification of MHC eluted peptides using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) with a particular recall to those presented in the context of the non classical human leukocytes antigen (HLA) class I molecule HLA-E. In addition we also discuss the advantages and disadvantages of the methods available in the literature to concentrate and fractionate the peptides prior to analysis by mass spectrometry. An application of our method for isolation and characterization of peptides presented in the context of HLA-E is finally reported.


Assuntos
Antígenos HLA/química , Antígenos de Histocompatibilidade Classe I/química , Vacinas de Subunidades/química , Animais , Cromatografia Líquida de Alta Pressão , Epitopos/imunologia , Antígenos HLA/imunologia , Antígenos HLA/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Humanos , Espectrometria de Massas , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Vacinas de Subunidades/imunologia , Vacinas de Subunidades/isolamento & purificação
7.
Reumatol. clín. (Barc.) ; 5(extr.1): 31-39, abr. 2009. tab, ilus
Artigo em Espanhol | IBECS | ID: ibc-78375

RESUMO

Con la instauración de las clínicas de artritis de reciente comienzo se valoran pacientes cada vez más precozmente en el curso de su enfermedad, por lo que un porcentaje importante de éstos aún no puede clasificarse dentro de un diagnóstico específico según los criterios de clasificación del American College of Rheumatology (ACR). En estos pacientes con artritis indiferenciada (AI) incluso más importante que establecer un diagnóstico es distinguir entre aquéllos que desarrollarán una artritis persistente y/o erosiva y, por tanto, serán candidatos a recibir tratamiento precoz con fármacos modificadores de la enfermedad (FAME) aunque no cumplan los criterios de clasificación del ACR, y los que tendrán un cuadro autolimitado. Los marcadores serológicos junto con las características clínicas en el momento de la presentación, integrados en modelos clínicos predictivos, son los instrumentos con los que actualmente cuenta el clínico para poder identificar a estos pacientes. Algunos estudios han demostrado las ventajas del tratamiento precoz en la AI (AU)


With the establishment of early arthritis clinics, patients can now be increasingly attended early in the course of their disease. This means that a significant proportion of these patients cannot be classified into a specific diagnosis using the traditional American College of Rheumatology (ACR) classification criteria. In these patients with undifferentiated arthritis (UA), even more important than assigning a diagnosis is the need to distinguish between patients who will develop a persistent and/or erosive disease and will be candidates for prompt treatment with disease-modifying antirheumatic drugs (DMARD), and patients in whom the disease is self-limiting. Serologic markers in combination with clinical features at presentation, integrated into predictive models, are the tools currently available to the clinician for identifying these patients. Several studies have demonstrated the advantages of early treatment in UA (AU)


Assuntos
Humanos , Artrite/tratamento farmacológico , Diagnóstico Precoce , Artrite Reumatoide/tratamento farmacológico , Prognóstico , Diagnóstico Diferencial , Antígenos HLA/isolamento & purificação , Diagnóstico por Imagem
8.
Dig Dis Sci ; 54(2): 360-8, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18629643

RESUMO

The association between anticentromere antibody (ACA) and hepatitis C virus (HCV) infection remains unclear. We subjected eight patients with HCV-related chronic liver disease (CLD) seropositive for ACA to a battery of clinical and laboratory tests. The patient cohort was dominated by females, and four of the eight (50%) patients had a concomitant autoimmune disease. All of the patients had high titers of ACA (>or=1:320). The histological activity index scores in chronic hepatitis C (CH-C) patients with ACA were significantly higher than those in CH-C patients without antinuclear antibody (ANA) (12.8 +/- 1.8 vs. 8.3 +/- 4.5, P = 0.0372). The frequency of human leukocyte antigen (HLA) DR-8 in patients with HCV-related CLD seropositive for ACA was significantly higher than that in patients with CH-C seronegative for ANA (71 vs. 18%, P = 0.0108). These findings suggest that ACA is induced by chronic HCV infection in association with HLA DR-8, and that CH-C patients with ACA exhibit more severe hepatic fibrosis and inflammation than CH-C patients without ANA.


Assuntos
Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/imunologia , Centrômero/imunologia , Hepatite C Crônica/imunologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Antígenos HLA/isolamento & purificação , Hepatite C Crônica/sangue , Hepatite C Crônica/patologia , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Fenótipo
9.
J Immunol ; 181(9): 6371-83, 2008 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-18941228

RESUMO

Viral peptides are presented by HLA class I on infected cells to activate CD8(+) T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8(+) T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines.


Assuntos
Antígenos HLA/imunologia , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Marcação por Isótopo/métodos , Vírus Vaccinia/imunologia , Vaccinia/imunologia , Vaccinia/prevenção & controle , Apresentação do Antígeno/imunologia , Linhagem Celular Transformada , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Epitopos de Linfócito T/fisiologia , Antígenos HLA/isolamento & purificação , Antígenos HLA-A/imunologia , Antígenos HLA-A/metabolismo , Antígeno HLA-A2 , Antígenos HLA-B/imunologia , Antígenos HLA-B/metabolismo , Antígeno HLA-B7 , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Humanos , Memória Imunológica , Células K562 , Ligantes , Proteínas Oncogênicas Virais/administração & dosagem , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas Virais/metabolismo , Proteínas Oncogênicas Virais/fisiologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologia , Vaccinia/metabolismo , Vírus Vaccinia/metabolismo , Proteínas Virais/administração & dosagem , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Proteínas Virais/fisiologia , Latência Viral/imunologia
10.
Transplantation ; 86(7): 912-8, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18852655

RESUMO

BACKGROUND: Transplant patients often produce human leukocyte antigen (HLA) antibodies against their donors and produce more specificities than can be accounted for by HLA antigen mismatches. We theorize that the presence of extra, otherwise unexplainable specificities could be accounted for if antibodies reacted to more than one epitope (primary and mimetic) on distinct HLA molecules. The theory states that mimetic epitopes consist of the same three amino acids that comprise the primary, sterically placed approximately the same distance apart as are the corresponding amino acids of the primary. METHODS: A mimetic epitope table containing all primary epitopes and corresponding mimetic epitopes was built. Then, the HLA specificities of monoclonal and single patient antibodies were determined. These specificities that could not be defined by unique position or amino acid epitopes alone were then used to query the mimetic epitope table. RESULTS: A single antibody from a transplant patient and three monoclonal antibodies produced reactions that can best be explained as the result of one antibody reacting to the same amino acids at two distinct sites on the molecule. Those position and amino acid combinations (pos/aa) are the primary and mimetic epitopes. Using computerized methods of searching, mimetic epitopes were found in five additional kidney transplant patients who produced nondonor-specific antibodies in addition to donor-specific antibodies. CONCLUSIONS: Epitopes on the HLA molecule that mimic the primary epitope have been found. We suggest that these mimetic epitopes explain the additional antibodies often found on HLA immunization resulting from allograft rejections, pregnancies, and transfusions.


Assuntos
Anticorpos Monoclonais/sangue , Antígenos HLA/sangue , Isoanticorpos/sangue , Transplante de Rim/imunologia , Leucócitos/imunologia , Adsorção , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Epitopos/sangue , Epitopos/isolamento & purificação , Antígenos HLA/isolamento & purificação , Humanos , Isoanticorpos/isolamento & purificação
11.
J Reprod Immunol ; 75(1): 11-22, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17485119

RESUMO

In recent years, the number of patients receiving in vitro fertilization (IVF) has been increasing, though the rate of successful implantations has remained at 10-20%. A major goal of this procedure is to afford the ability to select embryos with the most potential for implantation and development. Previous studies claimed to have detected soluble HLA-G (sHLA-G) protein in culture supernatant from 2 to 3-day embryos using ELISA methods, and concluded that sHLA-G protein levels were associated with successful implantation. This result, if substantiated could provide an important tool for IVF. In this study, we have re-examined these experiments by attempting to detect sHLA-G in the medium from 2 to 3-day embryos (84 samples) and 4 to 6-day embryos (25 samples) in which a part of blastocyst has started to differentiate into trophoblasts. Using a highly specific and sensitive ELISA, no sHLA-G protein was detectable in any sample, despite the fact that 27 of the 109 samples were from successfully implanted embryos. These results indicate that 2-6-day embryos do not secrete sHLA-G detectable by ELISA, and therefore that sHLA-G in culture medium is not a useful for successful implantation at this stage of development.


Assuntos
Embrião de Mamíferos/imunologia , Ensaio de Imunoadsorção Enzimática , Antígenos HLA/análise , Antígenos de Histocompatibilidade Classe I/análise , Meios de Cultura/análise , Técnicas de Cultura Embrionária , Implantação do Embrião , Desenvolvimento Embrionário , Feminino , Fertilização In Vitro , Antígenos HLA/isolamento & purificação , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Humanos , Gravidez , Sensibilidade e Especificidade
12.
Transplantation ; 83(7): 982-8, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17460571

RESUMO

BACKGROUND: The development of highly sensitive and specific assays for detecting and characterizing HLA-specific antibodies has contributed to an appreciation of the extensive involvement of those antibodies in graft injury and dysfunction. However, understanding the regulatory processes of the humoral response to transplantation and the mechanisms underlying therapeutic agents and protocols for preventing and treating sensitization requires a way to study HLA-specific B cells. METHODS: Lymphocyte preparations enriched for B cells were stained with one or more of three different HLA tetramers. Tetramer-positive (tet+) B cells were enumerated and evaluated for an association of their frequencies with known sensitization. In some cases, tet+ B cells were isolated and placed in culture with supplements known to activate B cells in a nonspecific fashion. RESULTS: For all tetramers used, the frequencies of tet+ B cells were significantly higher (4.1%-5.5%) among sensitized patients than among nonsensitized patients (1.6%-3.2%, P<0.001). Binding of the tetramers occurred by the surface immunoglobulin antigen receptor with little or no binding to antibody captured in the Fc receptor. Cultured tet+ B cells produced antibodies specific for epitopes of the tetramer antigen. There appeared to be a certain amount of crossreactivity in the binding of tetramers. The frequency of CD27+ cells among tet+ B cells was higher, on average (34.4%-38.8%) than among all B cells (26.2%) whereas the frequencies of CD38 were comparable in the two groups. CONCLUSIONS: Staining with HLA tetramers provides a means for identifying, quantifying, and isolating HLA-specific B cells.


Assuntos
Linfócitos B/imunologia , Antígenos HLA/imunologia , Antígenos HLA/isolamento & purificação , Falência Renal Crônica/imunologia , Formação de Anticorpos , Antígenos CD/imunologia , Antígenos CD19/imunologia , Técnicas de Cultura de Células , Feminino , Antígenos HLA/química , Antígeno HLA-B7/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Masculino , Ligação Proteica , Valores de Referência , Linfócitos T/imunologia
13.
J Immunol Methods ; 320(1-2): 119-31, 2007 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-17306825

RESUMO

The development of MHC/peptide multimers has facilitated the visualization and purification of antigen-specific T cells. However, the persistence of multimers leads to prolonged T cell receptor signaling and subsequently to altered T-cell function. We have recently developed a new type of MHC/peptide multimers, which can be dissociated from the T cell. Herein, we have generated and tested for the first time reversible HLA/peptide multimers, termed Streptamers, for the isolation of human T cells. The Streptamer technique demonstrates the specificity and sensitivity of conventional HLA/peptide tetramers with regards to the sorting of human T lymphocytes. This is shown for T cells directed against immunogenic peptides derived from viral and tumor-associated antigens. We show that antigen-specific cytotoxic T cells remain functionally active following Streptamer dissociation, whereas lytic function and proliferation of the T cells is impaired in the presence of conventional tetramers. These novel HLA/peptide Streptamer reagents allow the isolation of antigen-specific T cells with preserved function and, therefore, facilitate the development of adoptive T cell transfer regimens for the treatment of patients with cancer or infectious diseases.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos Virais/imunologia , Antígenos HLA/isolamento & purificação , Linfócitos T Citotóxicos/imunologia , Células Cultivadas , Citomegalovirus/imunologia , Antígenos HLA/química , Antígenos HLA/imunologia , Humanos , Antígeno MART-1 , Proteínas de Neoplasias/imunologia , Linfócitos T Citotóxicos/fisiologia
14.
Curr Protoc Immunol ; Chapter 16: Unit 16.3, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-18432987

RESUMO

Identification of peptides presented in human leukocyte antigen (HLA) class I molecules after viral infection is of strategic importance for immunology and vaccine development. A powerful strategy aimed at the rapid, unambiguous identification of naturally processed HLA class I-associated peptides, which are induced by viral infection, is presented here. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues for the human leukocyte antigen allele of interest. Subsequently, these cells are mixed with an equal number of noninfected cells, which are cultured in normal medium. Finally, peptides are acid-eluted from immunoprecipitated HLA molecules and subjected to two-dimensional nanoscale liquid chromatography-mass spectrometry analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules.


Assuntos
Antígenos HLA/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Epitopos Imunodominantes , Técnicas Imunológicas , Marcação por Isótopo , Apresentação do Antígeno , Distribuição Binomial , Cromatografia por Troca Iônica/métodos , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Epitopos Imunodominantes/isolamento & purificação , Processamento de Proteína Pós-Traducional , Processamento de Sinais Assistido por Computador , Espectrometria de Massas por Ionização por Electrospray/métodos , Viroses/imunologia
16.
Artigo em Inglês | MEDLINE | ID: mdl-16511266

RESUMO

HLA-G is a nonclassical class I major histocompatibility complex (MHC) molecule that is primarily expressed at the foetal-maternal interface. Although the role of HLA-G has not been fully elucidated, current evidence suggests it protects the foetus from the maternal immune response. In this report, HLA-G (44 kDa) is characterized by expression in Escherichia coli. The inclusion bodies were refolded in complex with a peptide derived from histone H2A (RIIPRHLQL), purified and subsequently crystallized. Correct refolding was determined using two conformation-dependent antibodies. Cobalt ions were shown to be an essential ingredient for obtaining diffraction-quality crystals. The crystals, which diffracted to 1.9 A resolution, belonged to space group P3(2)2(1), with unit-cell parameters a = b = 77.15, c = 151.72 A.


Assuntos
Cobalto/química , Antígenos HLA/química , Antígenos HLA/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Substituição de Aminoácidos/genética , Cátions Bivalentes/química , Linhagem Celular Tumoral , Cisteína/genética , Antígenos HLA/genética , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Serina/genética , Solubilidade
17.
Biochim Biophys Acta ; 1764(5): 985-8, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16290109

RESUMO

HLA-G is a non-classical MHC class I, which binds to inhibitory receptors, such as Leukocyte Ig-like receptors, to induce a wide range of tolerogenic immunological effects. HLA-G can be expressed as a disulfide-liked dimer both in solution and at the cell surface. However, the three-dimensional structure of the HLA-G dimer is unknown. Here, we report the crystallization of the disulfide-linked dimer form of HLA-G by adding dithiothreitol (DTT), enabling a 3.2-A data set to be collected. We also show that DTT promotes disulfide bond exchange of refolded HLA-G, whose free cysteine was protected, thus facilitating its dimerization. This technique could also be applied for disulfide-mediated dimer/multimer formation of refolded proteins harbouring free cysteines.


Assuntos
Dissulfetos/química , Antígenos HLA/química , Antígenos HLA/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Cristalização , Dimerização , Ditiotreitol , Antígenos HLA-G , Humanos
18.
J Immunol ; 171(4): 1715-21, 2003 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-12902470

RESUMO

Blastocyst MHC is a recently identified mouse MHC class Ib gene, which is selectively expressed in blastocyst and placenta, and may be the mouse homolog of HLA-G gene the products of which have been implicated in protection of fetal trophoblasts from maternal NK cells and evasion of some tumor cells from NK cell attack. In this study, we identified two blastocyst MHC gene transcripts encoding a full-length alpha-chain (bc1) and an alternatively spliced form lacking the alpha2 domain (bc2), which may be homologous to HLA-G1 and HLA-G2, respectively. Both placenta and a teratocarcinoma cell line predominantly expressed the bc2 transcript. When these cDNAs were expressed in TAP-deficient RMA-S or TAP-sufficient RMA cells, only bc1 protein was expressed on the surface of RMA cells, but both bc1 and bc2 proteins were retained in the cytoplasm of RMA-S cells. Significantly, the RMA-S cells expressing either bc1 or bc2 were protected from lysis by NK cells in vitro. This protection was at least partly mediated by up-regulation of Qa-1(b) expression on the surface of RMA-S cells, which engaged the CD94/NKG2A inhibitory receptor on NK cells. More importantly, the bc1- or bc2-expressing RMA-S cells were significantly protected from NK cell-mediated rejection in vivo. These results suggested a role for blastocyst MHC in protecting TAP-deficient trophoblasts and tumor cells from NK cell attack in vivo.


Assuntos
Blastocisto/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Antígenos H-2/fisiologia , Antígenos HLA/fisiologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Células Matadoras Naturais/imunologia , Linfoma de Células T/imunologia , Processamento Alternativo/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD/fisiologia , Citotoxicidade Imunológica/genética , Feminino , Rejeição de Enxerto/genética , Antígenos H-2/biossíntese , Antígenos H-2/genética , Antígenos HLA/biossíntese , Antígenos HLA/isolamento & purificação , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Humanos , Imunidade Celular/genética , Lectinas Tipo C/fisiologia , Linfoma de Células T/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Subfamília C de Receptores Semelhantes a Lectina de Células NK , Subfamília D de Receptores Semelhantes a Lectina de Células NK , Transplante de Neoplasias , Proteínas Nucleares , Gravidez , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/fisiologia , Receptores Imunológicos/fisiologia , Receptores de Células Matadoras Naturais , Homologia de Sequência de Aminoácidos , Transfecção , Células Tumorais Cultivadas
19.
Tissue Antigens ; 61(6): 443-50, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12823768

RESUMO

Genomic typing of polymorphic loci may be hampered by ambiguous typing results. Moreover, robust methods for simultaneous sequencing of two alleles present in a given sample may be difficult to establish. We used denaturing high-performance liquid chromatography (DHPLC) for physical separation of HLA-A alleles before sequence-based genomic typing (SBT). Physical separation was achieved by resolution of heteroduplexes between the sample alleles and a modified reference probe by DHPLC followed by selective reamplification of the sample alleles present in heteroduplexes. Complementary strands of the reference probe and sample alleles for heteroduplex induction were obtained by lambda-exonuclease digestion. HLA-A genotyping of 101 individuals using DHPLC-SBT yielded better typing resolution compared with serological typing and genotyping by the sequence-specific primer-polymerase chain reaction (SSP-PCR) method. Physical separation of alleles using a modified reference probe allows for development of fully automated methods for genomic typing of highly polymorphic loci such as HLA.


Assuntos
Alelos , Cromatografia Líquida de Alta Pressão/métodos , DNA/análise , Antígenos HLA/genética , Antígenos HLA/isolamento & purificação , Sequência de Bases , Primers do DNA , Éxons , Estudos de Viabilidade , Amplificação de Genes , Análise Heteroduplex , Heterozigoto , Teste de Histocompatibilidade , Humanos , Desnaturação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
20.
Eur J Biochem ; 269(4): 1199-208, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11856346

RESUMO

Subunits (alpha, beta and gamma) of the interleukin-2 receptor complex (IL-2R) are involved in both proliferative and activation-induced cell death (AICD) signaling of T cells. In addition, the signaling beta and gamma chains are shared by other cytokines (e.g. IL-7, IL-9, IL-15). However, the molecular mechanisms responsible for recruiting/sorting the alpha chains to the signaling chains at the cell surface are not clear. Here we show, in four cell lines of human adult T cell lymphoma/leukemia origin, that the three IL-2R subunits are compartmented together with HLA glycoproteins and CD48 molecules in the plasma membrane, by means of fluorescence resonance energy transfer (FRET), confocal microscopy and immuno-biochemical techniques. In addition to the beta and gamma(c) chains constitutively expressed in detergent-resistant membrane fractions (DRMs) of T cells, IL-2Ralpha (CD25) was also found in DRMs, independently of its ligand-occupation. Association of CD25 with rafts was also confirmed by its colocalization with GM-1 ganglioside. Depletion of membrane cholesterol using methyl-beta-cyclodextrin substantially reduced co-clustering of CD25 with CD48 and HLA-DR, as well as the IL-2 stimulated tyrosine-phosphorylation of STATs (signal transducer and activator of transcription). These data indicate a GPI-microdomain (raft)-assisted recruitment of CD25 to the vicinity of the signaling beta and gamma(c) chains. Rafts may promote rapid formation of a high affinity IL-2R complex, even at low levels of IL-2 stimulus, and may also form a platform for the regulation of IL-2 induced signals by GPI-proteins (e.g. CD48). Based on these data, the integrity of these GPI-microdomains seems critical in signal transduction through the IL-2R complex.


Assuntos
Leucemia de Células T/metabolismo , Linfoma de Células T/metabolismo , Microdomínios da Membrana , Receptores de Interleucina-2/isolamento & purificação , Antígenos CD/isolamento & purificação , Antígeno CD48 , Gangliosídeo G(M1)/isolamento & purificação , Glicoproteínas/isolamento & purificação , Antígenos HLA/isolamento & purificação , Humanos , Subunidade alfa de Receptor de Interleucina-2 , Receptores de Interleucina/isolamento & purificação , Transdução de Sinais , Células Tumorais Cultivadas
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