Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
J Autoimmun ; 146: 103203, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38643729

RESUMO

Lupus erythematosus (LE) is a heterogeneous, antibody-mediated autoimmune disease. Isolate discoid LE (IDLE) and systematic LE (SLE) are traditionally regarded as the two ends of the spectrum, ranging from skin-limited damage to life-threatening multi-organ involvement. Both belong to LE, but IDLE and SLE differ in appearance of skin lesions, autoantibody panels, pathological changes, treatments, and immunopathogenesis. Is discoid lupus truly a form of LE or is it a completely separate entity? This question has not been fully elucidated. We compared the clinical data of IDLE and SLE from our center, applied multi-omics technology, such as immune repertoire sequencing, high-resolution HLA alleles sequencing and multi-spectrum pathological system to explore cellular and molecular phenotypes in skin and peripheral blood from LE patients. Based on the data from 136 LE patients from 8 hospitals in China, we observed higher damage scores and fewer LE specific autoantibodies in IDLE than SLE patients, more uCDR3 sharing between PBMCs and skin lesion from SLE than IDLE patients, elevated diversity of V-J recombination in IDLE skin lesion and SLE PBMCs, increased SHM frequency and class switch ratio in IDLE skin lesion, decreased SHM frequency but increased class switch ratio in SLE PBMCs, HLA-DRB1*03:01:01:01, HLA-B*58:01:01:01, HLA-C*03:02:02:01, and HLA-DQB1*02:01:01:01 positively associated with SLE patients, and expanded Tfh-like cells with ectopic germinal center structures in IDLE skin lesions. These findings suggest a significant difference in the immunopathogenesis of skin lesions between SLE and IDLE patients. SLE is a B cell-predominate systemic immune disorder, while IDLE appears limited to the skin. Our findings provide novel insights into the pathogenesis of IDLE and other types of LE, which may direct more accurate diagnosis and novel therapeutic strategies.


Assuntos
Autoanticorpos , Lúpus Eritematoso Discoide , Lúpus Eritematoso Sistêmico , Pele , Humanos , Lúpus Eritematoso Discoide/imunologia , Lúpus Eritematoso Discoide/patologia , Feminino , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/diagnóstico , Masculino , Autoanticorpos/imunologia , Autoanticorpos/sangue , Pele/patologia , Pele/imunologia , Pele/metabolismo , Adulto , Pessoa de Meia-Idade , Alelos , Antígenos HLA/genética , Antígenos HLA/imunologia , Adulto Jovem , Multiômica
2.
Physiol Genomics ; 52(6): 255-268, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32437232

RESUMO

Precision medicine requires the translation of basic biological understanding to medical insights, mainly applied to characterization of each unique patient. In many clinical settings, this requires tools that can be broadly used to identify pathology and risks. Patients often present to the intensive care unit with broad phenotypes, including multiple organ dysfunction syndrome (MODS) resulting from infection, trauma, or other disease processes. Etiology and outcomes are unique to individuals, making it difficult to cohort patients with MODS, but presenting a prime target for testing/developing tools for precision medicine. Using multitime point whole blood (cellular/acellular) total transcriptomics in 27 patients, we highlight the promise of simultaneously mapping viral/bacterial load, cell composition, tissue damage biomarkers, balance between syndromic biology versus environmental response, and unique biological insights in each patient using a single platform measurement. Integration of a transcriptome workflow yielded unexpected insights into the complex interplay between host genetics and viral/bacterial specific mechanisms, highlighted by a unique case of virally induced genetics (VIG) within one of these 27 patients. The power of RNA-Seq to study unique patient biology while investigating environmental contributions can be a critical tool moving forward for translational sciences applied to precision medicine.


Assuntos
Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Perfilação da Expressão Gênica/métodos , Pneumonia Viral/genética , Pneumonia Viral/virologia , Medicina de Precisão/métodos , COVID-19 , Humanos , Pandemias , Transcrição Gênica , Carga Viral
3.
J Transl Med ; 17(1): 47, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777078

RESUMO

BACKGROUND: Immunology research, particularly next generation sequencing (NGS) of the immune T-cell receptor ß (TCRß) repertoire, has advanced progression in several fields, including treatment of various cancers and autoimmune diseases. This study aimed to identify the TCR repertoires from dry blood spots (DBS), a method that will help collecting real-world data for biomarker applications. METHODS: Finger-prick blood was collected onto a Whatman filter card. RNA was extracted from DBS of the filter card, and fully automated multiplex PCR was performed to generate a TCRß chain library for next generation sequencing (NGS) analysis of unique CDR3s (uCDR3). RESULTS: We demonstrated that the dominant clonotypes from the DBS results recapitulated those found in whole blood. According to the statistical analysis and laboratory confirmation, 40 of 2-mm punch disks from the filter cards were enough to detect the shared top clones and have strong correlation in the uCDR3 discovery with whole blood. uCDR3 discovery was neither affected by storage temperatures (room temperature versus - 20 °C) nor storage durations (1, 14, and 28 days) when compared to whole blood. About 74-90% of top 50 uCDR3 clones of whole blood could also be detected from DBS. A low rate of clonotype sharing, 0.03-1.5%, was found among different individuals. CONCLUSIONS: The DBS-based TCR repertoire profiling method is minimally invasive, provides convenient sampling, and incorporates fully automated library preparation. The system is sensitive to low RNA input, and the results are highly correlated with whole blood uCDR3 discovery allowing study scale-up to better understand the relationship and mutual influences between the immune and diseases.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Regiões Determinantes de Complementaridade/genética , Humanos , Preservação Biológica , RNA/isolamento & purificação , Temperatura
5.
Front Immunol ; 11: 582010, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33117392

RESUMO

Severe COVID-19 is associated with profound lymphopenia and an elevated neutrophil to lymphocyte ratio. We applied a novel dimer avoidance multiplexed polymerase chain reaction next-generation sequencing assay to analyze T (TCR) and B cell receptor (BCR) repertoires. Surprisingly, TCR repertoires were markedly diminished during the early onset of severe disease but recovered during the convalescent stage. Monitoring TCR repertoires could serve as an indicative biomarker to predict disease progression and recovery. Panoramic concurrent assessment of BCR repertoires demonstrated isotype switching and a transient but dramatic early IgA expansion. Dominant B cell clonal expansion with decreased diversity occurred following recovery from infection. Profound changes in T cell homeostasis raise critical questions about the early events in COVID-19 infection and demonstrate that immune repertoire analysis is a promising method for evaluating emergent host immunity to SARS-CoV-2 viral infection, with great implications for assessing vaccination and other immunological therapies.


Assuntos
Linfócitos B/imunologia , Betacoronavirus/imunologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Contagem de Linfócito CD4 , COVID-19 , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfopenia/patologia , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , SARS-CoV-2
6.
Artigo em Inglês | MEDLINE | ID: mdl-19724129

RESUMO

Proliferating cell nuclear antigen (PCNA) is a DNA sliding clamp which confers processivity on replicative DNA polymerases. PCNA also acts as a sliding platform that enables the association of many DNA-processing proteins with DNA in a non-sequence-specific manner. In this investigation, the PCNA from the hyperthermophilic archaeon Thermococcus thioreducens (TtPCNA) was cloned, overexpressed in Escherichia coli and purified to greater than 90% homogeneity. TtPCNA crystals were obtained by sitting-drop vapor-diffusion methods and the best ordered crystal diffracted to 1.86 A resolution using synchrotron radiation. The crystals belonged to the hexagonal space group P6(3), with unit-cell parameters a = b = 89.0, c = 62.8 A. Crystals of TtPCNA proved to be amenable to complete X-ray analysis and future structure determination.


Assuntos
Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/isolamento & purificação , Thermococcus/química , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida
7.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 65(Pt 11): 1131-5, 2009 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-19923734

RESUMO

Proliferating cell nuclear antigen (PCNA) is a DNA-clamping protein that is responsible for increasing the processivity of the replicative polymerases during DNA replication and repair. The PCNA from the eurypsychrophilic archaeon Methanococcoides burtonii DSM 6242 (MbPCNA) has been targeted for protein structural studies. A recombinant expression system has been created that overproduces MbPCNA with an N-terminal hexahistidine affinity tag in Escherichia coli. As a result, recombinant MbPCNA with a molecular mass of 28.3 kDa has been purified to at least 95% homogeneity and crystallized by vapor-diffusion equilibration. Preliminary X-ray analysis revealed a trigonal hexagonal R3 space group, with unit-cell parameters a = b = 102.5, c = 97.5 angstrom. A singleMbPCNA crystal was subjected to complete diffraction data-set collection using synchrotron radiation and reflections were measured to 2.40 angstrom resolution. The diffraction data were of suitable quality for indexing and scaling and an unrefined molecular-replacement solution has been obtained.


Assuntos
Proteínas Arqueais/química , Methanosarcinaceae/química , Antígeno Nuclear de Célula em Proliferação/química , Proteínas Recombinantes/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Cristalização , Cristalografia por Raios X , Dados de Sequência Molecular , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Difração de Raios X
8.
BMC Biotechnol ; 8: 44, 2008 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-18445293

RESUMO

BACKGROUND: Gene synthesis technologies are an important tool for structural biology projects, allowing increased protein expression through codon optimization and facilitating sequence alterations. Existing methods, however, can be complex and not always reproducible, prompting researchers to use commercial suppliers rather than synthesize genes themselves. RESULTS: A PCR-based gene synthesis method, referred to as SeqTBIO, is described to efficiently assemble the coding regions of two novel hyperthermophilic proteins, PAZ (Piwi/Argonaute/Zwille) domain, a siRNA-binding domain of an Argonaute protein homologue and a deletion mutant of a family A DNA polymerase (PolA). The gene synthesis procedure is based on sequential assembly such that homogeneous DNA products can be obtained after each synthesis step without extensive manipulation or purification requirements. Coupling the gene synthesis procedure to in vivo homologous recombination techniques allows efficient subcloning and site-directed mutagenesis for error correction. The recombinant proteins of PAZ and PolA were subsequently overexpressed in E. coli and used for protein crystallization. Crystals of both proteins were obtained and they were suitable for X-ray analysis. CONCLUSION: We demonstrate, by using PAZ and PolA as examples, the feasibility of integrating the gene synthesis, error correction and subcloning techniques into a non-automated gene to crystal pipeline such that genes can be designed, synthesized and implemented for recombinant expression and protein crystallization.


Assuntos
Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Escherichia coli/fisiologia , Reação em Cadeia da Polimerase/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Cristalização , Proteínas de Escherichia coli/genética , Estudos de Viabilidade , Proteínas Recombinantes/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA