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2.
Nat Commun ; 10(1): 2935, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270335

RESUMO

Trace elements play important roles in human health, but little is known about their functions in humoral immunity. Here, we show an important role for iron in inducing cyclin E and B cell proliferation. We find that iron-deficient individuals exhibit a significantly reduced antibody response to the measles vaccine when compared to iron-normal controls. Mice with iron deficiency also exhibit attenuated T-dependent or T-independent antigen-specific antibody responses. We show that iron is essential for B cell proliferation; both iron deficiency and α-ketoglutarate inhibition could suppress cyclin E1 induction and S phase entry of B cells upon activation. Finally, we demonstrate that three demethylases, KDM2B, KDM3B and KDM4C, are responsible for histone 3 lysine 9 (H3K9) demethylation at the cyclin E1 promoter, cyclin E1 induction and B cell proliferation. Thus, our data reveal a crucial role of H3K9 demethylation in B cell proliferation, and the importance of iron in humoral immunity.


Assuntos
Linfócitos B/imunologia , Proliferação de Células , Histonas/química , Histonas/imunologia , Imunidade Humoral , Lisina/imunologia , Animais , Linfócitos B/química , Linfócitos B/citologia , Ciclo Celular , Células Cultivadas , Ciclina E/genética , Ciclina E/imunologia , Desmetilação , Proteínas F-Box/genética , Proteínas F-Box/imunologia , Histonas/genética , Ferro/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/imunologia , Ativação Linfocitária , Lisina/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologia , Regiões Promotoras Genéticas , Linfócitos T/citologia , Linfócitos T/imunologia
3.
J Clin Exp Hematop ; 59(1): 1-16, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30918139

RESUMO

The microenvironment influences the behavior of follicular lymphoma (FL) but the specific roles of the immunomodulatory BTLA and TNFRSF14 (HVEM) are unknown. Therefore, we examined their immunohistochemical expression in the intrafollicular, interfollicular and total histological compartments in 106 FL cases (57M/49F; median age 57-years), and in nine relapsed-FL with transformation to DLBCL (tFL). BTLA expression pattern was of follicular T-helper cells (TFH) in the intrafollicular and of T-cells in the interfollicular compartments. The mantle zones were BTLA+ in 35.6% of the cases with similar distribution of IgD. TNFRSF14 expression pattern was of neoplastic B lymphocytes (centroblasts) and "tingible body macrophages". At diagnosis, the averages of total BTLA and TNFRSF14-positive cells were 19.2%±12.4STD (range, 0.6%-58.2%) and 46.7 cells/HPF (1-286.5), respectively. No differences were seen between low-grade vs. high-grade FL but tFL was characterized by low BTLA and high TNFRSF14 expression. High BTLA correlated with good overall survival (OS) (total-BTLA, Hazard Risk=0.479, P=0.022) and with high PD-1 and FOXP3+Tregs. High TNFRSF14 correlated with poor OS and progression-free survival (PFS) (total-TNFRSF14, HR=3.9 and 3.2, respectively, P<0.0001), with unfavorable clinical variables and higher risk of transformation (OR=5.3). Multivariate analysis including BTLA, TNFRSF14 and FLIPI showed that TNFRSF14 and FLIPI maintained prognostic value for OS and TNFRSF14 for PFS. In the GSE16131 FL series, high TNFRSF14 gene expression correlated with worse prognosis and GSEA showed that NFkB pathway was associated with the "High-TNFRSF14/dead-phenotype".In conclusion, the BTLA-TNFRSF14 immune modulation pathway seems to play a role in the pathobiology and prognosis of FL.


Assuntos
Linfoma Folicular/diagnóstico , Linfoma Difuso de Grandes Células B/diagnóstico , Receptores Imunológicos/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/química , Linfócitos B/patologia , Transformação Celular Neoplásica , Feminino , Humanos , Fatores Imunológicos , Linfoma Folicular/mortalidade , Linfoma Difuso de Grandes Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Análise de Sobrevida , Linfócitos T/química
4.
Mol Cell ; 74(2): 268-283.e5, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30902546

RESUMO

Linker histone H1 has been correlated with transcriptional inhibition, but the mechanistic basis of the inhibition and its reversal during gene activation has remained enigmatic. We report that H1-compacted chromatin, reconstituted in vitro, blocks transcription by abrogating core histone modifications by p300 but not activator and p300 binding. Transcription from H1-bound chromatin is elicited by the H1 chaperone NAP1, which is recruited in a gene-specific manner through direct interactions with activator-bound p300 that facilitate core histone acetylation (by p300) and concomitant eviction of H1 and H2A-H2B. An analysis in B cells confirms the strong dependency on NAP1-mediated H1 eviction for induction of the silent CD40 gene and further demonstrates that H1 eviction, seeded by activator-p300-NAP1-H1 interactions, is propagated over a CCCTC-binding factor (CTCF)-demarcated region through a distinct mechanism that also involves NAP1. Our results confirm direct transcriptional inhibition by H1 and establish a gene-specific H1 eviction mechanism through an activator→p300→NAP1→H1 pathway.


Assuntos
Fator de Ligação a CCCTC/genética , Proteína p300 Associada a E1A/genética , Proteínas/genética , Transcrição Genética , Acetilação , Linfócitos B/química , Sítios de Ligação , Fator de Ligação a CCCTC/química , Antígenos CD40/genética , Cromatina/química , Cromatina/genética , Proteína p300 Associada a E1A/química , Código das Histonas , Histonas/química , Histonas/genética , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Nucleossomos/química , Nucleossomos/genética , Regiões Promotoras Genéticas , Ligação Proteica/genética , Proteínas/química , tRNA Metiltransferases
5.
mBio ; 10(1)2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670616

RESUMO

Friend virus (FV) is a naturally occurring mouse retrovirus that infects dividing cells of the hematopoietic lineage, including antigen-presenting cells (APCs). The infection of APCs by viruses often induces their dysfunction, and it has been shown that FV infection reduces the ability of dendritic cells (DCs) to prime critical CD8+ T cell responses. Nonetheless, mice mount vigorous CD8+ T cell responses, so we investigated whether B cells might serve as alternative APCs during FV infection. Direct ex vivo analysis of B cells from FV-infected mice revealed that infected but not uninfected B cells upregulated expression of the costimulatory molecules CD80, CD86, and CD40, as well as major histocompatibility complex class II (MHC-II) molecules. Furthermore, in vitro studies showed that, compared to uninfected B cells from the same mice, the FV-infected B cells had significantly enhanced APC function, as measured by their capacity to prime CD8+ T cell activation and proliferation. Thus, in contrast to DCs, infection of B cells with FV enhanced their APC capacity and ability to stimulate the CD8+ T cell responses essential for virus control. FV infections also induce the activation and expansion of regulatory T cells (Tregs), so it was of interest to determine the impact of Tregs on B cell activation. The upregulation of costimulatory molecule expression and APC function of B cells was even more strongly enhanced by in vivo depletion of regulatory T cells than infection. Thus, Tregs exert potent homeostatic suppression of B cell activation that is partially overcome by FV infection.IMPORTANCE The primary role of B cells in immunity is considered the production of pathogen-specific antibodies, but another, less-well-studied, function of B cells is to present foreign antigens to T cells to stimulate their activation and proliferation. Dendritic cells (DCs) are considered the most important antigen-presenting cells (APCs) for CD8+ T cells, but DCs lose APC function when infected with Friend virus (FV), a model retrovirus of mice. Interestingly, B cells were better able to stimulate CD8+ T cell responses when they were infected with FV. We also found that the activation status of B cells under homeostatic conditions was potently modulated by regulatory T cells. This study illustrates an important link between B cell and T cell responses and illustrates an additional mechanism by which regulatory T cells suppress critical T cell responses during viral infections.


Assuntos
Apresentação do Antígeno , Linfócitos B/imunologia , Vírus da Leucemia Murina de Friend/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos B/química , Antígeno B7-1/análise , Antígeno B7-2/análise , Antígenos CD40/análise , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Antígenos de Histocompatibilidade Classe II/análise , Leucemia Experimental/imunologia , Leucemia Experimental/virologia , Ativação Linfocitária , Camundongos , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologia
6.
AIDS Res Hum Retroviruses ; 35(1): 33-39, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30298747

RESUMO

In this study, we aimed to quantify KREC (kappa-deleting recombination excision circle) levels and naive B cell output in healthy HIV-uninfected children, compared with HIV-infected South African children, before and after starting ART (antiretroviral therapy). Samples were acquired from a Child Wellness Clinic (n = 288 HIV-uninfected South African children, 2 weeks-12 years) and the Children with HIV Early Antiretroviral Therapy (CHER) trial (n = 153 HIV-infected South African children, 7 weeks-8 years). Naive B cell output was estimated using a mathematical model combining KREC levels to reflect B cell emigration into the circulation, flow cytometry measures of naive unswitched B cells to quantify total body naive B cells, and their rates of proliferation using the intracellular marker Ki67. Naive B cell output increases from birth to 1 year, followed by a decline and plateau into late childhood. HIV-infected children on or off ART had higher naive B cell outputs than their uninfected counterparts (p = .01 and p = .04). This is the first study to present reference ranges for measurements of KRECs and naive B cell output in healthy and HIV-infected children. Comparison between HIV-uninfected healthy children and HIV-infected children suggests that HIV may increase naive B cell output. Further work is required to fully understand the mechanisms involved and clinical value of measuring naive B cell output in children.


Assuntos
Antirretrovirais/uso terapêutico , Linfócitos B/imunologia , Proliferação de Células , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Imunidade Celular , Linfócitos B/química , Criança , Pré-Escolar , Estudos de Coortes , DNA/análise , Feminino , Citometria de Fluxo , Humanos , Lactente , Recém-Nascido , Antígeno Ki-67/análise , Masculino , Modelos Teóricos , África do Sul
7.
J Vis Exp ; (141)2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30531718

RESUMO

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.


Assuntos
Linfócitos B/fisiologia , Movimento Celular/fisiologia , Quimiotaxia/fisiologia , Imagem com Lapso de Tempo/métodos , Animais , Linfócitos B/química , Células Cultivadas , Tecido Linfoide/química , Tecido Linfoide/citologia , Tecido Linfoide/fisiologia , Camundongos , Baço/química , Baço/citologia , Baço/fisiologia
8.
J Vis Exp ; (141)2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30531724

RESUMO

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.


Assuntos
Linfócitos B/química , Rastreamento de Células/métodos , Imagem Molecular/métodos , Proteínas/análise , Linfócitos B/metabolismo , Linhagem Celular Transformada , Humanos , Interferometria/métodos , Microscopia/métodos , Nanotecnologia/métodos , Proteínas/metabolismo
9.
Clin Lab Med ; 38(4): 595-605, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30420055

RESUMO

HLA epitope matching provides a better approach to stratify patients at risk of developing antibody-mediated rejection compared with counting HLA mismatches. However, several immunologic parameters are not incorporated into these algorithms used to assess HLA epitopes, raising questions about the predictive value of these programs. Therefore, it is imperative to obtain more 3D structural data of antibody-antigen binding to "train" these computer algorithms. Also, mechanistic studies should be performed to prove these theoretic "epitopes." Most important, more information is needed to ensure these predictive computer algorithms are equitable and safe to use in clinical diagnostics before wide-scale implementation.


Assuntos
Epitopos , Antígenos HLA , Teste de Histocompatibilidade , Transplante , Algoritmos , Linfócitos B/química , Linfócitos B/imunologia , Biologia Computacional , Humanos , Linfócitos T/química , Linfócitos T/imunologia
10.
Elife ; 72018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30387712

RESUMO

A subset of atypical memory B cells accumulates in malaria and several infections, autoimmune disorders and aging in both humans and mice. It has been suggested these cells are exhausted long-lived memory B cells, and their accumulation may contribute to poor acquisition of long-lasting immunity to certain chronic infections, such as malaria and HIV. Here, we generated an immunoglobulin heavy chain knock-in mouse with a BCR that recognizes MSP1 of the rodent malaria parasite, Plasmodium chabaudi. In combination with a mosquito-initiated P. chabaudi infection, we show that Plasmodium-specific atypical memory B cells are short-lived and disappear upon natural resolution of chronic infection. These cells show features of activation, proliferation, DNA replication, and plasmablasts. Our data demonstrate that Plasmodium-specific atypical memory B cells are not a subset of long-lived memory B cells, but rather short-lived activated cells, and part of a physiologic ongoing B-cell response.


Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Memória Imunológica , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium chabaudi/imunologia , Animais , Subpopulações de Linfócitos B/química , Linfócitos B/química , Citometria de Fluxo , Técnicas de Introdução de Genes , Imunoglobulina G/genética , Malária/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Doenças dos Roedores/imunologia
11.
PLoS Comput Biol ; 14(10): e1006388, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30332400

RESUMO

B cells develop high affinity receptors during the course of affinity maturation, a cyclic process of mutation and selection. At the end of affinity maturation, a number of cells sharing the same ancestor (i.e. in the same "clonal family") are released from the germinal center; their amino acid frequency profile reflects the allowed and disallowed substitutions at each position. These clonal-family-specific frequency profiles, called "substitution profiles", are useful for studying the course of affinity maturation as well as for antibody engineering purposes. However, most often only a single sequence is recovered from each clonal family in a sequencing experiment, making it impossible to construct a clonal-family-specific substitution profile. Given the public release of many high-quality large B cell receptor datasets, one may ask whether it is possible to use such data in a prediction model for clonal-family-specific substitution profiles. In this paper, we present the method "Substitution Profiles Using Related Families" (SPURF), a penalized tensor regression framework that integrates information from a rich assemblage of datasets to predict the clonal-family-specific substitution profile for any single input sequence. Using this framework, we show that substitution profiles from similar clonal families can be leveraged together with simulated substitution profiles and germline gene sequence information to improve prediction. We fit this model on a large public dataset and validate the robustness of our approach on two external datasets. Furthermore, we provide a command-line tool in an open-source software package (https://github.com/krdav/SPURF) implementing these ideas and providing easy prediction using our pre-fit models.


Assuntos
Substituição de Aminoácidos/genética , Aminoácidos/metabolismo , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismo , Aminoácidos/genética , Animais , Linfócitos B/química , Linfócitos B/metabolismo , Células Clonais , Biologia Computacional , Bases de Dados de Proteínas , Humanos , Modelos Imunológicos , Receptores de Antígenos de Linfócitos B/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
EMBO Rep ; 19(10)2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30126925

RESUMO

The Myc family of oncogenic transcription factors regulates myriad cellular functions. Myc proteins contain a basic region/helix-loop-helix/leucine zipper domain that mediates DNA binding and heterodimerization with its partner Max. Among the Myc proteins, c-Myc is the most widely expressed and relevant in primary B lymphocytes. There is evidence suggesting that c-Myc can perform some of its functions in the absence of Max in different cellular contexts. However, the functional in vivo interplay between c-Myc and Max during B lymphocyte differentiation is not well understood. Using in vivo and ex vivo models, we show that while c-Myc requires Max in primary B lymphocytes, several key biological processes, such as cell differentiation and DNA replication, can initially progress without the formation of c-Myc/Max heterodimers. We also describe that B lymphocytes lacking Myc, Max, or both show upregulation of signaling pathways associated with the B-cell receptor. These data suggest that c-Myc/Max heterodimers are not essential for the initiation of a subset of important biological processes in B lymphocytes, but are required for fine-tuning the initial response after activation.


Assuntos
Linfócitos B/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Diferenciação Celular/genética , Proteínas Proto-Oncogênicas c-myc/genética , Sequência de Aminoácidos/genética , Animais , Linfócitos B/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Replicação do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Sequências Hélice-Alça-Hélice/genética , Humanos , Zíper de Leucina/genética , Camundongos , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-myc/química , Ativação Transcricional/genética
13.
AIDS ; 32(12): 1571-1578, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-29734222

RESUMO

OBJECTIVE: Recently, a seemingly novel innate immune cell subset bearing features of natural killer and B cells was identified in mice. So-called NKB cells appear as first responders to infections, but whether this cell population is truly novel or is in fact a subpopulation of B cells and exists in higher primates remains unclear. The objective of this study was to identify NKB cells in primates and study the impact of HIV/SIV infections. DESIGN AND METHODS: NKB cells were quantified in both naive and lentivirus infected rhesus macaques and humans by excluding lineage markers (CD3, CD127) and positive Boolean gating for CD20, NKG2A/C and/or NKp46. Additional phenotypic measures were conducted by RNA-probe and traditional flow cytometry. RESULTS: Circulating cytotoxic NKB cells were found at similar frequencies in humans and rhesus macaques (range, 0.01-0.2% of total lymphocytes). NKB cells were notably enriched in spleen (median, 0.4% of lymphocytes), but were otherwise systemically distributed in tonsil, lymph nodes, colon, and jejunum. Expression of immunoglobulin was highly variable, but heavily favoured IgM and IgA rather than IgG. Interestingly, NKB cell frequencies expanded in PBMC and colon during SIV infection, as did IgG expression, but were generally unaltered in HIV-infected humans. CONCLUSION: These results suggest a cell type expressing both natural killer and B-cell features exists in rhesus macaques and humans and are perturbed by HIV/SIV infection. The full functional niche remains unknown, but the unique phenotype and systemic distribution could make NKB cells unique targets for immunotherapeutics or vaccine strategies.


Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Trato Gastrointestinal/patologia , Infecções por HIV/patologia , Receptores de Células Matadoras Naturais/análise , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Adulto , Animais , Subpopulações de Linfócitos B/química , Linfócitos B/química , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina M/metabolismo , Contagem de Linfócitos , Macaca mulatta , Pessoa de Meia-Idade
15.
J Cell Biol ; 217(7): 2565-2582, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29685902

RESUMO

B lymphocytes use B cell receptors (BCRs) to sense the chemical and physical features of antigens. The activation of isotype-switched IgG-BCR by mechanical force exhibits a distinct sensitivity and threshold in comparison with IgM-BCR. However, molecular mechanisms governing these differences remain to be identified. In this study, we report that the low threshold of IgG-BCR activation by mechanical force is highly dependent on tethering of the cytoplasmic tail of the IgG-BCR heavy chain (IgG-tail) to the plasma membrane. Mechanistically, we show that the positively charged residues in the IgG-tail play a crucial role by highly enriching phosphatidylinositol (4,5)-biphosphate (PI(4,5)P2) into the membrane microdomains of IgG-BCRs. Indeed, manipulating the amounts of PI(4,5)P2 within IgG-BCR membrane microdomains significantly altered the threshold and sensitivity of IgG-BCR activation. Our results reveal a lipid-dependent mechanism for determining the threshold of IgG-BCR activation by mechanical force.


Assuntos
Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Fenômenos Mecânicos , Receptores de IgG/imunologia , Animais , Linfócitos B/química , Membrana Celular/química , Membrana Celular/imunologia , Humanos , Camundongos , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de IgG/química , Transdução de Sinais/imunologia
16.
Methods Mol Biol ; 1707: 69-80, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29388100

RESUMO

The production of antibodies requires the expansion and selection of high-affinity B cell clones. This process is initiated by antigen uptake through the B cell receptor (BCR), which recognizes and binds antigen displayed on the surface of an antigen-presenting cell (APC). To acquire the antigen, B cells use myosin contractility to physically pull BCR-antigen clusters from the APC membrane. These mechanical forces influence association and dissociation rates of BCR-antigen bonds, resulting in affinity-dependent acquisition of antigen by B cells. Mechanical regulation of B cell antigen acquisition from APCs remains poorly understood, although the recent development of DNA-based force sensors has enabled the measurement of mechanical forces generated in B cell-APC contacts. In this chapter, we describe a protocol to design, synthesize, and purify DNA-based force sensors to measure B cell antigen extraction forces using fluorescence microscopy.


Assuntos
Antígenos/química , Linfócitos B/química , Comunicação Celular , Sinapses Imunológicas/química , Receptores de Antígenos de Linfócitos B/química , Estresse Mecânico , Animais , Antígenos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Humanos , Sinapses Imunológicas/imunologia , Receptores de Antígenos de Linfócitos B/imunologia
17.
Methods Mol Biol ; 1707: 183-192, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29388108

RESUMO

Single-particle tracking has been used extensively to advance our understanding of the plasma membrane and the mechanisms controlling the movement of cell surface proteins. These studies provide fundamental insights into the regulation of membrane receptor activation and the assembly of signaling clusters. Here, we describe a method to label and track B cell receptor (BCR) and other cell surface proteins and how this method can be adapted to simultaneously track two molecular species or examine the movement of membrane proteins in relation to membrane microdomains. We recently used this method to study the role of the actin cytoskeleton in the regulation of B cell receptor dynamics at the cell surface.


Assuntos
Linfócitos B/imunologia , Microdomínios da Membrana/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Imagem Individual de Molécula/métodos , Animais , Linfócitos B/química , Linfócitos B/citologia , Humanos , Microdomínios da Membrana/química , Transporte Proteico/imunologia , Receptores de Antígenos de Linfócitos B/química
18.
Methods Mol Biol ; 1707: 235-241, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29388112

RESUMO

B-lymphocytes have the ability to repair their plasma membranes following injury, such as by bacterial cholesterol-dependent cytolysins. The repair process includes the removal of the pore from the inflicted region of the plasma membrane via lipid raft-mediated internalization. Lipid rafts are critical for B cell receptor (BCR) activation. Cholesterol-dependent pore forming bacterial toxins provide a useful tool for examining the role of lipid rafts in B cell activation and the underlying cellular mechanisms. This method serves as a great alternative of known cholesterol disruption reagents such as filipin, nystatin, and methyl-ß-cyclodextrin. Here, we describe a method of damaging primary murine B cell plasma membranes with the Streptococcus pyogenes cytolysin, Streptolysin O (SLO), and monitoring levels of damage, repair and BCR activation.


Assuntos
Linfócitos B/química , Colesterol/química , Ativação Linfocitária , Microdomínios da Membrana/química , Receptores de Antígenos de Linfócitos B/química , Streptococcus pyogenes/química , Estreptolisinas/química , Animais , Linfócitos B/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Colesterol/imunologia , Humanos , Microdomínios da Membrana/imunologia , Camundongos , Receptores de Antígenos de Linfócitos B/imunologia , Streptococcus pyogenes/imunologia , Estreptolisinas/imunologia
19.
Artigo em Inglês | MEDLINE | ID: mdl-29412869

RESUMO

Guanine-quadruplexes (G4) are stable tetra-stranded DNA structures that may cause DNA replication stress and inhibit gene expression. Defects in unwinding these structures by DNA helicases may result in telomere shortening and DNA damage. Furthermore, due to mutations in WRN helicase genes in Werner syndrome, G4 motifs are likely to be key elements in the expression of premature aging phenotypes. The methylation of DNA plays a significant role in the stability and occurrence of G4. Thus, G4 frequency and DNA methylation mechanisms may be affected by excesses or deficiencies in methyl donors such as folate. B-Lymphocytes from Werner patients (n = 5) and healthy individuals (n = 5) were cultured in RPMI medium under condition of folate deficiency (20 nM) or sufficiency (200 nM) for 14 days. Cells were fixed on microscope slides for immunofluorescent staining to measure G4 frequency and γH2AX (a marker of DNA strand breaks) intensity, using automated quantitative imaging fluorescent microscopy. There was a significant increase (p < 0.05) in G4 levels in Werner syndrome patients compared to healthy controls. Werner and control cells grown in 20 nM folate media also showed significant increases in G4 (p < 0.001) and γH2AX (p < 0.01) signals compared with the same cells grown in 200 nM folate. Control cells grown in 20 nM folate also showed a significant reduction in DNA methylation levels (P < 0.05). The results of this study suggest that the occurrence of DNA G4 structures can be modulated in vitro via nutrients with important roles in methylation.


Assuntos
Linfócitos B/citologia , DNA/química , Ácido Fólico/farmacologia , Síndrome de Werner/genética , Adulto , Linfócitos B/química , Linfócitos B/efeitos dos fármacos , Estudos de Casos e Controles , Células Cultivadas , DNA/efeitos dos fármacos , Dano ao DNA , Metilação de DNA/efeitos dos fármacos , Feminino , Quadruplex G/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome de Werner/sangue , Síndrome de Werner/metabolismo
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