Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.323
Filtrar
1.
Fish Shellfish Immunol ; 93: 612-622, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31408730

RESUMO

In teleost fish, IgM+ B cells play important roles in innate and adaptive immunity. Different IgM+ B cells are detected in teleost, named IgMlo and IgMhi B cell subsets, according to the distinct expression levels of membrane IgM (mIgM). However, the study on the heterogeneity in IgM+ B cell subsets remains poorly understood. In this study, the comparative transcriptomic profiles of IgM-, IgMlo and IgMhi from peripheral blood of Nile tilapia (Oreochromis niloticus) were carried out by using RNA-sequencing technique. A total of 6045 and 5470 differentially expressed genes (DEGs) were detected in IgMlo and IgMhi cells, respectively, as compared with IgM- lymphocytes, whereas 3835 genes were differentially expressed when IgMlo compared to IgMhi cells. Analysis of the KEGG database indicated that the DEGs were enriched in immune system categories and signaling transduction and interaction in IgM- vs IgMhi, IgM- vs IgMlo and IgMlo vs IgMhi. Comparatively, in IgMlo vs IgMhi, GO enrichment analysis indicated that the DEGs enriched in nucleic acid binding transcription factor activity. Analysis of crucial transcription factors for B cell differentiation indicated that IgMlo and IgMhi cell clusters belonged to the different B cell subsets. The data generated in this study may provide insights into understanding the heterogeneity of IgM+ cells in teleost, and suggest that IgM+ B cells play a crucial role in innate immunity.


Assuntos
Ciclídeos/genética , Ciclídeos/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Imunidade Inata/genética , Imunoglobulina M/imunologia , Transcrição Genética/imunologia , Animais , Perfilação da Expressão Gênica/veterinária , Imunoglobulina M/genética , Leucócitos/imunologia , /veterinária
2.
Curr Med Sci ; 39(3): 371-378, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31209805

RESUMO

Clinical trials have shown beneficial effects of probiotics on inflammatory bowel diseases (IBD), although the exact mechanism remains unknown. VSL#3, a mixture of 8 probiotic bacteria, has been confirmed to have adjunctive therapeutic effects on colitis. T follicular helper (Tfh) cells, a new separate subset of CD4+ T helper cells, have been proved to play a vital role in autoimmunity. The present study aimed to identify the beneficial effect of the probiotic mixture VSL#3 on the mouse model of colitis by regulating Tfh cells. Dextran sulfate sodium (DSS) was used to induce chronic colitis in C57BL/6 mice. VSL#3 (3×109 live bacteria) was given to C57BL/6 mice every other day for 60 days by gavage. The disease activity index (DAI), histological activity index (HAI), colon length and myeloperoxidase (MPO) activity were detected. Immunofluorescence was used to visualize the location of Tfh cells. Immunoglobulins, Tfh cells and plasma cells were quantified by enzyme-linked immunosorbent assay (ELISA), flow cytometry, real-time PCR or Western blotting. The results showed that after DSS treatment, the humoral immunity was disordered in C57BL/6 mice, with increased IgM, IgG and IgA levels in colonic mucus and increased Tfh cells in mesenteric lymph nodes (MLN). VSL#3 treatment showed anti-inflammatory effects as evidenced by reduced DAI score, HAI score and MPO activity. IgM, IgG and IgA levels were significantly reduced in colon mucus, and the number of Tfh cells was markedly decreased in MLN after VSL#3 treatment. It was concluded that VSL#3 alleviates DSS-induced colitis by downregulating Tfh cells, and Tfh cells may become a potential therapeutic target for IBD.


Assuntos
Colite/dietoterapia , Colo/imunologia , Linfonodos/imunologia , Probióticos/farmacologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Doença Crônica , Colite/genética , Colite/imunologia , Colite/patologia , Colo/patologia , Sulfato de Dextrana , Expressão Gênica , Imunidade Humoral , Imunoglobulina A/genética , Imunoglobulina G/genética , Imunoglobulina M/genética , Linfonodos/patologia , Contagem de Linfócitos , Masculino , Mesentério/imunologia , Mesentério/patologia , Camundongos , Camundongos Endogâmicos C57BL , Peroxidase/genética , Peroxidase/imunologia , Linfócitos T Auxiliares-Indutores/patologia , Resultado do Tratamento
3.
Mol Immunol ; 112: 59-71, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31078117

RESUMO

B-cell survival depends on signals induced by binding of B-cell activating factor (BAFF) to its receptor (BAFF-R). In this study, the full-length cDNAs of cat BAFF (cBAFF) and BAFF-R (cBAFF-R) were amplified from the spleen by reverse transcription PCR. The open reading frame of cBAFF cDNA encodes a protein of 285 amino acids containing a predicted transmembrane domain and a furin protease cleavage site, similar to mammalian, avian, and reptile BAFFs. The cBAFF-R gene encodes a 189 amino acid protein. Real-time quantitative PCR analyses revealed that the two genes are predominantly expressed in the spleen. csBAFF, EGFP/csBAFF, and cBAFF-R were efficiently expressed in Escherichia coli BL21 (DE3), as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analyses. After purification, the EGFP/csBAFF fusion protein showed a fluorescence spectrum similar to that of EGFP. Confocal laser scanning microscopy showed that EGFP/csBAFF bound to its receptor. In vitro, csBAFF promoted the survival of cat and mouse splenic B cells with/without a priming agent (Staphylococcus aureus Cowan 1, SAC) or anti-mouse IgM. Furthermore, it stimulated the survival of mouse B cells, similar to msBAFF. Recombinant cBAFF-R blocked the function of sBAFF in vitro. These findings indicate that csBAFF plays an important role in the survival of cat B cells and has functional cross reactivity between cats and other mammals, and suggest a role for the BAFF-BAFF-R system in regulating B-cell survival. Therefore, BAFF and BAFF-R show promise for enhancing the immune systems of animals.


Assuntos
Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/genética , Linfócitos B/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Gatos , Sobrevivência Celular/genética , Clonagem Molecular , Reações Cruzadas/genética , DNA Complementar/genética , Escherichia coli/genética , Imunoglobulina M/genética , Mamíferos/genética , Camundongos , Estrutura Molecular , Fases de Leitura Aberta/genética , Proteínas Recombinantes/genética , Baço/metabolismo , Staphylococcus aureus/genética
4.
Protein Pept Lett ; 26(5): 348-356, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30816077

RESUMO

BACKGROUND: The Bursa of Fabricius is an acknowledged central humoral immune organ unique to birds, which provides an ideal research model on the immature B cell development. OBJECTIVE: In this article, our motivation is to study the role on sIgM and establish the molecular basis and functional processes of Bursal Hexapeptide (BHP) in avian immature B cells DT40 cell lines. METHODS: In this article, we detected the expressions of sIgM mRNA with qPCR in DT40 cells with BHP treatment, and investigated the gene expression profiles of BHP-treated DT40 cells, employing microarray analyses. Also, to validate the differentially expressed genes, we performed KEGG pathway and Gene Ontology analysis in the BHP-treated DT40 cells. Finally, we comparatively analyzed the similar regulated genes and their involved immune functional processes between DT40 cell and mouse immature B cell line WEHI231 cell with BHP treatment. RESULTS: Following the proposed framework, we proved that the BHP enhanced the mRNA expression levels of IgM in DT40 cells, and induced 460 upregulated genes and 460 downregulated genes in BHP-treated DT40 cells. The pathway analysis showed that the differentially regulated genes in DT40 cell line with BHP treatment were involved in 12 enrichment pathways, in which Toll-like receptor signaling pathway was the vital pathways, and cytokine-cytokine receptor interaction and Jak-STAT signaling pathway were another two important pathways in BHP-treated DT40 cells. Moreover, BHP induced the immune related biological processes in BHP-treated DT40 cells, including T cell related, cytokine related, lymphocyte related, and innate immune response GO terms. Finally, the comparatively analysis showed that there were two downregulated genes GATA3 and IFNG to be found co-existed among the differentially expressed genes in BHP-treated DT40 cell and WEHI231 cells, which shared some same immune related functional processes in both cell lines. CONCLUSION: After the applying the framework, we proved the inducing roles and the gene expression profiles of BHP on avian immature B cells, and verified some molecular basis from the KEGG and GO analysis. These results provided the insight for mechanism on immature B cell differentiation, and offer the essential direction for the vaccine improvement.


Assuntos
Oligopeptídeos/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Animais , Bolsa de Fabricius , Linhagem Celular , Galinhas , Imunidade Inata , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Camundongos , Oligopeptídeos/farmacologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/imunologia , RNA Mensageiro/metabolismo , Transdução de Sinais , Especificidade da Espécie , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo
5.
Fish Shellfish Immunol ; 88: 266-271, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30849499

RESUMO

The normal microbiota plays a key role in the health of host, but little is known of how the fish immune system recognizes and responds to indigenous bacteria/probiotics. Our previous studies have showed that heat-inactivated indigenous Bacillus pumilus SE5 activate the TLR2 signaling pathways and modulate the intestinal microbiota in grouper (Epinephelus coioides), suggesting microbial-associated molecular patterns (MAMPs) involved. In this study, whole cell wall (CW) and two possible MAMPs, peptidoglycan (PG) and lipoteichoic acid (LTA) have been extracted from B. pumilus SE5 and their effects on intestinal immune related genes expression and microbiota were evaluated in a 60 days feeding trial. Significantly elevated expression of TLR1, TLR2, TLR5 and MyD88 was observed in fish fed the CW, PG and LTA containing diets, and the highest expression was observed in groups PG and LTA. At the same time, significantly upregulated expression of antimicrobial effectors, such as antimicrobial peptides (epinecidin-1, hepcidin-1 and ß-defensin), C-type Lectin and IgM was observed in fish fed PG and LTA containing diets. This induced activation of intestinal immunity was consistent with the microbiota data showing that CW, PG and LTA originated from SE5 modulated the overall structure of intestinal microbiota, and the relative abundance of potentially pathogenic Vibrio decreased significantly while beneficial Lactobacillus increased significantly in fish fed PG and LTA. In conclusion, both the PG and LTA originated from B. pumilus SE5 could activate TLRs/MyD88 signaling and expression of wide-ranging antibacterial effectors, and therefore shape the intestinal microbiota in grouper.


Assuntos
Bacillus pumilus/química , Bass/imunologia , Bass/microbiologia , Microbioma Gastrointestinal , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bass/genética , Bass/metabolismo , Parede Celular , Expressão Gênica , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/imunologia , Intestinos/microbiologia , Lactobacillus , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Lipopolissacarídeos/farmacologia , Peptidoglicano/farmacologia , Ácidos Teicoicos/farmacologia , Vibrio
6.
Fish Shellfish Immunol ; 87: 650-658, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30753920

RESUMO

The serum IgM concentration of ballan wrasse is relatively high, estimated to approximately 13 mg/ml in adult wild fish of 800 g. The present study revealed an unusual high abundance of IgM mRNA in the gut of ballan wrasse. Initially, transcripts encoding IgM, IgT, IgD, TCRα, TCRδ and CD3ε were quantified by RT-qPCR in several tissues of wild caught fish (approx. 800 g), indicating an elevated immune activity in hindgut and an extraordinarily high expression of IgM. Subsequently, a new RT-qPCR analysis was performed on the entire intestine, cut into four different segments, of reared fish (32-100 g). The analysis indicated immune activity along the entire intestine, but not as strong as in the hindgut. Furthermore, similar to the larger fish, the relative abundance of IgM transcripts was higher in the hindgut than in kidney and spleen, although the absolute level of IgM was in general higher in the larger fish. The secreted form of IgM was completely dominant in comparison to the membrane bound form of IgM and the other analysed genes. IgM was purified from gut mucus and external mucosal surfaces by magnetic beads coated with protein A. Mucus IgM reacted with rabbit antisera raised against serum IgM and contained subunits of the same size. Regarding the elevated immune activity in the intestine it is tempting to speculate on a possible compensatory strategy in this lineage of stomach-less fish, and that natural antibodies have an important role in the first line defence.


Assuntos
Imunidade Adaptativa/genética , Proteínas de Peixes/genética , Imunoglobulinas/genética , Perciformes/genética , Perciformes/imunologia , Receptores de Antígenos de Linfócitos T/genética , Animais , Proteínas de Peixes/imunologia , Trato Gastrointestinal/metabolismo , Perfilação da Expressão Gênica/veterinária , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Imunoglobulinas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Transcrição Genética/imunologia
7.
PLoS One ; 14(1): e0209860, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30608947

RESUMO

Immuno-PCR (iPCR) is one of the methods used for the detection of a wide range of analytes and features the high sensitivity of the polymerase chain reaction (PCR) method. iPCR uses antibodies coupled to DNA, followed by the amplification of the attached DNA using RT-PCR. Two major types of antibody-DNA conjugates are currently used, which are obtained as a result of non-covalent (biotin-streptavidin) or covalent interactions. Using a strain-promoted azide-alkyne cycloaddition (SPAAC), we synthesized covalent DNA-antibody conjugates, optimized the reaction conditions, and developed an efficient protocol for the purification of conjugates, with which all unreacted antibodies and oligonucleotides are separated. Covalent DNA-antibody conjugates were tested with iPCR assays that were previously developed for the detection of IgE and IgM antibodies with the use of the supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin. The results show that the modification of antibodies with amino groups did not allow us to obtain monolabeled antibodies or antibodies with a strictly defined number of DNA-labels. The degree of labeling determined by the dyes introduced through the azido group reflects the actual labeling degree statistically. If the average labeling degree for azido groups is 1.1, the conjugates contain 25% mono-labeled antibodies, 50% double-labeled antibodies, and 25% unlabeled ones. The specificity of the monoclonal antibody to human IgE (BE5) changed after conjugation with the oligonucleotide. The sensitivity of iPCR in the detection of IgM antibodies produced against the LeC disaccharide using a covalent conjugate was similar to that of a supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin, but the new procedure is two steps shorter.


Assuntos
Imunoglobulina E/genética , Imunoglobulina M/genética , Reação em Cadeia da Polimerase/métodos , Animais , Anticorpos Monoclonais/genética , Antígenos/imunologia , Biotina , Biotinilação , DNA/genética , Humanos , Imunoglobulina E/análise , Imunoglobulina M/análise , Camundongos , Oligonucleotídeos/genética , Sensibilidade e Especificidade , Estreptavidina
8.
Fish Shellfish Immunol ; 87: 275-285, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30668998

RESUMO

In this study, for better understanding the humoral immunity of rock bream (Oplegnathus fasciatus), 2 transcripts of immunoglobulin M (IgM) heavy chain gene including membrane bound (m-IgM) and secretory (s-IgM) forms were sequenced and analyzed their tissue distribution and differential expression in rock bream under rock bream iridovirus (RBIV) infection and vaccination since RBIV has caused mass mortality in rock bream aquaculture in Korea. Consequently, s-IgM cDNA was 1902 bp in length encoding a leader region, a variable region, four constant regions (CH1, CH2, CH3, CH4) and a C-terminal region while m-IgM cDNA was 1689 bp in length encoding shorter three constant regions (CH1, CH2, CH3) and two transmembrane regions. The predicted s-IgM and m-IgM represent a high structural similarity to other species including human. In tissue distribution analysis in healthy fish, the highest expression of s-IgM was observed in head kidney followed by body kidney, spleen, and mid gut whereas m-IgM expression was the highest in blood followed by head kidney and spleen. In vitro, s-IgM expression was up-regulated by LPS in head kidney and spleen cells at 24 h with no change of m-IgM expression. In vivo upon vaccination, s-IgM expression was up-regulated in liver and blood but not in head kidney while m-IgM expression was only up-regulated in head kidney. After challenge with RBIV, s-IgM expression level was higher in vaccinated fish than in unvaccinated fish and m-IgM expression was up-regulated in head kidney of vaccinated group. In conclusion, differential expression of m-IgM and s-IgM may indicate their differential functions to produce the most effective IgM during adaptive immune response. Although it is not able to assess specific IgM at protein level due to a lack of antibody against rock bream IgM, the present study on s-IgM and m-IgM gene expressions upon infection and vaccination will be useful in developing efficient vaccines in the future.


Assuntos
Imunidade Adaptativa/genética , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Vírus de DNA/prevenção & controle , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Imunoglobulina M/química , Iridoviridae/imunologia , Filogenia , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Alinhamento de Sequência/veterinária , Vacinação/veterinária , Vacinas Virais/imunologia
10.
Fish Shellfish Immunol ; 84: 233-243, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30300742

RESUMO

Three different immunoglobulin (Ig) isotypes, namely IgM, IgD, and IgT/IgZ have been described in most teleost, among which IgM and IgT are considered crucial in systematic and mucosal immunity, respectively. However, some teleost have no IgT/IgZ and it is unclear how other Ig isotypes interact to perform immune-protective roles in both systematic and mucosal sites. In this study, the complete cDNA sequences of IgM and IgD heavy chains were cloned and analyzed from yellow catfish (Pelteobagrus fulvidraco). The full-length cDNA of Pf-IgM and Pf-IgD heavy chains contained an open reading frame (ORF) of 1710 and 2991 bp encoding a predicted protein of 570 and 997 amino acids, respectively. Tissue-specific expression analysis indicated that both IgM and IgD were highly expressed in kidney and spleen, and higher expression levels were found at zygote and 13th day post hatching during early development. Multiple sequence alignment and phylogenetic analysis showed IgM and IgD of yellow catfish are closely related to other fish of Siluriformes. Moreover, we also constructed the infection model of yellow catfish with bacteria (Flavobacterium columnare G4) for the first time to study the function of Pf-IgM and Pf-IgD heavy chain genes in immune response. Quantitative real-time PCR (qRT-PCR) showed that significantly up-regulated expression of Pf-IgM was not only detected in liver and spleen, but also in mucosal tissues including skin and intestine, while Pf-IgD was just significantly increased in liver and spleen, which might suggest the main immune-protecting roles of IgM in mucosal tissues of yellow catfish.


Assuntos
Peixes-Gato/genética , Peixes-Gato/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Infecções por Flavobacteriaceae/imunologia , Flavobacterium/fisiologia , Perfilação da Expressão Gênica/veterinária , Imunoglobulina D/genética , Imunoglobulina D/imunologia , Imunoglobulina M/química , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Filogenia , Alinhamento de Sequência/veterinária
11.
Biochem Biophys Res Commun ; 509(2): 435-440, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30594398

RESUMO

We recently found that the membrane-bound receptor activator of NF-κB ligand (RANKL) on osteoblasts works as a receptor to stimulate osteoblast differentiation, however, the reason why the RANKL-binding molecules stimulate osteoblast differentiation has not been well clarified. Since the induction of cell-surface receptor clustering is known to lead to cell activation, we hypothesized that the induction of membrane-RANKL clustering on osteoblasts might stimulate osteoblast differentiation. Immunoblotting showed that the amount of RANKL on the membrane was increased by the RANKL-binding peptide OP3-4, but not by osteoprotegerin (OPG), the other RANKL-binding molecule, in Gfp-Rankl-transfected ST2 cells. Observation under a high-speed atomic force microscope (HS-AFM) revealed that RANKL molecules have the ability to form clusters. The induction of membrane-RANKL-OPG-Fc complex clustering by the addition of IgM in Gfp-Rankl-transfected ST2 cells could enhance the expression of early markers of osteoblast differentiation to the same extent as OP3-4, while OPG-Fc alone could not. These results suggest that the clustering-formation of membrane-RANKL on osteoblasts could stimulate early osteoblast differentiation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Oligopeptídeos/farmacologia , Osteoblastos/efeitos dos fármacos , Peptidomiméticos/farmacologia , Ligante RANK/genética , Animais , Sítios de Ligação , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Camundongos , Microscopia de Força Atômica , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Ligação Proteica , Ligante RANK/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Fatores de Tempo
12.
J Immunol ; 201(12): 3492-3496, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30420436

RESUMO

Generation of neoantigens by citrullination is implicated in the production of anti-citrullinated protein Abs in rheumatoid arthritis, but citrullination is also a physiological process. To verify whether citrullin-specific B cells are immunologically ignorant or tolerant in normal conditions, transgenic (Tg) mice expressing IgM with the V region of an anti-cyclic citrullinated peptide (CCP) mAb cloned from a rheumatoid arthritis patient were generated. CCP-specific B cells developed in the anti-CCP IgM Tg mice with an alteration of bone marrow B cell fractions, and the number of mature B cells decreased compared with wild-type or the control anti-influenza nucleoprotein-specific IgM Tg mice. In addition, B cells in anti-CCP IgM Tg mice are functionally anergic. Thus, tolerance is induced in CCP-specific B cells in vivo, suggesting that the immune systems are naturally exposed to citrullinated Ags, and anti-CCP Ab production requires additional steps beyond the generation of neoantigens by citrullination.


Assuntos
Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Imunoglobulina M/metabolismo , Peptídeos Cíclicos/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Anticorpos Monoclonais/genética , Células Cultivadas , Anergia Clonal , Humanos , Tolerância Imunológica , Imunoglobulina M/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos B/genética
13.
Eur J Immunol ; 48(12): 2068-2071, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30315705

RESUMO

The IgMi mouse fails to secrete antibodies or class switch its BCR from IgM. Our study reveals that other cellular compartments, including B-cell subsets, DC subsets, GC B cells and TFH cells are perturbed in the IgMi mouse, thus presenting important additional considerations when using the mouse to explore the role of secreted antibody.


Assuntos
Anticorpos/metabolismo , Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Células Dendríticas/imunologia , Centro Germinativo/imunologia , Imunoglobulina M/genética , Linfócitos T/imunologia , Animais , Anticorpos/genética , Formação de Anticorpos/genética , Diferenciação Celular , Switching de Imunoglobulina/genética , Imunoglobulina M/metabolismo , Ativação Linfocitária , Proteínas de Membrana/metabolismo , Camundongos
14.
Front Immunol ; 9: 2175, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30333823

RESUMO

The precise impact of the somatotrope axis upon the immune system is still highly debated. We have previously shown that mice with generalized ablation of growth hormone (GH) releasing hormone (GHRH) gene (Ghrh -/-) have normal thymus and T-cell development, but present a marked spleen atrophy and B-cell lymphopenia. Therefore, in this paper we have investigated vaccinal and anti-infectious responses of Ghrh -/- mice against S. pneumoniae, a pathogen carrying T-independent antigens. Ghrh -/- mice were unable to trigger production of specific IgM after vaccination with either native pneumococcal polysaccharides (PPS, PPV23) or protein-PPS conjugate (PCV13). GH supplementation of Ghrh -/- mice restored IgM response to PPV23 vaccine but not to PCV13 suggesting that GH could exert a specific impact on the spleen marginal zone that is strongly implicated in T-independent response against pneumococcal polysaccharides. As expected, after administration of low dose of S. pneumoniae, wild type (WT) completely cleared bacteria after 24 h. In marked contrast, Ghrh -/- mice exhibited a dramatic susceptibility to S. pneumoniae infection with a time-dependent increase in lung bacterial load and a lethal bacteraemia already after 24 h. Lungs of infected Ghrh -/- mice were massively infiltrated by inflammatory macrophages and neutrophils, while lung B cells were markedly decreased. The inflammatory transcripts signature was significantly elevated in Ghrh -/- mice. In this animal model, the somatotrope GHRH/GH/IGF1 axis plays a vital and unsuspected role in vaccine and immunological defense against S. pneumoniae.


Assuntos
Linfócitos B/imunologia , Hormônio Liberador de Hormônio do Crescimento/imunologia , Hormônio do Crescimento/deficiência , Vacinas Pneumocócicas/imunologia , Transdução de Sinais/imunologia , Streptococcus pneumoniae/imunologia , Animais , Linfócitos B/patologia , Hormônio do Crescimento/imunologia , Hormônio Liberador de Hormônio do Crescimento/genética , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/imunologia , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Transdução de Sinais/genética
16.
Front Immunol ; 9: 2115, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30319606

RESUMO

Vaccination induces "public" antibody clonotypes common to all individuals of a species, that may mediate universal protection against pathogens. Only few studies tried to trace back the origin of these public B-cell clones. Here we used Illumina sequencing and computational modeling to unveil the mechanisms shaping the structure of the fish memory antibody response against an attenuated Viral Hemorrhagic Septicemia rhabdovirus. After vaccination, a persistent memory response with a public VH5JH5 IgM component was composed of dominant antibodies shared among all individuals. The rearrangement model showed that these public junctions occurred with high probability indicating that they were already favored before vaccination due to the recombination process, as shown in mammals. In addition, these clonotypes were in the naïve repertoire associated with larger similarity classes, composed of junctions differing only at one or two positions by amino acids with comparable properties. The model showed that this property was due to selective processes exerted between the recombination and the naive repertoire. Finally, our results showed that public clonotypes greatly expanded after vaccination displayed several VDJ junctions differing only by one or two amino acids with similar properties, highlighting a convergent response. The fish public memory antibody response to a virus is therefore shaped at three levels: by recombination biases, by selection acting on the formation of the pre-vaccination repertoire, and by convergent selection of functionally similar clonotypes during the response. We also show that naive repertoires of IgM and IgT have different structures and sharing between individuals, due to selection biases. In sum, our comparative approach identifies three conserved features of the antibody repertoire associated with public memory responses. These features were already present in the last common ancestors of fish and mammals, while other characteristics may represent species-specific solutions.


Assuntos
Linfócitos B/imunologia , Peixes/imunologia , Septicemia Hemorrágica Viral/prevenção & controle , Novirhabdovirus/imunologia , Vacinas Virais/imunologia , Animais , Linfócitos B/metabolismo , Células Clonais/imunologia , Células Clonais/metabolismo , Septicemia Hemorrágica Viral/imunologia , Septicemia Hemorrágica Viral/virologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Memória Imunológica/imunologia , Recombinação V(D)J/imunologia , Vacinação , Vacinas Virais/administração & dosagem
17.
Front Immunol ; 9: 2194, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30319643

RESUMO

The exploitation of various human immunodeficiency virus type-1 (HIV-1) vaccines has posed great challenges for the researchers in precisely evaluating the vaccine-induced immune responses, however, the understanding of vaccination response suffers from the lack of unbiased characterization of the immune landscape. The rapid development of high throughput sequencing (HTS) makes it possible to scrutinize the extremely complicated immunological responses during vaccination. In the current study, three vaccines, namely N36, N51, and 5-Helix based on the HIV-1 gp41 pre-hairpin fusion intermediate were applied in rhesus macaques. We assessed the longitudinal vaccine responses using HTS, which delineated the evolutionary features of both T cell and B cell receptor repertoires with extreme diversities. Upon vaccination, we unexpectedly found significant discrepancies in the landscapes of T-cell and B-cell repertoires, together with the detection of significant class switching and the lineage expansion of the B cell receptor or immunoglobulin heavy chain (IGH) repertoire. The vaccine-induced expansions of lineages were further evaluated for mutation rate, lineage abundance, and lineage size features in their IGH repertoires. Collectively, these findings conclude that the N51 vaccine displayed superior performance in inducing the class-switch of B cell isotypes and promoting mutations of IgM B cells. In addition, the systematic HTS analysis of the immune repertoires demonstrates its wide applicability in enhancing the understanding of immunologic changes during pathogen challenge, and will guide the development, evaluation, and exploitation of new generation of diagnostic markers, immunotherapies, and vaccine strategies.


Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos B/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Linfócitos T/imunologia , Vacinas contra a AIDS/administração & dosagem , Animais , Linfócitos B/metabolismo , Modelos Animais de Doenças , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunogenicidade da Vacina , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Macaca mulatta , Masculino , Mutação , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos T/metabolismo
18.
Mol Immunol ; 103: 1-6, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30172112

RESUMO

Sequencing of immunoglobulin germline gene loci is a challenging process, e.g. due to their repetitiveness and complexity, hence limiting the insight in the germline gene repertoire of humans and other species. Through next generation sequencing technology, it is possible to generate immunoglobulin transcript data sets large enough to computationally infer the germline genes from which the transcripts originate. Multiple tools for such inference have been developed and they can be used for construction of individual germline gene databases, and for discovery of new immunoglobulin germline genes and alleles. However, there are challenges associated with these methods, many of them related to the biological process through which immunoglobulin coding genes are generated. The junctional diversity introduced during rearrangement of the immunoglobulin heavy chain variable (IGHV), diversity and joining genes specifically complicates the inference of the junction regions, with implications for inference of the 3'-end of IGHV genes. With the aim of coping with such diversity, an inference software package may not be able to identify novel alleles harbouring a difference in these regions compared to their closest relatives in the starting database. In this study, we were able to computationally infer one such previously uncharacterized allele, IGHV3-7*02 A318G. However, this was possible only if a strategy was used in which different variants of IGHV3-7*02 were included in the inference-initiating database. Importantly, the presence of the novel allele, but not the standard IGHV3-7*02 sequence, in the genotype was strongly supported by the actual sequences that were assigned to the allele. We thus showed that the starting database used will impact the germline gene inference process, and that difference in the 3'-end of IGHV genes may remain undetected unless specific, non-standard procedures are used to address this matter. We suggest that inferred genes/alleles should be confirmed e.g. by examination of the nucleotide composition of the 3'-bases of the inference-supporting sequence reads.


Assuntos
Biologia Computacional/métodos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Família Multigênica , Alelos , Sequência de Bases , Rearranjo Gênico , Genes de Imunoglobulinas/genética , Genótipo , Células Germinativas/imunologia , Células Germinativas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Imunoglobulina M/genética , Região Variável de Imunoglobulina/química , Homologia de Sequência do Ácido Nucleico
19.
BMC Genomics ; 19(1): 694, 2018 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-30241501

RESUMO

BACKGROUND: Natural antibodies (NAb) are an important component of the innate immune system, and fight infections as a part of the first line defence. NAb are poly-reactive and can respond non-specifically to antigens. Therefore, NAb may be a key trait when evaluating an animal's potential natural disease resistance. Variation in NAb is caused by both genetic and environmental factors. In this study genetic parameters of NAb were estimated and a genome-wide association study (GWAS) was performed to gain further understanding on the genes that are responsible for the observed genetic variation of NAb in Canadian Holsteins. RESULTS: In total, blood samples of 1327 cows from 64 farms were studied. NAb binding to keyhole limpet hemocyanin (KLH) were determined via indirect ELISA. Immunoglobulin (Ig) isotypes, IgG and IgM, were evaluated. From the sample population, 925 cows were genotyped for 45,187 markers and each individual marker was tested to detect genetic variation in NAb levels. The relationships among animals was accounted for with genomic relationship. Results show heritabilities of 0.27 ± 0.064 (IgG) and 0.31 ± 0.065 (IgM). In total, 23 SNPs were found to be associated with IgG, but no SNPs were associated with IgM (FDR p-value < 0.05). The significant SNPs were located on autosomal chromosomes 1, 20 and 21 of the cow genome. Functional annotation analysis of the positional candidate genes revealed two sets of genes with biologically relevant functions related to NAb. In one set, seven genes with crucial roles in the production of antibody in B cells were associated with the trafficking of vesicles inside the cells between organelles. In the second set, two genes among positional candidate genes were associated with isotype class-switching and somatic hypermutation of B cells. CONCLUSIONS: This study demonstrated the possibility of increasing NAb through selective breeding. In addition, the effects of two candidate pathways are proposed for further investigation of NAb production in Holsteins.


Assuntos
Anticorpos/sangue , Bovinos/genética , Estudo de Associação Genômica Ampla/métodos , Imunoglobulina G/genética , Imunoglobulina M/genética , Polimorfismo de Nucleotídeo Único , Animais , Anticorpos/genética , Anticorpos/imunologia , Canadá , Bovinos/sangue , Feminino , Genótipo , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino
20.
Proc Natl Acad Sci U S A ; 115(41): E9630-E9639, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30257949

RESUMO

Plasma cell differentiation involves coordinated changes in gene expression and functional properties of B cells. Here, we study the role of Mzb1, a Grp94 cochaperone that is expressed in marginal zone (MZ) B cells and during the terminal differentiation of B cells to antibody-secreting cells. By analyzing Mzb1 -/- Prdm1 +/gfp mice, we find that Mzb1 is specifically required for the differentiation and function of antibody-secreting cells in a T cell-independent immune response. We find that Mzb1-deficiency mimics, in part, the phenotype of Blimp1 deficiency, including the impaired secretion of IgM and the deregulation of Blimp1 target genes. In addition, we find that Mzb1 -/- plasmablasts show a reduced activation of ß1-integrin, which contributes to the impaired plasmablast differentiation and migration of antibody-secreting cells to the bone marrow. Thus, Mzb1 function is required for multiple aspects of plasma cell differentiation.


Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Integrina beta1/metabolismo , Chaperonas Moleculares/metabolismo , Plasmócitos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Animais , Células da Medula Óssea/citologia , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Integrina beta1/genética , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Plasmócitos/citologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA