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1.
Int J Mol Sci ; 25(7)2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38612661

RESUMO

Flow cytometry is a mainstay technique in cell biology research, where it is used for phenotypic analysis of mixed cell populations. Quantitative approaches have unlocked a deeper value of flow cytometry in drug discovery research. As the number of drug modalities and druggable mechanisms increases, there is an increasing drive to identify meaningful biomarkers, evaluate the relationship between pharmacokinetics and pharmacodynamics (PK/PD), and translate these insights into the evaluation of patients enrolled in early clinical trials. In this review, we discuss emerging roles for flow cytometry in the translational setting that supports the transition and evaluation of novel compounds in the clinic.


Assuntos
Pesquisa Translacional Biomédica , Ciência Translacional Biomédica , Humanos , Citometria de Fluxo , Projetos de Pesquisa , Descoberta de Drogas
2.
JCI Insight ; 2(19)2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28978806

RESUMO

Patients deficient in the guanine nucleotide exchange factor DOCK8 have decreased numbers and impaired in vitro function of Tregs and make autoantibodies, but they seldom develop autoimmunity. We show that, similarly, Dock8-/- mice have decreased numbers and impaired in vitro function of Tregs but do not develop autoimmunity. In contrast, mice with selective DOCK8 deficiency in Tregs develop lymphoproliferation, autoantibodies, and gastrointestinal inflammation, despite a normal percentage and in vitro function of Tregs, suggesting that deficient T effector cell function might protect DOCK8-deficient patients from autoimmunity. We demonstrate that DOCK8 associates with STAT5 and is important for IL-2-driven STAT5 phosphorylation in Tregs. DOCK8 localizes within the lamellar actin ring of the Treg immune synapse (IS). Dock8-/- Tregs have abnormal TCR-driven actin dynamics, decreased adhesiveness, an altered gene expression profile, an unstable IS with decreased recruitment of signaling molecules, and impaired transendocytosis of the costimulatory molecule CD86. These data suggest that DOCK8 enforces immunological tolerance by promoting IL-2 signaling, TCR-driven actin dynamics, and the IS in Tregs.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/imunologia , Tolerância Imunológica/imunologia , Sinapses Imunológicas/imunologia , Interleucina-2/imunologia , Linfócitos T Reguladores/imunologia , Animais , Autoanticorpos/biossíntese , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/imunologia , Gastroenterite/imunologia , Fatores de Troca do Nucleotídeo Guanina/deficiência , Inflamação/imunologia , Linfonodos/imunologia , Camundongos Knockout , Fosforilação/imunologia , Fator de Transcrição STAT5/metabolismo , Aumento de Peso/imunologia
3.
J Allergy Clin Immunol ; 139(4): 1293-1301.e4, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27609654

RESUMO

BACKGROUND: The B-cell receptor transmembrane activator and calcium modulator ligand interactor (TACI) is important for T-independent antibody responses. One in 200 blood donors are heterozygous for the TACI A181E mutation. OBJECTIVE: We sought to investigate the effect on B-cell function of TACI A181E heterozygosity in reportedly healthy subjects and of the corresponding TACI A144E mutation in mice. METHODS: Nuclear factor κB (NF-κB) activation was measured by using the luciferase assay in 293T cells cotransfected with wild-type and mutant TACI. TACI-driven proliferation, isotype switching, and antibody responses were measured in B cells from heterozygous TACI A144E knock-in mice. Mouse mortality was monitored after intranasal pneumococcal challenge. RESULTS: Levels of natural antibodies to the pneumococcal polysaccharide component phosphocholine were significantly lower in A181E-heterozygous than TACI-sufficient Swedish blood donors never immunized with pneumococcal antigens. Although overexpressed hTACI A181E and mTACI A144E acted as dominant-negative mutations in transfectants, homozygosity for A144E in mice resulted in absent TACI expression in B cells, indicating that the mutant protein is unstable when naturally expressed. A144E heterozygous mice, such as TACI+/- mice, expressed half the normal level of TACI on their B cells and exhibited similar defects in a proliferation-inducing ligand-driven B-cell activation, antibody responses to TNP-Ficoll, production of natural antibodies to phosphocholine, and survival after intranasal pneumococcal challenge. CONCLUSION: These results suggest that TACI A181E heterozygosity results in TACI haploinsufficiency with increased susceptibility to pneumococcal infection. This has important implications for asymptomatic TACI A181E carriers.


Assuntos
Pneumonia Pneumocócica/genética , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Técnicas de Introdução de Genes , Células HEK293 , Haploinsuficiência , Heterozigoto , Humanos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia Pneumocócica/imunologia , Reação em Cadeia da Polimerase , Proteína Transmembrana Ativadora e Interagente do CAML/imunologia
4.
J Clin Invest ; 126(10): 3837-3851, 2016 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-27599296

RESUMO

Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor-driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Movimento Celular , Proteínas do Citoesqueleto , Células HEK293 , Humanos , Sinapses Imunológicas/metabolismo , Linfonodos/citologia , Mecanotransdução Celular , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Mapas de Interação de Proteínas , Multimerização Proteica , Transporte Proteico , Linfócitos T/fisiologia
5.
J Allergy Clin Immunol ; 134(6): 1365-1374, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25218284

RESUMO

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8) deficiency is typified by recurrent infections, increased serum IgE levels, eosinophilia, and a high incidence of allergic and autoimmune manifestations. OBJECTIVE: We sought to determine the role of DOCK8 in the establishment and maintenance of human B-cell tolerance. METHODS: Autoantibodies were measured in the plasma of DOCK8-deficient patients. The antibody-coding genes from new emigrant/transitional and mature naive B cells were cloned and assessed for their ability to bind self-antigens. Regulatory T (Treg) cells in the blood were analyzed by means of flow cytometry, and their function was tested by examining their capacity to inhibit the proliferation of CD4(+)CD25(-) effector T cells. RESULTS: DOCK8-deficient patients had increased levels of autoantibodies in their plasma. We determined that central B-cell tolerance did not require DOCK8, as evidenced by the normally low frequency of polyreactive new emigrant/transitional B cells in DOCK8-deficient patients. In contrast, autoreactive B cells were enriched in the mature naive B-cell compartment, revealing a defective peripheral B-cell tolerance checkpoint. In addition, we found that Treg cells were decreased and exhibited impaired suppressive activity in DOCK8-deficient patients. CONCLUSIONS: Our data support a critical role for DOCK8 in Treg cell homeostasis and function and the enforcement of peripheral B-cell tolerance.


Assuntos
Linfócitos B/imunologia , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/imunologia , Síndromes de Imunodeficiência/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Autoanticorpos/sangue , Criança , Pré-Escolar , Feminino , Humanos , Tolerância Imunológica/imunologia , Lactente , Contagem de Linfócitos , Masculino
6.
PLoS One ; 9(5): e97628, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24830368

RESUMO

Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Progenitoras Mieloides/citologia , Proteínas de Ligação a RNA/metabolismo , Animais , Medula Óssea/patologia , Células da Medula Óssea/citologia , Linhagem da Célula , Proliferação de Células , Sobrevivência Celular , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Granulócitos/citologia , Homeostase , Imunofenotipagem , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ribonucleoproteínas/metabolismo
7.
J Immunol ; 192(6): 2651-8, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24532577

RESUMO

The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and Toll/IL-IR domain-containing adapter-inducing IFN-ß (TRIF)-related adaptor molecule (TRAM), which recruits TRIF. Following stimulation with LPS plus IL-4, Tram(-/-) and Trif(-/-) B cells completely failed to express Cε germline transcripts (GLT) and secrete IgE. In contrast, Myd88(-/-) B cells had normal expression of Cε GLT but reduced IgE secretion in response to LPS plus IL-4. Following LPS plus IL-4 stimulation, Cγ1 GLT expression was modestly reduced in Tram(-/-) and Trif(-/-) B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram(-/-), Trif(-/-), and Myd88(-/-) B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40 plus IL-4. Following stimulation with LPS plus IL-4, Trif(-/-) B cells failed to sustain NF-κB p65 nuclear translocation beyond 3 h and had reduced binding of p65 to the Iε promoter. Addition of the NF-κB inhibitor, JSH-23, to wild-type B cells 15 h after LPS plus IL-4 stimulation selectively blocked Cε GLT expression and IgE secretion but had little effect on Cγ1 GLT expression and IgG secretion. These results indicate that sustained activation of NF-κB driven by TRIF is essential for LPS plus IL-4-driven activation of the Cε locus and class switching to IgE.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Switching de Imunoglobulina/imunologia , Imunoglobulina E/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citidina Desaminase/genética , Citidina Desaminase/imunologia , Citidina Desaminase/metabolismo , Immunoblotting , Switching de Imunoglobulina/efeitos dos fármacos , Imunoglobulina E/genética , Imunoglobulina E/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Cadeias épsilon de Imunoglobulina/genética , Cadeias épsilon de Imunoglobulina/imunologia , Cadeias épsilon de Imunoglobulina/metabolismo , Cadeias gama de Imunoglobulina/genética , Cadeias gama de Imunoglobulina/imunologia , Cadeias gama de Imunoglobulina/metabolismo , Interleucina-4/imunologia , Interleucina-4/farmacologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Fenilenodiaminas/imunologia , Fenilenodiaminas/farmacologia , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Receptores de Interleucina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismo
8.
Mol Cell Biol ; 33(17): 3505-14, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23816884

RESUMO

RNA processing and transport are mediated by cotranscriptionally assembled ribonucleoprotein (RNP) complexes. RNPs have been postulated to help specify coordinated gene expression, but the requirements for specific RNP complexes in mammalian development and tissue homeostasis have not been extensively evaluated. THO is an evolutionarily conserved RNP complex that links transcription with nuclear export. THO is not essential for Saccharomyces cerevisiae viability, but it is essential for early mouse embryonic development. Embryonic lethality has limited the characterization of THO requirements in adult tissues. To overcome this limitation, a mouse model has been generated that allows widespread inducible deletion of Thoc1, which encodes an essential protein subunit of THO. Widespread Thoc1 deletion disrupts homeostasis within the small intestine but does not have detectable effects in other epithelial tissues such as the related mucosa of the large intestine. Thoc1 loss compromises the proliferation and lineage-generating capacity of small intestinal stem cells, disrupting the supply of differentiated cells in this rapidly renewing tissue. These findings demonstrate that the effects of THO deficiency in the adult mouse are tissue and cell type dependent.


Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/genética , Deleção de Genes , Intestino Delgado/citologia , Proteínas de Ligação a RNA/genética , Células-Tronco/citologia , Animais , Sobrevivência Celular , Proteínas de Ligação a DNA/metabolismo , Feminino , Intestino Delgado/metabolismo , Intestino Delgado/ultraestrutura , Lactação , Glândulas Mamárias Animais/fisiologia , Glândulas Mamárias Animais/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/metabolismo , Células-Tronco/metabolismo
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