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1.
Toxicol In Vitro ; 46: 155-162, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29017774

RESUMO

Many natural and industrial proteins are known to have properties that can result in type I hypersensitivity, however, to date, no validated test system exists that can predict the sensitizing potential of these allergens. Thus, the objective of this study was to develop a protocol based on the myeloid cell-based Genomic Allergen Rapid Detection (GARD) assay that can be used to assess and predict the capacity of protein allergens known to induce sensitization in the respiratory tract. Cellular responses induced by eight selected proteins were assessed using transcriptional profiling, flow cytometry and multiplex cytokine analysis. 391 potential biomarkers were identified as a predictive signature and a series of cross-validations supported the validity of the model. These results together with biological pathway analysis of the transcriptomic data indicate that the investigated cell system is able to capture relevant events linked to type I hypersensitization.


Assuntos
Alérgenos/toxicidade , Proteínas/toxicidade , Sistema Respiratório/efeitos dos fármacos , Testes de Toxicidade/métodos , Alternativas aos Testes com Animais , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Biomarcadores , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Dermatite Alérgica de Contato/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Sistema Respiratório/imunologia
2.
J Biol Chem ; 292(32): 13312-13322, 2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28637874

RESUMO

Albumin is the most abundant plasma protein involved in the transport of many compounds, such as fatty acids, bilirubin, and heme. The endothelial cellular neonatal Fc receptor (FcRn) has been suggested to play a central role in maintaining high albumin plasma levels through a cellular recycling pathway. However, direct mapping of this process is still lacking. This work presents the use of wild-type and engineered recombinant albumins with either decreased or increased FcRn affinity in combination with a low or high FcRn-expressing endothelium cell line to clearly define the FcRn involvement, intracellular pathway, and kinetics of albumin trafficking by flow cytometry, quantitative confocal microscopy, and an albumin-recycling assay. We found that cellular albumin internalization was proportional to FcRn expression and albumin-binding affinity. Albumin accumulation in early endosomes was independent of FcRn-binding affinity, but differences in FcRn-binding affinities significantly affected the albumin distribution between late endosomes and lysosomes. Unlike albumin with low FcRn-binding affinity, albumin with high FcRn-binding affinity was directed less to the lysosomes, suggestive of FcRn-directed albumin salvage from lysosomal degradation. Furthermore, the amount of recycled albumin in cell culture media corresponded to FcRn-binding affinity, with a ∼3.3-fold increase after 1 h for the high FcRn-binding albumin variant compared with wild-type albumin. Together, these findings uncover an FcRn-dependent endosomal cellular-sorting pathway that has great importance in describing fundamental mechanisms of intracellular albumin recycling and the possibility to tune albumin-based therapeutic effects by FcRn-binding affinity.


Assuntos
Endossomos/metabolismo , Endotélio Vascular/metabolismo , Lisossomos/metabolismo , Microvasos/metabolismo , Receptores Fc/agonistas , Albumina Sérica/metabolismo , Substituição de Aminoácidos , Linhagem Celular Transformada , Microanálise por Sonda Eletrônica , Endossomos/ultraestrutura , Endotélio Vascular/citologia , Endotélio Vascular/ultraestrutura , Corantes Fluorescentes , Regulação da Expressão Gênica , Variação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Radioisótopos do Iodo , Cinética , Lisossomos/ultraestrutura , Microscopia Confocal , Microvasos/citologia , Microvasos/ultraestrutura , Engenharia de Proteínas , Transporte Proteico , Receptores Fc/genética , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Albumina Sérica/genética
3.
Immunopharmacol Immunotoxicol ; 35(4): 505-13, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23841452

RESUMO

Enteroantigens (eAgs) drive tolerogenic and inflammatory immune responses in the gut and are of importance for sustained immune homeostasis in colonic mucosa. Decline of regulatory activity in the gut mucosa might result in chronic colitis. B7-H4 is a co-inhibitory receptor expressed by professional antigen-presenting cells. By delivering signal 2 during T cell activation, it inhibits T cell proliferation and inflammation. In this study, we have used a newly developed B7-H4-Ig fusion protein and evaluated its effect on eAg-activated effector and regulatory T cells (Treg) in vitro and in vivo. T cells were recovered from the mesenteric lymph nodes (MLNs) of untreated or B7-H4-Ig-treated BALB/c mice. Treatment of cells in vitro did neither affect the proliferation of effector T cells nor the function of Tregs. In vivo, B7-H4 treatment increased the total number of MLN-derived CD4⁺ and CD8⁺ T cell subsets as well as the functional activity of MLN-derived Tregs, whereas the proliferative activity of eAg or alloantigen specific effector T cells was not influenced, although treatment resulted in less secretion of inflammatory cytokines and chemokines from these cells. B7-H4-Ig treatment of severe combined immune-deficient (SCID) mice undergoing T cell transfer colitis did not influence the course of disease probably reflecting the lack of Tregs in this model of chronic colitis. In conclusion, we show that treatment with B7-H4-Ig in vivo changes lymphocyte homeostasis and increases the regulatory potential in normal mice, but does not affect the course of disease development in SCID mice undergoing T cell transfer colitis.


Assuntos
Homeostase/imunologia , Imunoglobulina G/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T Reguladores/imunologia , Inibidor 1 da Ativação de Células T com Domínio V-Set/farmacologia , Animais , Colite/tratamento farmacológico , Colite/imunologia , Feminino , Humanos , Imunoglobulina G/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Proteínas Recombinantes de Fusão/genética , Inibidor 1 da Ativação de Células T com Domínio V-Set/genética
4.
Int Immunol ; 18(5): 817-25, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16574668

RESUMO

We have previously shown that conventional as well as germ-free CD4+ T cells depleted of CD25+ cells from the gut-associated lymphoid tissue and the periphery proliferate specifically in response to enterobacterial antigen exposure whereas unfractionated CD4+ T cells are not reactive under these conditions. Here we show that the majority of the enteroantigen-specific CD4+ CD25- T cells are naive cells expressing a CD45RB high, CD62L high and CD44 low phenotype. These cells are also present in the thymus and data from adult thymectomized mice show that they represent late (>6 weeks) thymic emigrants. Upon enteroantigen activation, the CD4+ CD25- T cells secrete IL-4, IL-5, IL-10, granulocyte macrophage colony-stimulating factor, tumor necrosis factor-alpha and IFN-gamma. Clonotype mapping of the TCRBV regions 1-18 of enteroantigen-reactive CD4+ CD25- T cells by TCR clonotype mapping revealed the polyclonal nature of this subset. In conclusion, we have for the first time demonstrated the presence of an evolutionary, functionally conserved subset of CD4+ T cells, which are reactive against enterobacterial antigens. This subset resides both in the thymus and the periphery; it is not dependent on previous antigen experience and represents late thymic emigrants, which by enteroantigen-induced activation express a mixed Th 1-Th 2 phenotype. At homeostatic conditions, CD25+ T cells maintain peripheral tolerance in this CD4+ T cell subset.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Receptores de Interleucina-2/imunologia , Animais , Antígenos CD8/imunologia , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Timo/citologia , Timo/imunologia
5.
Immunology ; 113(4): 499-508, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15554928

RESUMO

Regulatory T (Treg) cells, derived from co-cultures of unfractionated CD4(+) T cells and immature dendritic cells (DC), suppress enteroantigen-induced proliferation of CD4(+) CD25(-) T cells. The DC-induced Treg cells are a mixture of CD25(+) (10-20%) and CD25(-) (80-90%) T cells. However, all the suppressor activity in vitro and in vivo resides in the CD25(+) T-cell subset. The CD25(+) DC-induced Treg cells can inhibit enteroantigen-induced proliferation in vitro through a transwell membrane, and their function does not appear to depend on previous activation. DC-induced CD25(+) Treg cells display a naive phenotype, expressing high levels of CD45RB and l-selectin (CD62L). In addition, the DC-induced Treg cells mediate a stronger suppressive activity than prototype CD25(+) regulatory T cells. The DC-induced Treg cells, and hereof purified CD25(+) and CD25(-) T-cell fractions, were co-injected into severe combined immunodeficiency (SCID) mice with colitis-inducing CD4(+) CD25(-) T cells. Both unfractionated CD4(+) and purified CD25(+) Treg cells fully protected the recipients against the development of colitis. In contrast, co-transfer of fractionated CD25(-) T cells offered no protection against disease development. Enterobacterial antigen-exposed CD4(+) T cells of the protected mice secreted higher levels of interleukin-10 and lower levels of interferon-gamma than the unprotected mice. The present data demonstrate DC-induced CD4(+) CD25(+) Treg cells, which phenotypically and functionally differ from the generally accepted prototype of CD25(+) Treg cells. These data may initiate new procedures for the expansion of Treg cells for clinical use.


Assuntos
Colite/imunologia , Células Dendríticas/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Divisão Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Feminino , Imunofenotipagem , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Receptores de Interleucina-2/análise
6.
Eur J Immunol ; 34(3): 695-704, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14991599

RESUMO

Unfractionated CD4+ T cells from the gut-associated lymphoid tissue (GALT) and peripheral lymph nodes are unresponsive when exposed to enterobacterial antigens in vitro. Under similar conditions, CD4+ T cells depleted in vivo or in vitro of CD4+CD25+ T cells proliferate extensively. The CD4+CD25- T cell reactivity depends on MHC class II presentation, specific TCR stimulation, CD4 ligation, and antigen processing by antigen-presenting cells. The CD4+CD25- T cells respond to autologous and heterologous enterobacterial antigens, but not to antigens from the feces of germ-free mice. Surprisingly, CD4+CD25- T cells obtained from the GALT of germ-free mice also proliferate when exposed to enterobacterial antigens, and adding back the conventional or germ-free CD4+CD25+ T cells to the enteroantigen-stimulated CD4+CD25- T cells abolishes proliferation. As judged from carboxyfluorescein diacetate succinimidyl ester-labeling experiments, 4-5% of the CD4+CD25- T cells respond to enteroantigen. The data show for the first time that CD4+CD25- T cells with reactivity towards the enterobacterial flora and regulatory CD4+CD25- T cells are present in both conventional and germ-free mice. The data suggest that a significant proportion of the peripheral pool of CD4+CD25- T cells express anti-enterobacterial reactivity, which, due to the presence of regulatory CD4+CD25+ T cells, is kept in a quiescent state.


Assuntos
Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Mucosa Intestinal/imunologia , Ativação Linfocitária , Receptores de Interleucina-2/análise , Subpopulações de Linfócitos T/imunologia , Animais , Apresentação de Antígeno , Linfócitos T CD4-Positivos/classificação , Células Cultivadas , Citocinas/biossíntese , Feminino , Antígenos de Histocompatibilidade Classe II/metabolismo , Tolerância Imunológica , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Antígenos de Linfócitos T/imunologia , Organismos Livres de Patógenos Específicos , Células Th2/imunologia
7.
Int Immunol ; 14(7): 701-11, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12096029

RESUMO

Efficient induction of T cell responses is normally assumed to require both TCR-mediated signaling and engagement of co-stimulatory molecules, in particular CD28. However, the importance of CD28 co-stimulation in induction and maintenance of antiviral T cell responses is not clearly established. For this reason antiviral CD4(+) and CD8(+) T cell responses in CD28-deficient mice were studied using two different viruses [vesicular stomatitis virus and lymphocytic choriomeningitis virus (LCMV)]. Intracellular cytokine staining and/or MHC-peptide tetramers were used to enumerate antigen-specific T cells. In addition, we used DNA constructs encoding viral epitopes to probe the importance of the epitope itself. Our results reveal that while the context of antigen presentation (live virus versus DNA construct) is a critical factor in determining the requirement for CD28 co-stimulation, epitope and virus dose play little if any role. Direct visualization of antigen-specific cells also confirms the notion that CD28 is more critical for the generation of antiviral T(h)1 cells than for T(c)1 cells generated in response to the same virus (LCMV). Most importantly, the present study reveals that CD28 generally is essential for the host to respond optimally over a broad set of conditions, and our results may imply that the relatively CD28 independent activation of LCMV-specific CD8(+) T cells may represent an extreme situation related to the non-cytolytic nature of this virus allowing the delivery of a uniquely strong and prolonged signal 1.


Assuntos
Infecções por Arenaviridae/imunologia , Antígenos CD28/fisiologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Infecções por Rhabdoviridae/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos CD28/genética , Antígenos CD40/genética , Epitopos/genética , Epitopos/imunologia , Imunização , Ativação Linfocitária , Vírus da Coriomeningite Linfocítica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmídeos/administração & dosagem , Plasmídeos/genética , Especificidade da Espécie , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vírus da Estomatite Vesicular Indiana/genética
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