RESUMO
Solid mixtures of ammonia and water, the so-called ammonia hydrates, are thought to be major components of solar and extra-solar icy planets. We present here a thorough characterization of the recently reported high pressure (P)-temperature (T) phase VII of ammonia monohydrate (AMH) using Raman spectroscopy, X-ray diffraction, and quasi-elastic neutron scattering (QENS) experiments in the ranges 4-10 GPa, 450-600 K. Our results show that AMH-VII exhibits common structural features with the disordered ionico-molecular alloy (DIMA) phase, stable above 7.5 GPa at 300 K: both present a substitutional disorder of water and ammonia over the sites of a body-centered cubic lattice and are partially ionic. The two phases however markedly differ in their hydrogen dynamics, and QENS measurements show that AMH-VII is characterized by free molecular rotations around the lattice positions which are quenched in the DIMA phase. AMH-VII is thus a peculiar crystalline solid in that it combines three types of disorder: substitutional, compositional, and rotational.
RESUMO
BACKGROUND: The gut-liver-axis presents the pathophysiological hallmark for multiple liver diseases and has been proposed to be modulated during stress and shock. Access to the gut-liver-axis needs crossing of the mucus and gut-vascular barrier. The role of ß-adrenoreceptor-activation for both barriers has not been defined and is characterized here. METHODS: Splanchnic ß-adrenergic stimulation was achieved by chronic intraperitoneal application of isoproterenol via alzet-pump in vivo. The intestinal permeability and gut-vascular barrier function was assessed in ileal loop experiments. The extravasation of predefined sizes of fluorescence isothiocyanate (FITC)-dextran molecules in ileal microcirculation was evaluated by intravital confocal laser endomicroscopy in vivo. Mucus parameters thickness, goblet cell count and mucin-expression were assessed by stereomicroscopy, immunostaining and RNA-sequencing respectively. Ileal lamina propria (LP) as well as mesenteric lymph node mononuclear cells was assessed by FACS. RESULTS: Healthy mice lack translocation of 4 kDa-FITC-dextran from the small intestine to the liver, whereas isoproterenol-treated mice demonstrate pathological translocation (PBT). Mucus layer is reduced in thickness with loss of goblet-cells and mucin-2-staining and -expression in isoproterenol-treated animals under standardized gnotobiotic conditions. Isoproterenol disrupts the gut vascular barrier displaying Ileal extravasation of large-sized 70- and 150 kDa-FITC-dextran. This pathological endothelial permeability and accessibility induced by isoproterenol associates with an augmented expression of plasmalemmal-vesicle-associated-protein-1 in intestinal vessel. Ileal LP after isoproterenol treatment contains more CD11c+-dendritic cells (DC) with increased appearance of CCR7+ DC in mesenteric lymph nodes. CONCLUSIONS: Isoproterenol impairs the intestinal muco-epithelial and endothelial-vascular barrier promoting PBT to the liver. This barrier dysfunction on multiple levels potentially can contribute to liver injury induced by catecholamines during states of increased ß-adrenergic drive.
Assuntos
Mucosa Intestinal , Isoproterenol , Muco , Animais , Isoproterenol/farmacologia , Fígado , Camundongos , PermeabilidadeRESUMO
In this study, we aimed to investigate some functional characteristics and the immunomodulatory properties of three strains of Lactobacillus plantarum of dairy origin which, in a previous screening, showed to be candidate probiotics. Genome sequencing and comparative genomics, which confirmed the presence of genes involved in folate and riboflavin production and in the immune response of dendritic cells (DCs), prompted us to investigate the ability of the three strains to accumulate the two vitamins and their immunomodulation properties. The ability of the three strains to release antioxidant components in milk was also investigated. Small amounts of folate and riboflavin were produced by the three strains, while they showed a good antioxidant capacity in milk with FRAP method. The immune response experiments well correlated with the presence of candidate genes influencing in DCs cytokine response to L. plantarum. Specifically, the amounts of secreted cytokins by DCs after stimulation with cells of Lp790, Lp813 and Lp998 resulted pro-inflammatory whereas stimulation with culture supernatants (postbiotics) inhibited the release of interleukin (IL)-12p70 and increased the release of the anti-inflammatory IL-10 cytokine. This study adds further evidence on the importance of L. plantarum in human health. Understanding how probiotics (or postbiotics) work in preclinical models can allow a rational choice of the different strains for clinical and/or commercial use.
Assuntos
Células Dendríticas/efeitos dos fármacos , Fatores Imunológicos/administração & dosagem , Lactobacillus plantarum/genética , Leite/microbiologia , Probióticos/administração & dosagem , Animais , Bovinos , Células Cultivadas , Células Dendríticas/imunologia , Genoma Bacteriano , Genômica , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Lactobacillus plantarum/classificação , Lactobacillus plantarum/imunologia , Lactobacillus plantarum/isolamento & purificação , FilogeniaRESUMO
The use of probiotics and synbiotics in the food industry or as food supplements for a balanced diet and improved gut homeostasis has been blooming for the past decade. As feedback from healthy consumers is rather enthusiastic, a lot of effort is currently directed in elucidating the mechanisms of interaction between beneficial microbes and barrier and immune function of the host. The use of probiotics or synbiotics for treating certain pathologies has also been examined, however, the outcome has not always been favourable. In most cases, the effect of the administered probiotic is evident when the bacteria are still alive at the time they reach the small and large intestine, suggesting that it is dependent on the metabolic activity of the bacteria. Indeed, in some occasions it has been shown that the culture supernatant of these bacteria mediates the immunomodulatory effect conferred to the host. Recent work on relevant probiotic strains has also led to the isolation and characterisation of certain probiotic-produced, soluble factors, here called postbiotics, which were sufficient to elicit the desired response. Here, we summarise these recent findings and propose the use of purified and well characterised postbiotic components as a safer alternative for clinical applications, especially in chronic inflammatory conditions like inflammatory bowel disease, where probiotics have not yet given encouraging results as far as induction of remission is concerned.
Assuntos
Produtos Biológicos/isolamento & purificação , Produtos Biológicos/uso terapêutico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Probióticos/química , Probióticos/uso terapêutico , Produtos Biológicos/farmacologia , Humanos , Probióticos/efeitos adversos , Probióticos/farmacologiaRESUMO
Thymic stromal lymphopoietin (TSLP) is produced by epithelial cells and keratinocytes, and is involved in immune homeostasis or inflammation. The mechanism through which TSLP regulates intestinal inflammation is unclear. Here, we report that mouse dendritic cells (DCs) express TSLP both in vitro and in vivo in response to Toll-like receptor ligation in a MyD88-dependent fashion. TSLP is produced by the CD103(+) subset of tolerogenic gut DCs and is downregulated during experimental colitis. TSLP produced by DCs acts directly on T cells by reducing their capacity to produce interleukin (IL)-17 and fostering the development of Foxp3(+) T cells. Consistently, TSLP protects against colitis development through a direct action on T cells, as adoptive transfer of naïve T cells from TSLPR(-/-) to SCID mice results in a more severe colitis, with increased frequency of IL-17-producing T cells and inflammatory cytokines. Hence, we describe a new anti-inflammatory role of TSLP in the gut.
Assuntos
Colite/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismo , Transferência Adotiva , Animais , Antígenos CD/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Colite/induzido quimicamente , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Sulfato de Dextrana/administração & dosagem , Progressão da Doença , Fatores de Transcrição Forkhead/metabolismo , Cadeias alfa de Integrinas/metabolismo , Interleucina-17/metabolismo , Intestinos/imunologia , Intestinos/patologia , Camundongos , Camundongos Knockout , Camundongos SCID , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Células Th17/imunologia , Células Th17/patologia , Linfopoietina do Estroma do TimoRESUMO
Modulation of death is a pathogen strategy to establish residence and promote survival in host cells and tissues. Shigella spp. are human pathogens that invade colonic mucosa, where they provoke lesions caused by their ability to manipulate the host cell responses. Shigella spp. induce various types of cell death in different cell populations. However, they are equally able to protect host cells from death. Here, we have investigated on the molecular mechanisms and cell effectors governing the balance between survival and death in epithelial cells infected with Shigella. To explore these aspects, we have exploited both, the HeLa cell invasion assay and a novel ex vivo human colon organ culture model of infection that mimics natural conditions of shigellosis. Our results definitely show that Shigella induces a rapid intrinsic apoptosis of infected cells, via mitochondrial depolarization and the ensuing caspase-9 activation. Moreover, for the first time we identify the eukaryotic stress-response factor growth arrest and DNA damage 45α as a key player in the induction of the apoptotic process elicited by Shigella in epithelial cells, revealing an unexplored role of this molecule in the course of infections sustained by invasive pathogens.
Assuntos
Apoptose , Proteínas de Ciclo Celular/metabolismo , Disenteria Bacilar/metabolismo , Células Epiteliais/citologia , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Shigella flexneri/fisiologia , Proteínas de Ciclo Celular/genética , Morte Celular , Colo/citologia , Colo/metabolismo , Colo/microbiologia , Disenteria Bacilar/microbiologia , Disenteria Bacilar/fisiopatologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células HeLa , Humanos , Técnicas In Vitro , Proteínas Nucleares/genética , Shigella flexneri/genéticaRESUMO
OBJECTIVE: In mice, a subpopulation of gut dendritic cells (DCs) expressing CD103 drives the development of regulatory T (T(reg)) cells. Further, it was recently described that the cross-talk between human intestinal epithelial cells (IECs) and DCs helps in maintaining gut immune homeostasis via the induction of non-inflammatory DCs. In this study, an analysis was carried out to determine whether IECs could promote the differentiation of CD103+ tolerogenic DCs, and the function of primary CD103+ DCs isolated from human mesenteric lymph nodes (MLNs) was evaluated. METHODS: Monocyte-derived DCs (MoDCs) and circulating CD1c+ DCs were conditioned or not with supernatants from Caco-2 cells or IECs isolated from healthy donors or donors with Crohn's disease and analysed for their ability to induce T(reg) cell differentiation. In some cases, transforming growth factor beta (TGFbeta), retinoic acid (RA) or thymic stromal lymphopoietin (TSLP) were neutralised before conditioning. CD103+ and CD103- DCs were sorted by fluorescence-activated cell sorting (FACS) from MLNs and used in T(reg) cell differentiation experiments. RESULTS: It was found that human IECs promoted the differentiation of tolerogenic DCs able to drive the development of adaptive Foxp3+ T(reg) cells. This control was lost in patients with Crohn's disease and paralleled a reduced expression of tolerogenic factors by primary IECs. MoDCs differentiated with RA or IEC supernatant upregulated the expression of CD103. Consistently, human primary CD103+ DCs isolated from MLNs were endowed with the ability to drive T(reg) cell differentiation. This subset of DCs expressed CCR7 and probably represents a lamina propria-derived migratory population. CONCLUSIONS: A population of tolerogenic CD103+ DCs was identified in the human gut that probably differentiate in response to IEC-derived factors and drive T(reg) cell development.
Assuntos
Diferenciação Celular , Células Dendríticas/citologia , Intestinos/citologia , Linfócitos T Reguladores/citologia , Antígenos CD/metabolismo , Células CACO-2/citologia , Doença de Crohn/imunologia , Doença de Crohn/patologia , Células Dendríticas/imunologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Humanos , Imunidade Celular , Cadeias alfa de Integrinas/metabolismo , Linfonodos/citologia , Ativação Linfocitária/imunologiaRESUMO
Intestinal dendritic cells (DCs) have been shown to display specialized functions, including the ability to promote gut tropism to lymphocytes, to polarize noninflammatory responses, and to drive the differentiation of adaptive Foxp3(+) regulatory T (T(reg)) cells. However, very little is known about what drives the mucosal phenotype of DCs. Here, we present evidence that the local microenvironment, and in particular intestinal epithelial cells (ECs), drive the differentiation of T(reg)-cell-promoting DCs, which counteracts Th1 and Th17 development. EC-derived transforming growth factor-beta (TGF-beta) and retinoic acid (RA), but not thymic stromal lymphopoietin (TSLP), were found to be required for DC conversion. After EC contact, DCs upregulated CD103 and acquired a tolerogenic phenotype. EC-conditioned DCs were capable of inducing de novo T(reg) cells with gut-homing properties that when adoptively transferred, protected mice from experimental colitis. Thus, we have uncovered an essential mechanism in which EC control of DC function is required for tolerance induction.
Assuntos
Colite/imunologia , Células Dendríticas/imunologia , Mucosa Intestinal/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Comunicação Celular/imunologia , Diferenciação Celular/imunologia , Colite/patologia , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Feminino , Tolerância Imunológica/imunologia , Interleucina-17/imunologia , Interleucina-17/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Tretinoína/imunologia , Tretinoína/metabolismo , Linfopoietina do Estroma do TimoRESUMO
AIM: To evaluate the status of activation of the intestinal dendritic cells (DCs) and T lymphocytes (T cells) from surgical specimens of human colon and adenocarcinoma, and the potential effect of administration of interleukin 2 (IL-2). METHODS: Patients undergoing colectomy for cancer were randomized to receive subcutaneous IL-2 (12million UI/day) (treated group; n=10) for 3days before operation or no treatment (control group, n=10). DCs and T cells were isolated and purified from the lamina propria (LP) of segments of normal colon and adenocarcinoma of both groups. Cell phenotype was determined by expression of membrane receptors. Interaction between DC and T cells was assesses by a mixed leukocyte reaction using naïve T cells co-cultured with DCs. CD4+ T-cell polarization was studied by intracellular staining with monoclonal antibodies for interleukin-4 and interferon-gamma. RESULTS: CD4+ T cells were significantly less in tumour than in LP (p<0.05) in both treated and control groups. IL-2 did not modify the number of any of the T-cell subsets analysed. In contrast, T cells isolated from LP and neoplasm of treated patients produced more interferon-gamma and less interleukin-4 (p<0.05 vs. controls). IL-2 administration significantly increased (p<0.05) the number of mature, myeloid and plasmocytoid DCs compared to controls. Allogeneic naïve T cells were polarized toward a Th1 type of response which appeared to be mediated by IL-2 activated DCs. CONCLUSIONS: systemic IL-2 treatment may have immunomodulatory properties on intestinal DC maturation and drive a Th1 mediated anti-neoplastic response.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/administração & dosagem , Linfócitos T CD4-Positivos/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Células Dendríticas/efeitos dos fármacos , Interleucina-2/administração & dosagem , Mucosa Intestinal/efeitos dos fármacos , Adenocarcinoma/cirurgia , Idoso , Linfócitos T CD4-Positivos/metabolismo , Colectomia , Neoplasias do Colo/cirurgia , Células Dendríticas/metabolismo , Feminino , Humanos , Mucosa Intestinal/citologia , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
The characterization of tumor antigens recognized by immune effector cells has opened the perspective of developing therapeutic vaccines in the field of breast cancer. The potential advantages of the vaccines are: (i) the induction of a robust immune response against tumors that are spontaneously weekly immunogenic; (ii) the tumor specificity for some antigens; (iii) the good tolerance and safety profile and (iv) the long-term immune memory, critical to prevent efficiently tumor recurrence. Most trials evaluating breast cancer vaccines have been carried out in patients with extended metastatic breast cancer, characterized by aggressive tumors, resistant to standard cytotoxic treatments, so that clinical efficacy was difficult to achieve. However, some significant immune responses against tumor antigens induced upon vaccinations were recorded. The aim of this review is to analyze the activity of vaccination strategies in current clinical trials. Data of clinical activity have been observed by using vaccines targeting HER2/neu protein, human telomerase reverse transcriptase, carcinoembryonic antigen and carbohydrate antigen given after stem cell rescue. The review discusses possible future directions for vaccine development and applications in the adjuvant setting.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Vacinas Anticâncer , Antígenos de Neoplasias/química , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Antígeno Carcinoembrionário/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Imunoterapia , Receptor ErbB-2/metabolismo , Telomerase/metabolismoRESUMO
Tracer methods are increasingly being exploited to examine the trafficking patterns of cells transferred into recipient models of diseases, to optimize immune cell therapies, and to assess cancer gene therapy and vaccines in various cancer models. In animal cancer models, noninvasive monitoring by imaging tumor response could significantly facilitate the development of immune cell therapies against cancer. Currently, ex vivo lymphocyte labeling is primarily done by direct labeling. Major advances in cell labeling procedures have led to the use of reporter constructs to assess gene expression in vivo. With this novel technique, the reporter gene marks the cell with a specific protein that distinguishes the cell and its cellular progeny from other cells after migration, homing and mitosis. Several in vivo imaging procedures, including positron emission tomography, single photon emission tomography and magnetic resonance imaging, have been rescaled for studies in small animals. Other methods initially used for in vitro bioluminescence and fluorescence studies have also been refined for in vivo studies. When combined, these methods allow to assess cell trafficking in a noninvasive fashion, beyond lymphocyte response to inflammation, including metastatic diffusion and stem cell transplantation.
Assuntos
Movimento Celular/imunologia , Diagnóstico por Imagem/métodos , Linfócitos/imunologia , Linfócitos/patologia , Neoplasias Experimentais/diagnóstico , Neoplasias Experimentais/imunologia , Animais , Genes Reporter/genética , Humanos , Linfócitos/diagnóstico por imagem , Neoplasias Experimentais/genética , Cintilografia , Coloração e Rotulagem/métodosRESUMO
Dendritic cells are potent activators of the immune response. They reside in tissues which interface the external environment, but we shall see that they do not perform only a passive role by monitoring microorganisms that have entered the body. Rather, DC can actively participate to microbial entry across mucosal surfaces by creeping between epithelial cells and by internalizing bacteria via their dendrites.
Assuntos
Bactérias , Células Dendríticas/fisiologia , Fagocitose , Animais , Extensões da Superfície Celular/fisiologia , Células Dendríticas/microbiologia , Células Dendríticas/ultraestrutura , Conteúdo Gastrointestinal/microbiologia , Humanos , Junções Intercelulares , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologiaRESUMO
Immature dendritic cells (DCs) sample their environment for antigens and after stimulation present peptide associated with major histocompatibility complex class II (MHC II) to naive T cells. We have studied the intracellular trafficking of MHC II in cultured DCs. In immature cells, the majority of MHC II was stored intracellularly at the internal vesicles of multivesicular bodies (MVBs). In contrast, DM, an accessory molecule required for peptide loading, was located predominantly at the limiting membrane of MVBs. After stimulation, the internal vesicles carrying MHC II were transferred to the limiting membrane of the MVB, bringing MHC II and DM to the same membrane domain. Concomitantly, the MVBs transformed into long tubular organelles that extended into the periphery of the cells. Vesicles that were formed at the tips of these tubules nonselectively incorporated MHC II and DM and presumably mediated transport to the plasma membrane. We propose that in maturing DCs, the reorganization of MVBs is fundamental for the timing of MHC II antigen loading and transport to the plasma membrane.
Assuntos
Apresentação de Antígeno , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Vesículas Transportadoras/metabolismo , Animais , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/ultraestrutura , Endocitose/fisiologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Transporte Proteico , Regulação para CimaRESUMO
Immature and mature dendritic cells (DC) have been well characterized functionally and phenotypically. Microorganisms or bacterial products such as lipopolysaccharide (LPS) and inflammatory molecules, including tumor necrosis factor (TNF-alpha), are both believed to activate the DC maturation program which allows DC to initiate and amplify innate and adaptive immune responses. However, there is increasing evidence that the functional state of DC, induced by different stimuli, may be relevant for the immune response outcome. Thus, we compared the transcriptional program of mature, transitional and immature DC, after either LPS or TNF-alpha stimulation. GeneChip oligonucleotide microarrays, representing approximately 6,500 murine genes and ESTs, were used for this analysis. A very diverse modulation of gene expression was observed with the two stimuli. Only LPS-treated cells showed a pattern of expression of genes compatible with a definitive growth arrest and with a suitable activation and control of the immune response.
Assuntos
Células Dendríticas/imunologia , Animais , Apresentação de Antígeno , Apoptose , Ciclo Celular , Diferenciação Celular , Linhagem Celular , Citocinas/biossíntese , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade/imunologia , Inflamação/genética , Inflamação/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/imunologia , RNA Mensageiro/biossíntese , Ativação Transcricional , Fator de Necrose Tumoral alfa/farmacologiaAssuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Interações Hospedeiro-Parasita/imunologia , Animais , Apresentação de Antígeno , Diferenciação Celular , Movimento Celular , Células Dendríticas/microbiologia , Células Dendríticas/virologia , Humanos , Tolerância Imunológica , Infecções/imunologia , Infecções/microbiologia , Infecções/parasitologia , Infecções/virologia , Transdução de Sinais , Linfócitos T/imunologiaRESUMO
Dendritic cells (DCs) are strong activators of primary T cell responses. Their priming ability is acquired upon encounter with maturation stimuli. To identify the genes that are differentially expressed upon maturation induced by exposure to Gram-negative bacteria, a kinetic study of DC gene expression was done with microarrays representing 11,000 genes and ESTs (expressed sequence tags). Approximately 3000 differentially expressed transcripts were identified. We found that functional interleukin 2 (IL-2) mRNA, which gave rise to IL-2 production, was transiently up-regulated at early time-points after bacterial encounter. In contrast, macrophages did not produce IL-2 upon bacterial stimulation. Thus, IL-2 is an additional key cytokine that confers unique T cell stimulatory capacity to DCs.
Assuntos
Células Dendríticas/imunologia , Interleucina-2/biossíntese , Ativação Linfocitária , Animais , Medula Óssea/crescimento & desenvolvimento , Células Cultivadas , Perfilação da Expressão Gênica , Bactérias Gram-Negativas/imunologia , Interleucina-2/genética , Interleucina-2/fisiologia , Cinética , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , Linfócitos T/imunologiaRESUMO
Dendritic cell (DC) maturation is essential for the initiation of T-dependent immune responses. Nuclear factor kappa B/Rel (NF kappa B/Rel) transcription factors are ubiquitously expressed signalling molecules, known to regulate the transcription of a large number of genes involved in immune responses, including cytokines such as IL-1, IL-6, TNF-alpha and cell surface molecules (MHC class I and II, B7.2). In this study, we have compared the activation of five members of the NF-kappa B family, p65, c-Rel, p50, RelB and p52, during DC maturation in response to lipopolysaccharide (LPS) and to Salmonella typhimurium. We have shown that although the translocation of NF-kappa B occurred very early, 30 min after treatment with both S. typhimurium and LPS, bacteria-induced NF-kappa B activation was more pronounced. Four out of five members, i.e. p65, c-Rel, p50 and RelB, were similarly activated upon the two stimuli but with different kinetics. Indeed, we have observed that p65, c-Rel and p50 were translocated early, whereas RelB was translocated later in DC activation. This differential regulation suggests that the various members of NF-kappa B family can mediate distinct functions of DC physiology.
Assuntos
Células Dendríticas/imunologia , Lipopolissacarídeos/imunologia , NF-kappa B/metabolismo , Salmonella typhimurium/imunologia , Transdução de Sinais , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Meios de Cultura , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Camundongos , Subunidade p50 de NF-kappa B , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Fator de Transcrição RelA , Fator de Transcrição RelB , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The C-saccharide analogue of the GalNAc (Tn epitope) has been covalently linked to the T cell epitope peptide (328)(-)(340)OVA using a chemoselective convergent synthetic approach. In this way, a non-hydrolyzable synthetic vaccine was obtained composed by a B epitope conjugated to a T cell epitope. This compound was tested in a proliferation assay with spleen cells from DO11.10 mice. The molecule was recognized by transgenic T cells although at a slightly lower efficiency if compared with the reference peptide OVA. An additional experiment with dendritic cells fixed with glutaraldehyde shows that the glycopeptide can bind to extracellular MHC molecules without need of internalization and processing and that the C-glycoside part does not interfere with TCR recognition. These observations constitute an important starting point for the use of this molecule as vaccine against the Tn-expressing TA3-Ha mouse mammary carcinoma.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Vacinas Anticâncer/síntese química , Vacinas Anticâncer/farmacologia , Glicopeptídeos/imunologia , Monossacarídeos/imunologia , Animais , Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/metabolismo , Vacinas Anticâncer/imunologia , Linhagem Celular , Reagentes de Ligações Cruzadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Glicopeptídeos/síntese química , Glicosídeos/química , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Monossacarídeos/química , Ovalbumina/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Dendritic cells (DCs) are considered the most potent antigen-presenting cells and probably the only ones able to prime naive T cells. Indeed, DCs are distributed in tissues that interface the external environment, where they act as sentinels for incoming bacteria, viruses, and fungi. We have previously analyzed the capacity of DCs to interact with bacteria, and we have shown that bacteria can act as "Trojan horses," delivering heterologous proteins to DCs in a processed form that allows extremely efficient loading of both MHC class I and class II molecules. In this study, we have optimized the usage of recombinant bacteria as an antigen delivery system for DCs, with the aim to develop a new DC vaccination strategy in antitumor immunity. We have focused on a low immunogenic antigen, the tyrosinase-related protein-2 (Trp-2), a self-antigen expressed in mouse and human melanoma for which induction of antitumor immunity has proven to be very ineffective. We have given mice injections of either Trp-2/recombinant bacteria-loaded DCs or with bacteria alone engineered to express the Trp-2 melanoma antigen. We have shown that only DCs loaded with recombinant bacteria, but not with wild-type bacteria, were able to induce Trp-2-specific CTLs and immunity against the B16 tumor. Immunity was obtained in experiments of tumor vaccination as well as in experiments of tumor therapy. When therapy with bacteria-loaded DCs was performed in B16 tumor-bearing mice, 60% of the treated mice were tumor free 2 months after the initial tumor growth.
Assuntos
Antígenos de Neoplasias/metabolismo , Células Dendríticas/metabolismo , Oxirredutases Intramoleculares/metabolismo , Animais , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Fenótipo , Salmonella typhimurium/metabolismo , Baço/citologia , Baço/metabolismo , Linfócitos T Citotóxicos/metabolismo , Fatores de TempoRESUMO
Penetration of the gut mucosa by pathogens expressing invasion genes is believed to occur mainly through specialized epithelial cells, called M cells, that are located in Peyer's patches. However, Salmonella typhimurium that are deficient in invasion genes encoded by Salmonella pathogenicity island 1 (SPI1) are still able to reach the spleen after oral administration. This suggests the existence of an alternative route for bacterial invasion, one that is independent of M cells. We report here a new mechanism for bacterial uptake in the mucosa tissues that is mediated by dendritic cells (DCs). DCs open the tight junctions between epithelial cells, send dendrites outside the epithelium and directly sample bacteria. In addition, because DCs express tight-junction proteins such as occludin, claudin 1 and zonula occludens 1, the integrity of the epithelial barrier is preserved.