RESUMO
BACKGROUND: Some genomic alterations in non-small cell lung cancer (NSCLC) are known to differ according to race, sex, or age. These studies have been limited in sample size and thus they cannot detect the differences precisely and comprehensively. METHODS: Tissue-based comprehensive genomic profiling was performed on 75,362 patients with NSCLC from the United States during routine clinical care. Additionally, we examined data of a Japanese NSCLC cohort with 1,019 patients. In the US cohort, 296 genes were examined for pathogenic alterations. Predominant genetic ancestry was inferred using a SNP-based approach, and patients were categorized into European (EUR), African (AFR), East Asian (EAS), Admixed American (AMR), and South Asian (SAS) ancestry groups. Patients were additionally stratified by histologic type, age (<40/≥40 years, <75/≥75 years), and sex. The prevalence of high tumor mutational burden (TMB-High) and microsatellite instability status was also calculated. RESULTS: Stratified by ancestry, EGFR alterations were significantly enriched in EAS versus other ancestry groups. The prevalence of ALK was significantly higher in the AMR, EAS, and SAS patients than in AFR and EUR patients. KRAS and STK11 were enriched in EUR and AFR patients versus other groups. TMB-High was significantly enriched in AFR patients versus all other groups. An analysis based on sex revealed differences in prevalence of alterations in 80 genes and TMB-High status. For example, EGFR, ALK, BRAF, and KRAS alterations were significantly enriched in females, whereas TP53, STK11, KEAP1, and TMB-High were significantly enriched in males. With respect to age, the prevalence of alterations in 41 genes, including ALK, RET, MET, EGFR, STK11, KEAP1, BRAF, and KRAS, as well as TMB-High, were significantly different between patients aged <40 years and those aged ≥40 years. CONCLUSIONS: Comprehensive analysis from a large real-world dataset revealed ancestry-associated differences in genomic alterations in NSCLC. Age- and sex-related differences in prevalence of genomic alterations and TMB-High status were also observed.
RESUMO
Colistin is a cationic cyclic antimicrobial peptide used as a last resort against multidrug-resistant gram-negative bacteria. To understand the factors involved in colistin susceptibility, we screened colistin-sensitive mutants from an E. coli gene-knockout library (Keio collection). The knockout of purA, whose product catalyzes the synthesis of adenylosuccinate from IMP in the de novo purine synthesis pathway, resulted in increased sensitivity to colistin. Adenylosuccinate is subsequently converted to AMP, which is phosphorylated to produce ADP, a substrate for ATP synthesis. The amount of ATP was lower in the purA-knockout mutant than that in the wild-type strain. ATP synthesis is coupled with proton transfer, and it contributes to the membrane potential. Using the membrane potential probe, 3,3'-diethyloxacarbocyanine iodide [DiOC2(3)], we found that the membrane was hyperpolarized in the purA-knockout mutant compared to that in the wild-type strain. Treatment with the proton uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), abolished the hyperpolarization and colistin sensitivity in the mutant. The purA-knockout mutant exhibited increased sensitivity to aminoglycosides, kanamycin, and gentamicin; their uptake requires a membrane potential. Therefore, the knockout of purA, an adenylosuccinate synthase, decreases ATP synthesis concurrently with membrane hyperpolarization, resulting in increased sensitivity to colistin.
Assuntos
Adenilossuccinato Sintase , Escherichia coli , Escherichia coli/genética , Colistina/farmacologia , Prótons , Antibacterianos/farmacologia , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Trifosfato de Adenosina , Testes de Sensibilidade MicrobianaRESUMO
AIMS: In Japan, the use of comprehensive genomic profiling (CGP) is only available for cancer patients who have no standard of care (SoC), or those who have completed SoC. This may lead to missed treatment opportunities for patients with druggable alterations. In this study, we evaluated the potential impact of CGP testing before SoC on medical costs and clinical outcome in untreated patients with advanced or recurrent biliary tract cancer (BTC), non-squamous non-small cell lung cancer (NSQ-NSCLC), or colorectal cancer (CRC) in Japan between 2022 and 2026. MATERIALS AND METHODS: We constructed a decision-tree model reflecting the healthcare environment of Japan, to estimate the clinical outcome and medical costs impact of CGP testing by comparing two groups (with vs without CGP testing before SoC). The epidemiological parameters, detection rates of druggable alterations, and overall survival were collected from literature and claims databases in Japan. Treatment options selected based on druggable alterations were set in the model based on clinical experts' opinions. RESULTS: In 2026, the number of untreated patients with advanced or recurrent BTC, NSQ-NSCLC, and CRC was estimated to be 8600, 32,103, and 24,896, respectively. Compared with the group without CGP testing before SoC, CGP testing before SoC increased druggable alteration detection and treatment rate with matched therapies in all three cancer types. The medical costs per patient per month were estimated to increase with CGP testing before SoC in the three cancer types by 19,600, 2900, and 2200 JPY (145, 21, and 16 USD), respectively. LIMITATIONS: Only those druggable alterations with matched therapies were considered in the analysis model, while the potential impact of other genomic alterations provided by CGP testing was not considered. CONCLUSIONS: The present study suggested that CGP testing before SoC may improve patient outcomes in various cancer types with a limited and controllable increase in medical costs.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Japão , Recidiva Local de Neoplasia/genética , GenômicaRESUMO
Dendritic cells (DCs) take up antigens derived from pathogens such as bacteria and viruses, and from tumor cells and induce the activation of antigen-specific T cells through major histocompatibility complex (MHC)-mediated antigen presentation. Mainstream cigarette smoke extract (CSE) has various effects, and the effects of its major components, nicotine and tar, have been analyzed extensively. Recently, the physiological effects of nicotine- and tar-removed CSE (cCSE) have also been reported. However, the effects of cCSE on DC-mediated immune responses remain unknown. In this study, we found that cCSE enhanced lipopolysaccharide (LPS)-stimulated induction of the expression of MHC-I and MHC-II on the cell surface of mouse bone marrow-derived DCs (BMDCs). In contrast, cCSE suppressed the induction of CD86 induced by stimulation with curdlan and interferon-γ (IFN-γ). In addition, cCSE suppressed the production of IL-12, IL-23, and IL-10 by LPS and curdlan stimulation. In the presence of cCSE, LPS-stimulated BMDCs showed enhanced activation of CD4 and CD8 T cells and increased IL-2 production from T cells by antigen presentation in a mixed-leukocyte reaction assay. In contrast, cCSE did not affect the activation of T cells by curdlan- or IFN-γ-stimulated BMDCs, and curdlan-stimulated BMDCs suppressed IL-17 production from T cells and enhanced IFN-γ production. These results suggest that cCSE has different effects on the activation signals induced by LPS, curdlan, and IFN-γ in BMDCs and modulates the antigen presentation function of BMDCs.
Assuntos
Apresentação de Antígeno , Fumar Cigarros , Camundongos , Animais , Nicotina/farmacologia , Nicotina/metabolismo , Lipopolissacarídeos/metabolismo , Medula Óssea/metabolismo , Interferon gama/metabolismo , Células Dendríticas , Camundongos Endogâmicos C57BLRESUMO
Dendritic cells (DCs) present foreign antigens to T cells via the major histocompatibility complex (MHC), thereby inducing acquired immune responses. ATP accumulates at sites of inflammation or in tumor tissues, which triggers local inflammatory responses. However, it remains to be clarified how ATP modulates the functions of DCs. In this study, we investigated the effects of extracellular ATP on mouse bone marrow-derived dendritic cells (BMDCs) as well as the potential for subsequent T cell activation. We found that high concentrations of ATP (1 mM) upregulated the cell surface expression levels of MHC-I, MHC-II, and co-stimulatory molecules CD80 and CD86 but not those of co-inhibitory molecules PD-L1 and PD-L2 in BMDCs. Increased surface expression of MHC-I, MHC-II, CD80, and CD86 was inhibited by a pan-P2 receptor antagonist. In addition, the upregulation of MHC-I and MHC-II expression was inhibited by an adenosine P1 receptor antagonist and by inhibitors of CD39 and CD73, which metabolize ATP to adenosine. These results suggest that adenosine is required for the ATP-induced upregulation of MHC-I and MHC-II. In the mixed leukocyte reaction assay, ATP-stimulated BMDCs activated CD4 and CD8T cells and induced interferon-γ (IFN-γ) production by these T cells. Collectively, these results suggest that high concentrations of extracellular ATP upregulate the expression of antigen-presenting and co-stimulatory molecules but not that of co-inhibitory molecules in BMDCs. Cooperative stimulation of ATP and its metabolite adenosine was required for the upregulation of MHC-I and MHC-II. These ATP-stimulated BMDCs induced the activation of IFN-γ-producing T cells upon antigen presentation.
Assuntos
Células Dendríticas , Linfócitos T , Camundongos , Animais , Apresentação de Antígeno , Ativação Linfocitária , Trifosfato de Adenosina/metabolismoRESUMO
Bacterial and viral respiratory infections can initiate acute lung injury and acute respiratory distress syndrome. Neutrophils and their granule enzymes, including neutrophil elastase, are key mediators of the pathophysiology of acute respiratory failure. Although intracellular neutrophil elastase functions as a host defensive factor against pathogens, its leakage into airway spaces induces degradation of host connective tissue components. This leakage disrupts host innate immune responses via proteolytic cleavage of Toll-like receptors and cytokines. Here, we investigated whether neutrophils possess proteases that cleave adaptive immune molecules. We found that expression of the human leukocyte antigen (HLA) class II molecule HLA-DP ß1 was decreased in THP-1-derived macrophages treated with supernatants from dead neutrophils. This decreased HLA-DP ß1 expression was counteracted by treatment with neutrophil elastase inhibitor, suggesting proteolytic cleavage of HLA-DP ß1 by neutrophil elastase. SDS-PAGE showed that neutrophil elastase cleaved recombinant HLA-DP α1, -DP ß1, -DQ α1, -DQ ß1, -DR α, and -DR ß1. Neutrophil elastase also cleaved HLA-DP ß1 on extracellular vesicles isolated from macrophages without triggering morphological changes. Thus, leakage of neutrophil elastase may disrupt innate immune responses, antigen presentation, and T cell activation. Additionally, inhibition of neutrophil elastase is a potential therapeutic option for treating bacterial and viral pneumonia.
Assuntos
Antígenos de Histocompatibilidade Classe II/metabolismo , Elastase de Leucócito/metabolismo , Pneumonia Pneumocócica/metabolismo , Proteólise , Animais , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Vesículas Extracelulares/metabolismo , Humanos , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Neutrófilos/metabolismo , Proteínas Recombinantes/metabolismo , Streptococcus pneumoniae/fisiologia , Células THP-1 , Traqueia/microbiologiaRESUMO
In addition to antigen presentation to CD4+ T cells, aggregation of cell surface major histocompatibility complex class II (MHC-II) molecules induces signal transduction in antigen presenting cells that regulate cellular functions. We previously reported that crosslinking of MHC-II induced the endocytosis of MHC-II, which was associated with decreased surface expression levels in murine dendritic cells (DCs) and resulted in impaired activation of CD4+ T cells. However, the downstream signal that induces MHC-II endocytosis remains to be elucidated. In this study, we found that the crosslinking of MHC-II induced intracellular Ca2+ mobilization, which was necessary for crosslinking-induced MHC-II endocytosis. We also found that these events were suppressed by inhibitors of Syk and phospholipase C (PLC). Treatments with a phorbol ester promoted MHC-II endocytosis, whereas inhibitors of protein kinase C (PKC) suppressed crosslinking-induced endocytosis of MHC-II. These results suggest that PKC could be involved in this process. Furthermore, crosslinking-induced MHC-II endocytosis was suppressed by inhibitors of clathrin-dependent endocytosis. Our results indicate that the crosslinking of MHC-II could stimulate Ca2+ mobilization and induce the clathrin-dependent endocytosis of MHC-II in murine DCs.
Assuntos
Clatrina/metabolismo , Células Dendríticas/imunologia , Endocitose/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteína Quinase C/metabolismo , Animais , Apresentação de Antígeno/imunologia , Células da Medula Óssea/citologia , Cálcio/metabolismo , Células Cultivadas , Clatrina/antagonistas & inibidores , Reagentes de Ligações Cruzadas/metabolismo , Endocitose/imunologia , Estrenos/farmacologia , Masculino , Camundongos , Ésteres de Forbol/farmacologia , Proteína Quinase C/antagonistas & inibidores , Pirrolidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Organismos Livres de Patógenos Específicos , Estaurosporina/farmacologia , Estilbenos/farmacologia , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
The use of non-human animal models for infection experiments is important for investigating the infectious processes of human pathogenic bacteria at the molecular level. Mammals, such as mice and rabbits, are also utilized as animal infection models, but large numbers of animals are needed for these experiments, which is costly, and fraught with ethical issues. Various non-mammalian animal infection models have been used to investigate the molecular mechanisms of various human pathogenic bacteria, including Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa. This review discusses the desirable characteristics of non-mammalian infection models and describes recent non-mammalian infection models that utilize Caenorhabditis elegans, silkworm, fruit fly, zebrafish, two-spotted cricket, hornworm, and waxworm.
Assuntos
Infecções Bacterianas/microbiologia , Caenorhabditis elegans/microbiologia , Modelos Animais de Doenças , Drosophila melanogaster/microbiologia , Gryllidae/microbiologia , Peixe-Zebra/microbiologia , Animais , Bactérias/patogenicidade , Bombyx/microbiologia , Humanos , Larva/microbiologia , Manduca/microbiologia , Mariposas/microbiologiaRESUMO
Extracellular ATP released from stimulated and/or damaged cells modulates physiological responses via stimulation of various purinoceptors. We previously showed that ATP potentiated the Ag-induced mast cell (MC) degranulation via purinoceptors pharmacologically similar to the ionotropic P2X4 receptor. In this study, we investigated the role of P2X4 receptor in MC degranulation induced by stimulation of IgE-FcεRI complex with Ag, using bone marrow-derived MCs (BMMCs) prepared from wild type and P2X4 receptor-deficient (P2rx4-/- ) mice. ATP significantly increased Ag-induced degranulation in BMMCs prepared from wild type mice. This effect of ATP was reduced in BMMCs prepared from P2rx4-/- mice. The potentiating effect of ATP was restored by expressing P2X4 receptor in P2rx4-/- BMMCs. The P2X4 receptor-mediated effects were maintained even after differentiating into the connective tissue-type MCs. P2X4 receptor stimulation did not affect the Ag-induced Ca2+ response but enhanced Ag-induced early signals, such as tyrosine phosphorylation of Syk and phospholipase C-γ. Interestingly, these effects of ATP on Syk phosphorylation were not impaired by pretreatment with Cu2+, an inhibitor of the P2X4 receptor channel, or removal of external Ca2+, suggesting that a mechanisms other than Ca2+ influx through ion channel activity may be involved. In vivo experiments revealed that systemic and intradermal passive anaphylaxis responses were significantly alleviated in P2rx4-/- mice. Taken together, the present data suggest that the P2X4 receptor plays an essential role in ATP-induced upregulation of MC degranulation in response to Ag, and also contributes to the Ag-induced allergic response in vivo.
Assuntos
Trifosfato de Adenosina/metabolismo , Antígenos/metabolismo , Degranulação Celular/fisiologia , Hipersensibilidade/metabolismo , Mastócitos/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Anafilaxia/metabolismo , Animais , Células da Medula Óssea/metabolismo , Cálcio/metabolismo , Imunoglobulina E/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de IgE/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Steroidal anti-inflammatory drugs are widely used for the treatment of chronic cutaneous inflammation, such as atopic dermatitis, although it remains unknown how they modulate cutaneous mast cell functions. We investigated the effects of prolonged treatment with a synthetic glucocorticoid, dexamethasone, on murine connective tissue-type mast cells using in vitro and in vivo models. Our connective tissue-type bone marrow-derived cultured mast cell model was found to be sensitive to mast cell secretagogues, such as compound 48/80 and substance P, and higher expression levels of α subunit of a trimeric G protein, Gi1, and several Mas-related G protein-coupled receptor (Mrgpr) subtypes were observed in comparison with immature cultured mast cells. Secretagogue-induced degranulation and up-regulation of these genes was suppressed when cultured in the presence of dexamethasone. The profiles of granule constituents were drastically altered by dexamethasone. Topical application of dexamethasone down-modulated secretagogue-induced degranulation and the expression levels of several Mrgpr subtypes in cutaneous tissue. These results suggest that mast cell-mediated IgE-independent cutaneous inflammation could be suppressed by steroidal anti-inflammatory drugs through the down-regulation of G αi1 and several Mrgpr subtypes in mast cells.
Assuntos
Degranulação Celular , Células do Tecido Conjuntivo/citologia , Dexametasona/farmacologia , Imunoglobulina E/metabolismo , Mastócitos/fisiologia , Células 3T3 , Animais , Células da Medula Óssea/citologia , Degranulação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histamina/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , RNA/metabolismo , Pele/irrigação sanguínea , Pele/efeitos dos fármacosRESUMO
Mast cells play a pivotal role in allergic reactions and inflammations. Aggregation of the high affinity IgE receptor (FcεRI) eventually leads to the release of granule components such as histamine, as well as the de novo synthesis of inflammatory cytokines and lipid mediators. These substances are involved in the development of allergy and inflammation. Therefore, efficient inhibitors of mast cell activation would be therapeutically beneficial. We previously demonstrated that the synthetic peptide derived from the NH2-terminal region (2-17: GNIFANLFKGLFGKKE) of a small GTPase ARF1 (ADP-ribosylation factor1) inhibited FcεRI-induced mast cell degranulation. However, detailed structure-activity relationship study of NH2-terminal portion of ARF1 peptide has not been done. In addition, it is still unclear whether the NH2-terminal peptide of ARF1 suppresses FcεRI-induced production of cytokines and lipid mediators such as leukotriene C4 (LTC4) from mast cells. Here we show that amino acid residues K10-K16 are necessary for ARF1 peptide to efficiently inhibit FcεRI-induced activation of bone marrow-derived mast cells (BMMCs), indicated by decreased mast cell degranulation, cytokine secretion and leukotriene release. Furthermore, we show that ARF1 peptide inhibits IgE-mediated passive cutaneous anaphylaxis reaction. Our results suggest that the peptide derived from ARF1 could be developed into a novel anti-allergic agent for therapeutic intervention in allergy and mast cell-related pathologies.
Assuntos
Fator 1 de Ribosilação do ADP/imunologia , Antialérgicos/imunologia , Degranulação Celular/imunologia , Mastócitos/imunologia , Peptídeos/imunologia , Receptores de IgE/imunologia , Animais , Citocinas/imunologia , Leucotrieno C4/imunologia , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Accumulating evidence suggests that mast cells play critical roles in disruption and maintenance of intestinal homeostasis, although it remains unknown how they affect the local microenvironment. Interleukin-9 (IL-9) was found to play critical roles in intestinal mast cell accumulation induced in various pathological conditions, such as parasite infection and oral allergen-induced anaphylaxis. Newly recruited intestinal mast cells trigger inflammatory responses and damage epithelial integrity through release of a wide variety of mediators including mast cell proteases. We established a novel culture model (IL-9-modified mast cells, MCs/IL-9), in which murine IL-3-dependent bone-marrow-derived cultured mast cells (BMMCs) were further cultured in the presence of stem cell factor and IL-9. In MCs/IL-9, drastic upregulation of Mcpt1 and Mcpt2 was found. Although histamine storage and tryptase activity were significantly downregulated in the presence of SCF and IL-9, this was entirely reversed when mast cells were cocultured with a murine fibroblastic cell line, Swiss 3T3. MCs/IL-9 underwent degranulation upon IgE-mediated antigen stimulation, which was found to less sensitive to lower concentrations of IgE in comparison with BMMCs. This model might be useful for investigation of the spatiotemporal changes of newly recruited intestinal mast cells.
Assuntos
Interleucina-9/farmacologia , Mastócitos/imunologia , Fator de Células-Tronco/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Células Cultivadas , Quimases/metabolismo , Histamina/metabolismo , Imunoglobulina E/imunologia , Interleucina-3/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/enzimologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Células Swiss 3T3RESUMO
Accumulating evidence suggests that histamine synthesis induced in several types of tumor tissues modulates tumor immunity. We found that a transient histamine synthesis was induced in CD11bâºGr-1⺠splenocytes derived from BALB/c mice transplanted with a syngeneic colon carcinoma, CT-26, when they were co-cultured with CT-26 cells. Significant levels of IFN-γ were produced under this co-culture condition. We explored the modulatory roles of histamine on IFN-γ production and found that several histamine receptor antagonists, such as pyrilamine, diphenhydramine, JNJ7777120, and thioperamide, could significantly suppress IFN-γ production. However, suppression of IFN-γ production by these antagonists was also found when splenocytes were derived from the Hdc-/- BALB/c mice. Suppressive effects of these antagonists were found on IFN-γ production induced by concanavalin A or the combination of an anti-CD3 antibody and an anti-CD28 antibody in a histamine-independent manner. Murine splenocytes were found to express H1 and H2 receptors, but not H3 and H4 receptors. IFN-γ production in the Hh1r-/- splenocytes induced by the combination of an anti-CD3 antibody and an anti-CD28 antibody was significantly suppressed by these antagonists. These findings suggest that pyrilamine, diphenhydramine, JNJ7777120, and thioperamide can suppress IFN-γ production in activated splenocytes in a histamine-independent manner.
Assuntos
Antagonistas dos Receptores Histamínicos/farmacologia , Interferon gama/biossíntese , Baço/metabolismo , Animais , Linhagem Celular Tumoral , Histamina/genética , Histamina/metabolismo , Interferon gama/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H2/genética , Receptores Histamínicos H2/metabolismo , Baço/patologiaRESUMO
Antigenic peptide-loaded MHC class II molecules (peptide-MHC class II) are constitutively expressed on the surface of professional antigen-presenting cells (APCs), including dendritic cells, B cells, macrophages and thymic epithelial cells, and are presented to antigen-specific CD4(+) T cells. The mechanisms of antigen uptake, the nature of the antigen processing compartments and the lifetime of cell surface peptide-MHC class II complexes can vary depending on the type of APC. It is likely that these differences are important for the function of each distinct APC subset in the generation of effective adaptive immune responses. In this Review, we describe our current knowledge of the mechanisms of uptake and processing of antigens, the intracellular formation of peptide-MHC class II complexes, the intracellular trafficking of peptide-MHC class II complexes to the APC plasma membrane and their ultimate degradation.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos/imunologia , Antígenos/metabolismo , Membrana Celular/imunologia , Membrana Celular/metabolismo , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Peptídeos/imunologia , Transporte ProteicoRESUMO
Mast cells are involved in various immunological responses, although it remains unknown how their terminal differentiation is regulated. We previously established a culture model that mimics the process of mast cell maturation in the cutaneous tissue and found that growth factor independent 1 (Gfi1) was up-regulated whereas its paralogue Gfi1b down-regulated. Here we investigated the roles of Gfi1 and Gfi1b in the process of mast cell maturation using a murine mast cell line, MC9. Gfi1 and Gfi1b cDNAs were stably expressed in MC9 cells using the recombinant lentivirus. Histamine synthesis was significantly induced by stem cell factor (SCF) alone, whereas tryptase expression was significantly augmented in the presence of both SCF and Swiss 3T3 cells. Since exogenously expressed Gfi1 and Gfi1b might affect their expression levels in MC9 cells, we investigated the relationship between the expression profiles of Gfi1/Gfi1b proteins and maturation indices, such as histamine synthesis and tryptase expression. The comparison suggested that histamine synthesis during the co-culture period was positively regulated by Gfi1b while augmented expression of tryptase was abolished by one-sided expression of Gfi1/Gfi1b. Our findings indicated the involvement of Gfi1 and Gfi1b in the process of murine mast cell maturation.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/genética , Histamina/genética , Mastócitos/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Ativação Transcricional , Triptases/genética , Células 3T3 , Animais , Linhagem Celular , Técnicas de Cocultura , DNA Complementar , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Histamina/biossíntese , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Fator de Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Triptases/metabolismoRESUMO
As sentinels of the immune system, dendritic cells (DCs) continuously generate and turnover antigenic peptide-MHC class II complexes (pMHC-II). pMHC-II generation is a complex process that involves many well-characterized MHC-II biosynthetic intermediates; however, the mechanisms leading to MHC-II turnover/degradation are poorly understood. We now show that pMHC-II complexes undergoing clathrin-independent endocytosis from the DC surface are efficiently ubiquitinated by the E3 ubiquitin ligase March-I in early endosomes, whereas biosynthetically immature MHC-II-Invariant chain (Ii) complexes are not. The inability of MHC-II-Ii to serve as a March-I substrate is a consequence of Ii sorting motifs that divert the MHC-II-Ii complex away from March-I(+) early endosomes. When these sorting motifs are mutated, or when clathrin-mediated endocytosis is inhibited, MHC-II-Ii complexes internalize by using a clathrin-independent endocytosis pathway and are now ubiquitinated as efficiently as pMHC-II complexes. These data show that the selective ubiquitination of internalizing surface pMHC-II in March-I(+) early endosomes promotes degradation of "old" pMHC-II and spares forms of MHC-II that have not yet loaded antigenic peptides or have not yet reached the DC surface.
Assuntos
Células Dendríticas/imunologia , Endossomos/metabolismo , Complexos Multiproteicos/metabolismo , Peptídeos/metabolismo , Biotinilação , Genes MHC da Classe II/genética , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Lisossomos/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Peptídeos/imunologia , Proteólise , RNA Interferente Pequeno/genética , UbiquitinaçãoRESUMO
Major histocompatibility complex class II molecules (MHC-II) on antigen presenting cells (APCs) engage the TCR on antigen-specific CD4 T cells, thereby providing the specificity required for T cell priming and the induction of an effective immune response. In this study, we have asked whether antigen-loaded dendritic cells (DCs) that have been in contact with antigen-specific CD4 T cells retain the ability to stimulate additional naïve T cells. We show that encounter with antigen-specific primed CD4 T cells induces the degradation of surface MHC-II in antigen-loaded DCs and inhibits the ability of these DCs to stimulate additional naïve CD4 T cells. Cross-linking with MHC-II mAb as a surrogate for T-cell engagement also inhibits APC function and induces MHC-II degradation by promoting the clustering of MHC-II present in lipid raft membrane microdomains, a process that leads to MHC-II endocytosis and degradation in lysosomes. Encounter of DCs with antigen-specific primed T cells or engagement of MHC-II with antibodies promotes the degradation of both immunologically relevant and irrelevant MHC-II molecules. These data demonstrate that engagement of MHC-II on DCs after encounter with antigen-specific primed CD4 T cells promotes the down-regulation of cell surface MHC-II in DCs, thereby attenuating additional activation of naïve CD4 T cells by these APCs.
Assuntos
Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colesterol/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Apresentação Cruzada/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos/imunologia , Proteólise/efeitos dos fármacosRESUMO
Appropriate culture models for tissue mast cells are required to determine how they are involved in regulation of local immune responses. We previously established a culture model for cutaneous mast cells, in which bone marrow-derived immature mast cells were co-cultured with Swiss 3T3 fibroblasts in the presence of stem cell factor. In this study, we focused on the roles of hyaluronan, which is produced by the feeder fibroblasts and forms the extracellular matrix during the co-culture period. Hyaluronan synthesis was found to be mediated by hyaluronan synthase 2 (HAS2) expressed in Swiss 3T3 cells. A decreases in the amount of hyaluronan, which was achieved by retroviral expression of short hairpin RNA for Has2 or by addition of hyaluronidase, significantly enhanced the proliferation of the cultured mast cells without any obvious effects on their maturation. Although we previously demonstrated that CD44 is required for proliferation of cutaneous mast cells, the deficiency of hyaluronan did not affect the proliferation of the cultured mast cells that lack CD44. These findings suggest that the extracellular matrix containing hyaluronan may have a potential to restrict proliferation of cutaneous mast cells in a CD44-independent manner.
Assuntos
Fibroblastos/metabolismo , Glucuronosiltransferase/metabolismo , Ácido Hialurônico/metabolismo , Mastócitos/citologia , Animais , Células da Medula Óssea/citologia , Proliferação de Células , Células Cultivadas , Feminino , Técnicas de Silenciamento de Genes , Glucuronosiltransferase/genética , Receptores de Hialuronatos/genética , Hialuronan Sintases , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Swiss 3T3RESUMO
SRPX2 (Sushi repeat-containing protein, X-linked 2) has recently emerged as a multifunctional protein that is involved in seizure disorders, angiogenesis and cellular adhesion. Here, we analyzed this protein biochemically. SRPX2 protein was secreted with a highly posttranslational modification. Chondroitinase ABC treatment completely decreased the molecular mass of purified SRPX2 protein to its predicted size, whereas heparitinase, keratanase and hyaluroinidase did not. Secreted SRPX2 protein was also detected using an anti-chondroitin sulfate antibody. These results indicate that SRPX2 is a novel chondroitin sulfate proteoglycan (CSPG). Furthermore, a binding assay revealed that hepatocyte growth factor dose-dependently binds to SRPX2 protein, and a ligand-glycosaminoglycans interaction was speculated to be likely in proteoglycans. Regarding its molecular architecture, SRPX2 has sushi repeat modules similar to four other CSPGs/lecticans; however, the molecular architecture of SRPX2 seems to be quite different from that of the lecticans. Taken together, we found that SRPX2 is a novel CSPG that is overexpressed in gastrointestinal cancer cells. Our findings provide key glycobiological insight into SRPX2 in cancer cells and demonstrate that SRPX2 is a new member of the cancer-related proteoglycan family.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Neoplasias Gastrointestinais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Linhagem Celular Tumoral , Condroitina ABC Liase/metabolismo , Neoplasias Colorretais/genética , Neoplasias Gastrointestinais/genética , Regulação Neoplásica da Expressão Gênica , Glicosaminoglicanos/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Proteínas de Membrana , Proteínas de Neoplasias , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Whole genome-scale integrated analyses of exon array and array-comparative genomic hybridization are expected to enable the identification of unknown genetic features of cancer cells. Here, we evaluated this approach in 22 gastric and colorectal cancer cell lines, focusing on protein kinase genes and genes belonging to the cadherin-catenin family. Regarding alternative splicing patterns, several cancer cell lines predominantly expressed isoform 1 of protein kinase A catalytic subunit beta (PRKACB). Paired gastric cancer specimens demonstrated that isoform 1 of PRKACB was a novel cancer-related variant transcript in gastric cancers. In addition, the exon array analysis clearly identified exon 3 or exon 3-4 skipping in catenin beta 1, a short intron insertion with exon 9 skipping in CDH1, and a deletional transcript of CDH13. These abnormal transcripts were shown to have arisen from small genomic deletions. Meanwhile, an integrated analysis of 11 gastric cancer cell lines revealed that four cell lines amplified fibroblast growth factor receptor 2, with truncated forms observed in two of the cell lines. Gene amplification, and not the truncated form, was found to determine the sensitivity to a fibroblast growth factor receptor inhibitor, indicating that our cell line panel might be useful for cell-based evaluations of specific inhibitors. Using an integrated analysis, we identified several abnormal transcripts and genomic alterations in gastric and colorectal cancer cells. Our approach might enable genetic changes to be identified more efficiently, and the present results warrant further investigation using clinical samples and integrated analyses.