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BACKGROUND AND PURPOSE: Gastrointestinal tumours overexpress voltage-gated calcium (CaV3) channels (CaV3.1, 3.2 and 3.3). CaV3 channels regulate cell growth and apoptosis colorectal cancer. Gossypol, a polyphenolic aldehyde found in the cotton plant, has anti-tumour properties and inhibits CaV3 currents. A systematic study was performed on gossypol blocking mechanism on CaV3 channels and its potential anticancer effects in colon cancer cells, which express CaV3 isoforms. EXPERIMENTAL APPROACH: Transcripts for CaV3 proteins were analysed in gastrointestinal cancers using public repositories and in human colorectal cancer cell lines HCT116, SW480 and SW620. The gossypol blocking mechanism on CaV3 channels was investigated by combining heterologous expression systems and patch-clamp experiments. The anti-tumoural properties of gossypol were estimated by cell proliferation, viability and cell cycle assays. Ca2+ dynamics were evaluated with cytosolic and endoplasmic reticulum (ER) Ca2+ indicators. KEY RESULTS: High levels of CaV3 transcripts correlate with poor prognosis in gastrointestinal cancers. Gossypol blockade of CaV3 isoforms is concentration- and use-dependent interacting with the closed, activated and inactivated conformations of CaV3 channels. Gossypol and CaV3 channels down-regulation inhibit colorectal cancer cell proliferation by arresting cell cycles at the G0/G1 and G2/M phases, respectively. CaV3 channels underlie the vectorial Ca2+ uptake by endoplasmic reticulum in colorectal cancer cells. CONCLUSION AND IMPLICATIONS: Gossypol differentially blocked CaV3 channel and its anticancer activity was correlated with high levels of CaV3.1 and CaV3.2 in colorectal cancer cells. The CaV3 regulates cell proliferation and Ca2+ dynamics in colorectal cancer cells. Understanding this blocking mechanism maybe improve cancer therapies.
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Pathophysiological response after acute myocardial infarction (AMI) is described as a three-stage model involving temporal phenotypic modifications of both immune cells and fibroblasts: a primary inflammatory phase, followed by a reparative phase and a fibrous scar maturation phase. Purinergic receptors, particularly the P2Y11 receptor, have been reported to be involved in the regulation of inflammation after ischemia and could act for the resolution of inflammation after AMI. For the first time, we characterized the immuno-inflammatory and P2Y11 expression profiles of peripheral blood mononuclear cells (PBMC) from AMI patients and analyzed the consequences of presenting these cells to cardiac fibroblasts in vitro. PBMC from 178 patients were collected at various times after reperfused ST-segment elevation AMI, from H0 to M12. Expression level of P2RY11 and genes involved in tolerogenic profile of dendritic cells and T cell polarization were evaluated by RT-PCR. P2Y11 protein expression was assessed by flow cytometry. PBMC and human cardiac fibroblasts (HCF) were cocultured and α-SMA/vimentin ratio was analyzed by flow cytometry. Within the first 48 h after AMI, expression levels of HMOX1, STAT3 and CD4 increased while IDO1 and TBX21/GATA3 ratio decreased. Concomitantly, the expression of P2RY11 increased in both T and B cells. In vitro, PBMC collected at H48 after AMI induced an increase in α-SMA/vimentin ratio in HCF. Our results suggest that human PBMC display an evolving inflammatory profile with reparative characteristics the first two days after AMI and secrete soluble mediators leading to the fibroblastic proteins modification, thus participating to myocardial fibrosis.
Assuntos
Leucócitos Mononucleares , Infarto do Miocárdio , Humanos , Leucócitos Mononucleares/metabolismo , Vimentina/metabolismo , Infarto do Miocárdio/metabolismo , Inflamação/metabolismo , Fenótipo , Fibroblastos/metabolismoRESUMO
Proteinase 3 (PR3) is the main target antigen of antineutrophil cytoplasmic antibodies (ANCAs) in PR3-ANCA-associated vasculitis. A small fraction of PR3 is constitutively exposed on the surface of quiescent blood neutrophils in a proteolytically inactive form. When activated, neutrophils expose an induced form of membrane-bound PR3 (PR3mb) on their surface as well, which is enzymatically less active than unbound PR3 in solution due to its altered conformation. In this work, our objective was to understand the respective role of constitutive and induced PR3mb in the immune activation of neutrophils triggered by murine anti-PR3 mAbs and human PR3-ANCA. We quantified immune activation of neutrophils by the measurement of the production of superoxide anions and secreted protease activity in the cell supernatant before and after treatment of the cells by alpha-1 protease inhibitor that clears induced PR3mb from the cell surface. Incubation of TNFα-primed neutrophils with anti-PR3 antibodies resulted in a significant increase in superoxide anion production, membrane activation marker exposition, and secreted protease activity. When primed neutrophils were first treated with alpha-1 protease inhibitor, we observed a partial reduction in antibody-induced neutrophil activation, suggesting that constitutive PR3mb is sufficient to activate neutrophils. The pretreatment of primed neutrophils with purified antigen-binding fragments used as competitor significantly reduced cell activation by whole antibodies. This led us to the conclusion that PR3mb promoted immune activation of neutrophils. We propose that blocking and/or elimination of PR3mb offers a new therapeutic strategy to attenuate neutrophil activation in patients with PR3-ANCA-associated vasculitis.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Anticorpos Anticitoplasma de Neutrófilos , Mieloblastina , Animais , Humanos , Camundongos , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/metabolismo , Mieloblastina/imunologia , Mieloblastina/metabolismo , Neutrófilos/metabolismo , Inibidores de Proteases/metabolismo , Superóxidos/metabolismoRESUMO
Recent evidence showed that in response to elevated sodium dietary intakes, many body tissues retain Na+ ions for long periods of time and can reach concentrations up to 200 mM. This could modulate the immune system and be responsible for several diseases. However, studies brought contrasted results and the effects of external sodium on human dendritic cell (DC) responses to danger signals remain largely unknown. Considering their central role in triggering T cell response, we tested how NaCl-enriched medium influences human DCs properties. We found that DCs submitted to high extracellular Na+ concentrations up to 200 mM remain viable and maintain the expression of specific DC markers, however, their maturation, chemotaxis toward CCL19, production of pro-inflammatory cytokines and ROS in response to LPS were also partially inhibited. In line with these results, the T-cell allostimulatory capacity of DCs was also inhibited. Finally, our data indicate that high NaCl concentrations triggered the phosphorylation of SGK1 and ERK1/2 kinases. These results raised the possibility that the previously reported pro-inflammatory effects of high NaCl concentrations on T cells might be counterbalanced by a downregulation of DC activation.
Assuntos
Lipopolissacarídeos , Cloreto de Sódio , Humanos , Cloreto de Sódio/farmacologia , Cloreto de Sódio/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Diferenciação Celular , Cloretos/metabolismo , Cloretos/farmacologia , Células Dendríticas , Citocinas/metabolismo , Sódio/metabolismo , Sódio/farmacologia , Células CultivadasRESUMO
BACKGROUND: Zika virus (ZIKV), a member of the Flaviviridae family, has caused massive outbreaks of infection in tropical areas over the last decade and has now begun spreading to temperate countries. Little is currently known about the specific host factors involved in the intracellular life cycle of ZIKV. Flaviviridae viruses interact closely with host-cell lipid metabolism and associated secretory pathways. Another Flaviviridae, hepatitis C virus, is highly dependent on apolipoprotein E (ApoE) for the completion of its infectious cycle. We therefore investigated whether ZIKV also interacted with this protein. METHODS: ZIKV infections were performed on both liver and microglia derived cell lines in order to proceed to colocalization analysis and immunoprecipitation assays of ApoE and Zika envelope glycoprotein (Zika E). Transmission electron microscopy combined to immunogold labeling was also performed on the infected cells and related supernatant to study the association of ApoE and Zika E protein in the virus-induced membrane rearrangements and secreted particles, respectively. Finally, the potential of neutralization of anti-ApoE antibodies on ZIKV particles was studied. RESULT: We demonstrated an interaction between ApoE and the Zika E protein. This specific interaction was observed in virus-induced host-cell membrane rearrangements, but also on newly formed intracellular particles. The partial neutralizing effect of anti-ApoE antibody and the immunogold labeling of the two proteins on secreted virions indicates that this interaction is conserved during ZIKV intracellular trafficking and release. CONCLUSIONS: These data suggest that another member of the Flaviviridae also interacts with ApoE, indicating that this could be a common mechanism for the viruses from this family.
Assuntos
Flaviviridae , Infecção por Zika virus , Zika virus , Anticorpos Antivirais , Apolipoproteínas E , Linhagem Celular , Humanos , Proteínas do Envelope Viral , Vírion/metabolismoRESUMO
Metastatic progression is a major burden for breast cancer patients and is associated with the ability of cancer cells to overcome stressful conditions, such as nutrients deprivation and hypoxia, and to gain invasive properties. Autophagy and epithelial-to-mesenchymal transition are critical contributors to these processes. Here, we show that the P2X4 purinergic receptor is upregulated in breast cancer biopsies from patients and it is primarily localised in endolysosomes. We demonstrate that P2X4 enhanced invasion in vitro, as well as mammary tumour growth and metastasis in vivo. The pro-malignant role of P2X4 was mediated by the regulation of lysosome acidity, the promotion of autophagy and cell survival. Furthermore, the autophagic activity was associated with epithelial-to-mesenchymal transition (EMT), and this role of P2X4 was even more pronounced under metabolic challenges. Pharmacological and gene silencing of P2X4 inhibited both autophagy and EMT, whereas its rescue in knocked-down cells led to the restoration of the aggressive phenotype. Together, our results demonstrate a previously unappreciated role for P2X4 in regulating lysosomal functions and fate, promoting breast cancer progression and aggressiveness.
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Neoplasias da Mama , Receptores Purinérgicos P2X4 , Autofagia/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X4/metabolismoRESUMO
Colorectal cancer (CRC) is the second leading cause of death worldwide, with 0.9 million deaths per year. The metastatic stage of the disease is identified in about 20% of cases at the first diagnosis and is associated with low patient-survival rates. Voltage-gated sodium channels (NaV) are abnormally overexpressed in several carcinomas including CRC and are strongly associated with the metastatic behavior of cancer cells. Acidification of the extracellular space by Na+/H+ exchangers (NHE) contributes to extracellular matrix degradation and cell invasiveness. In this study, we assessed the expression levels of pore-forming α-subunits of NaV channels and NHE exchangers in tumor and adjacent non-malignant tissues from colorectal cancer patients, CRC cell lines and primary tumor cells. In all cases, SCN5A (gene encoding for NaV1.5) was overexpressed and positively correlated with cancer stage and poor survival prognosis for patients. In addition, we identified an anatomical differential expression of SCN5A and SLC9A1 (gene encoding for NHE-1) being particularly relevant for tumors that originated on the sigmoid colon epithelium. The functional activity of NaV1.5 channels was characterized in CRC cell lines and the primary cells of colon tumors obtained using tumor explant methodologies. Furthermore, we assessed the performance of two new small-molecule NaV1.5 inhibitors on the reduction of sodium currents, as well as showed that silencing SCN5A and SLC9A1 substantially reduced the 2D invasive capabilities of cancer cells. Thus, our findings show that both NaV1.5 and NHE-1 represent two promising targetable membrane proteins against the metastatic progression of CRC.
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In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag-gRNA complex nor distinguish gRNA packaging in single particles. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5'-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein-protein interactions.
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Produtos do Gene gag , HIV-1 , Nucleoproteínas , Regiões 5' não Traduzidas , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Genômica , HIV-1/genética , HIV-1/metabolismo , Microscopia Eletrônica de Transmissão , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , RNA Guia de Cinetoplastídeos , RNA Viral/genética , RNA Viral/metabolismo , Montagem de Vírus/genéticaRESUMO
The SCN4B gene, coding for the NaVß4 subunit of voltage-gated sodium channels, was recently found to be expressed in normal epithelial cells and down-regulated in several cancers. However, its function in normal epithelial cells has not been characterized. In this study, we demonstrated that reducing NaVß4 expression in MCF10A non-cancer mammary epithelial cells generated important morphological changes observed both in two-dimensional cultures and in three-dimensional cysts. Most notably, the loss of NaVß4 induced a complete loss of epithelial organisation in cysts and increased proteolytic activity towards the extracellular matrix. Loss of epithelial morphology was associated with an increased degradation of ß-catenin, reduced E-cadherin expression and induction of mesenchymal markers N-cadherin, vimentin, and α-SMA expression. Overall, our results suggest that Navß4 may participate in the maintenance of the epithelial phenotype in mammary cells and that its downregulation might be a determining step in early carcinogenesis.
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Células Epiteliais/metabolismo , Glândulas Mamárias Animais/citologia , Subunidades Proteicas/metabolismo , Subunidade beta-4 do Canal de Sódio Disparado por Voltagem/metabolismo , Animais , Linhagem Celular , Polaridade Celular , Regulação para Baixo , Células Epiteliais/citologia , Feminino , Humanos , Mesoderma/metabolismo , Fenótipo , Proteólise , beta Catenina/metabolismoRESUMO
Granulomatosis with polyangiitis (GPA) is a rare but serious necrotizing auto-immune vasculitis. GPA is mostly associated with the presence of Anti-Neutrophil Cytoplasmic Antibody (ANCA) targeting proteinase 3 (PR3-ANCA), a serine protease contained in neutrophil granules but also exposed at the membrane. PR3-ANCAs have a proven fundamental role in GPA: they bind neutrophils allowing their auto-immune activation responsible for vasculitis lesions. PR3-ANCAs bind neutrophil surface on the one hand by their Fab binding PR3 and on the other by their Fc binding Fc gamma receptors. Despite current therapies, GPA is still a serious disease with an important mortality and a high risk of relapse. Furthermore, although PR3-ANCAs are a consistent biomarker for GPA diagnosis, relapse management currently based on their level is inconsistent. Indeed, PR3-ANCA level is not correlated with disease activity in 25% of patients suggesting that not all PR3-ANCAs are pathogenic. Therefore, the development of new biomarkers to evaluate disease activity and predict relapse and new therapies is necessary. Understanding factors influencing PR3-ANCA pathogenicity, i.e. their potential to induce auto-immune activation of neutrophils, offers interesting perspectives in order to improve GPA management. Most relevant factors influencing PR3-ANCA pathogenicity are involved in their interaction with neutrophils: level of PR3 autoantigen at neutrophil surface, epitope of PR3 recognized by PR3-ANCA, isotype and glycosylation of PR3-ANCA. We detailed in this review the advances in understanding these factors influencing PR3-ANCA pathogenicity in order to use them as biomarkers and develop new therapies in GPA as part of a personalized approach.
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Anticorpos Anticitoplasma de Neutrófilos/imunologia , Granulomatose com Poliangiite/imunologia , Mieloblastina/imunologia , Neutrófilos/imunologia , Anticorpos Anticitoplasma de Neutrófilos/metabolismo , Biomarcadores/metabolismo , Granulomatose com Poliangiite/metabolismo , Granulomatose com Poliangiite/terapia , Humanos , Mieloblastina/metabolismo , Neutrófilos/metabolismo , Peroxidase/imunologia , Peroxidase/metabolismo , Ligação Proteica , Recidiva , Fatores de RiscoRESUMO
Granulomatosis with polyangiitis (GPA) is a severe autoimmune vasculitis associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA) mainly targeting proteinase 3 (PR3), a neutrophilic serine proteinase. PR3-ANCA binding to membrane-bound PR3 on neutrophils induce their auto-immune activation responsible for vascular lesions. However, the correlation between PR3-ANCA level and disease activity remains inconsistent, suggesting the existence of non-pathogenic PR3-ANCA. In order to prove their existence, we immortalized B lymphocytes from blood samples of GPA patients in remission having persistent PR3-ANCA to isolate non-activating PR3-ANCA. We obtained for the first time a non-activating human IgG1κ anti-PR3 monoclonal antibody (mAb) named 4C3. This new mAb binds soluble PR3 with a high affinity and membrane-bound PR3 on an epitope close to the PR3 hydrophobic patch and in the vicinity of the active site. 4C3 is able to bind FcγRIIA and FcγRIIIB and has a G2F glycosylation profile on asparagine 297. 4C3 did not induce activation of neutrophils and could inhibit human polyclonal PR3-ANCA-induced activation suggesting that 4C3 is non-pathogenic. This characteristic relies on the recognized epitope on PR3 rather than to the Fc portion properties. The existence of non-pathogenic PR3-ANCA, which do not activate neutrophils, could explain the persistence of high PR3-ANCA levels in some GPA patients in remission and why PR3-ANCA would not predict relapse. Finally, these results offer promising perspectives particularly regarding the understanding of PR3-ANCA pathogenicity and the development of new diagnostic and therapeutic strategies in GPA.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Granulomatose com Poliangiite/imunologia , Mieloblastina/imunologia , Idoso , Anticorpos Anticitoplasma de Neutrófilos/metabolismo , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Linfócitos B/enzimologia , Sítios de Ligação de Anticorpos , Biomarcadores/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Mapeamento de Epitopos , Epitopos , Feminino , Glicosilação , Granulomatose com Poliangiite/diagnóstico , Granulomatose com Poliangiite/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , Ativação de Neutrófilo , Estudo de Prova de ConceitoRESUMO
The acquisition of invasive capacities by carcinoma cells, i.e. their ability to migrate through and to remodel extracellular matrices, is a determinant process leading to their dissemination and to the development of metastases. these cancer cell properties have often been associated with an increased Rho-ROCK signalling, and ROCK inhibitors have been proposed for anticancer therapies. In this study we used the selective ROCK inhibitor, Y-27632, to address the participation of the Rho-ROCK signalling pathway in the invasive properties of SW620 human colon cancer cells. Contrarily to initial assumptions, Y-27632 induced the acquisition of a pro-migratory cell phenotype and increased cancer cell invasiveness in both 3- and 2-dimensions assays. This effect was also obtained using the other ROCK inhibitor Fasudil as well as with knocking down the expression of ROCK-1 or ROCK-2, but was prevented by the inhibition of NaV1.5 voltage-gated sodium channel activity. Indeed, ROCK inhibition enhanced the activity of the pro-invasive NaV1.5 channel through a pathway that was independent of gene expression regulation. In conclusions, our evidence identifies voltage-gated sodium channels as new targets of the ROCK signalling pathway, as well as responsible for possible deleterious effects of the use of ROCK inhibitors in the treatment of cancers.
Assuntos
Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Invasividade Neoplásica/patologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Hepatitis B virus (HBV) core protein (HBc) is essential to the formation of the HBV capsid. HBc contains two domains: the N-terminal domain corresponding to residues 1-140 essential to form the icosahedral shell and the C-terminal domain corresponding to a basic and phosphorylated peptide, and required for DNA replication. The role of these two domains for HBV capsid assembly was essentially studied in vitro with HBc purified from mammalian or non-mammalian cell lysates, but their respective role in living cells remains to be clarified. We therefore investigated the assembly of the HBV capsid in Huh7 cells by combining fluorescence lifetime imaging microscopy/Förster's resonance energy transfer, fluorescence correlation spectroscopy and transmission electron microscopy approaches. We found that wild-type HBc forms oligomers early after transfection and at a sub-micromolar concentration. These oligomers are homogeneously diffused throughout the cell. We quantified a stoichiometry ranging from ~170 to ~230 HBc proteins per oligomer, consistent with the visualization of eGFP-containingHBV capsid shaped as native capsid particles by transmission electron microscopy. In contrast, no assembly was observed when HBc-N-terminal domain was expressed. This highlights the essential role of the C-terminal domain to form capsid in mammalian cells. Deletion of either the third helix or of the 124-135 residues of HBc had a dramatic impact on the assembly of the HBV capsid, inducing the formation of mis-assembled oligomers and monomers, respectively. This study shows that our approach using fluorescent derivatives of HBc is an innovative method to investigate HBV capsid formation.
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Vírus da Hepatite B/genética , Hepatite B/genética , Proteínas do Core Viral/genética , Montagem de Vírus/genética , Capsídeo/metabolismo , Replicação do DNA , Hepatite B/virologia , Vírus da Hepatite B/patogenicidade , Humanos , Domínios Proteicos/genética , RNA Viral/genética , Replicação Viral/genéticaRESUMO
HBc is a small protein essential for the formation of the icosahedral HBV capsid. Its multiple roles in the replication cycle make this protein a promising target for the development of antiviral molecules. Based on the structure of HBc, a series of HBV assembly inhibitors, also known as capsid assembly modulators, were identified. We investigated the effect of BAY 41-4109, a heteroaryldihydropyrimidine derivative that promotes the assembly of a non-capsid polymer. We showed, by confocal microscopy, that BAY 41-4109 mediated HBc aggregation, mostly in the cytoplasm of Huh7 cells. Image analysis revealed that aggregate size depended on BAY 41-4109 concentration and treatment duration. Large aggregates in the vicinity of the nucleus were enclosed by invaginations of the nuclear envelope. This deformation of the nuclear envelope was confirmed by transmission electron microscopy (TEM) and immuno-TEM. These two techniques also revealed that the HBc aggregates were accumulations of capsid-like shells with an electron-dense material consisting of HBV core fragments. These findings, shedding light on the ultrastructural organization of HBc aggregates, provide insight into the mechanisms of action of BAY 41-4109 against HBV and will serve as a basis for comparison with other HBV capsid assembly inhibitors.
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Antivirais/farmacologia , Capsídeo/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Microscopia Eletrônica/métodos , Agregados Proteicos/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/ultraestrutura , Vírus da Hepatite B/genética , Humanos , Montagem de Vírus/efeitos dos fármacosRESUMO
We aimed to determine the role of cytomegalovirus (CMV)-infected donor cells in the development of a CMV-specific immune response in kidney transplant recipients. We assessed the CMV pp65-specific immune response by using interferon-É£ ELISPOT and dextramers in peripheral blood mononuclear cells from 115 recipients (D+R- 31, D+R + 44, D-R + 40) late after transplantation (mean 59 ± 42 months). Receiving a kidney from a D+ donor resulted in a higher number of IFN-É£-producing anti-CMV T cells (P = .004). This effect disappeared with the absence of shared HLA class I specificities between donors and recipients (P = .430). To confirm the role of donor cells in stimulating the expansion of newly developed CMV-specific CD8+ T cells after transplantation, we compared the number of HLA-A2-restricted CMV-specific CD8+ T cells in primo-infected recipients who received an HLA-A2 or non-HLA-A2 graft. The median of anti-CMV pp65 T cells restricted by HLA-A2 was very low for patients who received a non-HLA-A2 graft vs an HLA-A2 graft (300 [0-14638] vs. 17972 [222-85594] anti-CMV pp65 CD8+ T cells/million CD8+ T cells, P = .001). This adds new evidence that CMV-infected kidney donor cells present CMV peptides and drive an inflation of memory CMV-specific CD8+ T cells, likely because of frequent CMV replications within the graft.
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Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Genes MHC Classe I/imunologia , Rejeição de Enxerto/etiologia , Transplante de Rim/efeitos adversos , Leucócitos Mononucleares/imunologia , Doadores de Tecidos , Antígenos Virais/imunologia , Estudos Transversais , Infecções por Citomegalovirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/imunologia , Fosfoproteínas/imunologia , Estudos Retrospectivos , Linfócitos T Citotóxicos/imunologia , Proteínas da Matriz Viral/imunologiaRESUMO
BACKGROUND: Inflammatory bowel disease (IBD) is one of the most common chronic gastrointestinal diseases, but the underlying molecular mechanisms remain largely unknown. Studies of monogenic diseases can provide insight into the pathogenesis of IBD. OBJECTIVE: We thought to determine the underlying molecular causes of IBD occurring in 2 unrelated families in association with an immune deficiency. METHODS: We performed genetic linkage analysis and candidate gene sequencing on 13 patients from a large consanguineous family affected by early-onset IBD, progressive immune deficiency, and, in some cases, autoimmunity and alopecia, a condition we named enteropathy-lymphocytopenia-alopecia. The candidate gene was also sequenced in an unrelated patient with a similar phenotype. We performed histologic analysis of patients' intestinal biopsy specimens and carried out functional assays on PBMCs. Gut organoids derived from a patient's biopsy specimen were analyzed. RESULTS: We identified biallelic missense mutations in tetratricopeptide repeat domain 7A (TTC7A) in all patients from both families. The resulting TTC7A depletion modified the proliferation, adhesion, and migratory capacities of lymphocytes through inappropriate activation of the RhoA signaling pathway. Normal function was restored by wild-type TTC7A expression or addition of a RhoA kinase inhibitor. The growth and polarity of gut epithelial organoids were also found to be dependent on the RhoA signaling pathway. CONCLUSIONS: We show that TTC7A regulates the actin cytoskeleton dynamics in lymphocytes through the RhoA signaling pathway and is required in both lymphocytes and epithelial cells for maintaining equilibrium between cell proliferation, migration, polarization, and cell death. Our study highlights variability in the phenotypic expression resulting from TTC7A deficiency and outlines that impairment of both epithelial cells and lymphocytes cooperatively causes IBD.
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Alopecia , Doenças Inflamatórias Intestinais , Linfopenia , Proteínas/genética , Proteínas/imunologia , Adolescente , Adulto , Alopecia/genética , Alopecia/imunologia , Alopecia/patologia , Criança , Pré-Escolar , Colo/patologia , Duodeno/patologia , Feminino , Humanos , Lactente , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/patologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Antro Pilórico/patologia , Adulto Jovem , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/imunologia , Proteína rhoA de Ligação ao GTP/imunologiaAssuntos
Homeostase/genética , Sistema Imunitário/fisiologia , Intestinos/fisiologia , Proteínas/fisiologia , Polaridade Celular/genética , Enterócitos/fisiologia , Humanos , Atresia Intestinal/complicações , Atresia Intestinal/genética , Atresia Intestinal/imunologia , Imunodeficiência Combinada Severa/complicações , Imunodeficiência Combinada Severa/genética , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
Human dendritic cells (hDCs) produce IL-2 and express IL-2R α-chain (CD25), but the role of IL-2 in DC functions is not well defined. A recent study suggested that the main function of CD25 on hDCs was to transpresent IL-2 to activate T lymphocytes. Our results demonstrate the expression of the three chains of the IL-2R on hDCs and that IL-2 induces STAT5 phosphorylation. Interestingly, use of inhibitors of p-STAT5 revealed that IL-2 increases LPS-induced IFN-γ through STAT5 phosphorylation. Finally, we report that IL-2 increases the ability of hDCs to activate helpless CD8(+) T cells, most likely because of IL-2-triggered IFN-γ synthesis, as we previously described. For the first time, to our knowledge, we disclose that IL-2 induces monocyte-derived hDC's functional maturation and activation through IL-2R binding. Interestingly, our study suggests a direct effect of anti-CD25 mAbs on hDCs that may contribute to their clinical efficacy.
Assuntos
Células Dendríticas/imunologia , Interferon gama/imunologia , Interleucina-2/imunologia , Fator de Transcrição STAT5/imunologia , Anticorpos/farmacologia , Células Dendríticas/citologia , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-2/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-2/imunologia , Lipopolissacarídeos/farmacologia , Masculino , Monócitos/citologia , Monócitos/imunologia , Fosforilação/efeitos dos fármacos , Fosforilação/imunologiaRESUMO
Multiple intestinal atresia (MIA) is a rare cause of bowel obstruction that is sometimes associated with a combined immunodeficiency (CID), leading to increased susceptibility to infections. The factors underlying this rare disease are poorly understood. We characterized the immunological and intestinal features of 6 unrelated MIA-CID patients. All patients displayed a profound, generalized lymphocytopenia, with few lymphocytes present in the lymph nodes. The thymus was hypoplastic and exhibited an abnormal distribution of epithelial cells. Patients also had profound disruption of the epithelial barrier along the entire gastrointestinal tract. Using linkage analysis and whole-exome sequencing, we identified 10 mutations in tetratricopeptide repeat domain7A (TTC7A), all of which potentially abrogate TTC7A expression. Intestinal organoid cultures from patient biopsies displayed an inversion of apicobasal polarity of the epithelial cells that was normalized by pharmacological inhibition of Rho kinase. Our data indicate that TTC7A deficiency results in increased Rho kinase activity, which disrupts polarity, growth, and differentiation of intestinal epithelial cells, and which impairs immune cell homeostasis, thereby promoting MIA-CID development.
Assuntos
Atresia Intestinal/genética , Mucosa Intestinal/patologia , Proteínas/genética , Imunodeficiência Combinada Severa/genética , Sequência de Bases , Polaridade Celular , Células Cultivadas , Criança , Consanguinidade , Análise Mutacional de DNA , Células Epiteliais/fisiologia , Exoma , Feminino , Estudos de Associação Genética , Ligação Genética , Humanos , Lactente , Atresia Intestinal/imunologia , Atresia Intestinal/mortalidade , Atresia Intestinal/patologia , Linfonodos/patologia , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/patologia , Masculino , Linhagem , Proteínas/metabolismo , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/mortalidade , Imunodeficiência Combinada Severa/patologia , Timo/anormalidades , Timo/patologia , Quinases Associadas a rho/metabolismoRESUMO
Regulatory T cells (Treg) play a crucial role in controlling immunity and transplant rejection. Two main groups of Treg have been described: antigen-induced Treg (iTreg) and natural Treg (nTreg). The ways to induce and the mechanisms of action of Treg subsets remained ill defined, particularly for their effects on CD8(+) T cells. CD8(+) T cells are major agents in the rejection of allografts; the aim of this study is to investigate the effects exerted on CD8(+) T cells by human CD4(+) iTreg induced by mycophenolic acid-treated dendritic cells. iTreg suppress the proliferation of CD8(+) T cells by allogeneic cell-cell interaction with mature dendritic cells and irrespectively of the TCR specificity of the CD8(+) T cells and cell-cell contact of iTreg with CD8(+) T cells. In our model, this suppression is independent of the action of IL-10 and TGF-ß1. iTreg were able to modify phenotype and inhibited IFN-γ and TNF-α secretion by CD8(+) T cells. Most interestingly, iTreg inhibit the synthesis of perforin and of granzymes A and B by CD8(+) T cells and impaired their cytotoxicity against allogeneic targets. In summary, our study showed the involvement of iTreg in the down-regulation of cytotoxic responses mediated by CD8(+) T cells in an allospecific context. Following studies that have shown the existence of a regulation control exerted by iTreg on CD4(+) T cells and dendritic cells, this work ultimately shows that this regulation can reach CD8(+) T-cell functions.