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OBJECTIVES: Giant cell arteritis (GCA) is a common vasculitis affecting patients aged 50 and older. GCA leads to chronic inflammation of large/medium-sized vessel walls with complications such as permanent vision loss and risk of stroke and aortic aneurysms. Early diagnosis is crucial and relies on temporal artery biopsy (TAB) and ultrasound imaging of temporal and axillary arteries. However, these methods have limitations. Serum biomarkers as autoantibodies have been reported but with inconclusive data for their use in the clinical setting. Additionally, C-reactive protein and erythrocyte sedimentation rate are non-specific and limited in reflecting disease activity, particularly in patients treated with IL-6 inhibitors. This study aimed to identify serum autoantibodies as new diagnostic biomarkers for GCA using a human protein array. METHODS: One commercial and one proprietary human protein array were used for antibody profiling of sera from patients with GCA (n=55), Takayasu (TAK n=7), and Healthy Controls (HC n=28). The identified candidate autoantigens were purified and tested for specific autoantibodies by ELISA. RESULTS: Antibodies against two proteins, VSIG10L (V-Set and Immunoglobulin Domain Containing 10 Like) and DCBLD1 (discoidin), were identified and found to be associated with GCA, with an overall prevalence of 43-57%, respectively, and high specificity as individual antibodies. A control series of TAK sera tested negative. CONCLUSIONS: Detecting GCA-specific autoantibodies may offer a new, non-invasive tool for improving our diagnostic power in GCA. Even though cell-mediated immune responses are crucial for GCA pathogenesis, this finding opens the way for investigating the additional role of humoral immune responses in the disease.
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Autoanticorpos , Autoantígenos , Biomarcadores , Arterite de Células Gigantes , Humanos , Arterite de Células Gigantes/imunologia , Arterite de Células Gigantes/sangue , Arterite de Células Gigantes/diagnóstico , Autoanticorpos/sangue , Biomarcadores/sangue , Autoantígenos/imunologia , Feminino , Idoso , Masculino , Pessoa de Meia-Idade , Estudos de Casos e Controles , Arterite de Takayasu/imunologia , Arterite de Takayasu/sangue , Arterite de Takayasu/diagnóstico , Valor Preditivo dos Testes , Análise Serial de Proteínas , Ensaio de Imunoadsorção EnzimáticaRESUMO
[This corrects the article DOI: 10.3389/fimmu.2023.1194087.].
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Colorectal cancer (CRC) is a leading cause of cancer-associated death. In the tumor site, the interplay between effector immune cells and cancer cells determines the balance between tumor elimination or outgrowth. We discovered that the protein TMEM123 is over-expressed in tumour-infiltrating CD4 and CD8 T lymphocytes and it contributes to their effector phenotype. The presence of infiltrating TMEM123+ CD8+ T cells is associated with better overall and metastasis-free survival. TMEM123 localizes in the protrusions of infiltrating T cells, it contributes to lymphocyte migration and cytoskeleton organization. TMEM123 silencing modulates the underlying signaling pathways dependent on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, which are required for synaptic force exertion. Using tumoroid-lymphocyte co-culture assays, we found that lymphocytes form clusters through TMEM123, anchoring to cancer cells and contributing to their killing. We propose an active role for TMEM123 in the anti-cancer activity of T cells within tumour microenvironment.
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Neoplasias Colorretais , Linfócitos do Interstício Tumoral , Humanos , Linfócitos T CD8-Positivos , Técnicas de Cocultura , Transdução de Sinais , Microambiente TumoralRESUMO
A molecular mimicry between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human proteins supports the possibility that autoimmunity takes place during coronavirus disease 2019 (COVID-19) contributing to tissue damage. For example, anti-phospholipid antibodies (aPL) have been reported in COVID-19 as a result of such mimicry and thought to contribute to the immunothrombosis characteristic of the disease. Consistently, active immunization with the virus spike protein may elicit the production of cross-reactive autoantibodies, including aPL. We prospectively looked at the aPL production in healthcare workers vaccinated with RNA- (BNT162b2, n. 100) or adenovirus-based vaccines (ChAdOx1, n. 50). Anti-cardiolipin, anti-beta2 glycoprotein I, anti-phosphatidylserine/prothrombin immunoglobulin G (IgG), IgA, and IgM before and after vaccination were investigated. Anti-platelet factor 4 immunoglobulins were also investigated as autoantibodies associated with COVID-19 vaccination. Additional organ (anti-thyroid) and non-organ (anti-nuclear) autoantibodies and IgG against human proteome were tested as further post-vaccination autoimmunity markers. The antibodies were tested one or three months after the first injection of ChAdOx1 and BNT162b2, respectively; a 12-month clinical follow-up was also performed. Vaccination occasionally induced low titers of aPL and other autoantibodies but did not affect the titer of pre-existing autoantibodies. No significant reactivities against a microarray of approximately 20,000 human proteins were found in a subgroup of ChAdOx1-vaccinees. Consistently, we did not record any clinical manifestation theoretically associated with an underlying autoimmune disorder. The data obtained after the vaccination (two doses for the RNA-based and one dose for the adenovirus-based vaccines), and the clinical follow-up are not supporting the occurrence of an early autoimmune response in this cohort of healthcare workers.
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COVID-19 , Anticorpos Antifosfolipídeos , Autoanticorpos , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Pessoal de Saúde , Humanos , Imunoglobulina G , RNA , SARS-CoV-2 , VacinaçãoRESUMO
BACKGROUND: Antibodies against cationic platelet chemokine, platelet factor 4 (PF4/CXCL4), have been described in heparin-induced thrombocytopenia (HIT), but also in patients positive for antiphospholipid antibodies (aPL) even in the absence of heparin treatment and HIT-related clinical manifestations. Anti-PF4 antibodies have been recently described also in subjects who developed thrombosis with thrombocytopenia syndrome (TTS) in association with adenoviral vector-based, but not with mRNA-based, COVID-19 vaccines. OBJECTIVE: To investigate whether COVID-19 vaccination affects the production of anti-PF4 antibodies in aPL-positive patients and in control groups. METHODS: Anti-PF4 immunoglobulins were detected in patients' and controls' serum samples by ELISA and their ability to activate normal platelets was assessed by the platelet aggregation test. RESULTS: Anti-PF4 were found in 9 of 126 aPL-positive patients, 4 of 50 patients with COVID-19, 9 of 49 with other infections, and 1 of 50 aPL-negative patients with systemic lupus erythematosus. Clinical manifestations of TTS were not observed in any aPL patient positive for anti-PF4, whose serum failed to cause platelet aggregation. The administration of COVID-19 vaccines did not affect the production of anti-PF4 immunoglobulins or their ability to cause platelet aggregation in 44 aPL-positive patients tested before and after vaccination. CONCLUSIONS: Heparin treatment-independent anti-PF4 antibodies can be found in aPL-positive patients and asymptomatic carriers, but their presence, titre as well as in vitro effect on platelet activation are not affected by COVID-19 vaccination.
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Anticorpos Antifosfolipídeos/análise , Vacinas contra COVID-19 , COVID-19 , Fator Plaquetário 4/imunologia , COVID-19/prevenção & controle , Humanos , VacinaçãoRESUMO
BACKGROUND: Patients with solid tumours have high COVID-19 mortality. Limited and heterogeneous data are available regarding the immunogenicity of SARS-CoV-2 mRNA vaccines in this population. METHODS AND FINDINGS: This is a prospective, single-centre cohort study aiming at evaluating seroconversion in terms of anti-spike antibodies in a population of patients with solid tumours undergoing cancer therapy within 2 months before the second vaccine dose, as compared with a cohort of controls. Subjects who were not SARS-CoV-2 naïve were excluded, and 171 patients were included in the final study population (150 vaccinated with BNT162b2, 87.7%; 21 with mRNA-1273, 12.3%) and compared with 2406 controls. The median follow-up time from the second dose of vaccination was 30 days (12-42; IQR: 26-34). Most patients had metastatic disease (138, 80.7%). Seroconversion rate was significantly lower in cancer patients than in controls (94.2% versus 99.8%, p < 0.001). At univariate logistic regression analysis, Odds ratio (OR) for seroconversion was also reduced in older individuals (>70 years). A multivariate logistic model confirmed cancer as the only significant variable in impairing seroconversion (OR 0.03, p < 0.001). In the cancer population, a multivariate analysis among clinical variables, including the type of cancer treatment, showed ECOG PS > 2 as the only one of impact (OR 0.07, p = 0.012). CONCLUSIONS: There is a fraction of 6% of patients with solid tumours undergoing cancer treatment, mainly with poorer performance status, who fail to obtain seroconversion after SARS-CoV-2 mRNA vaccines. These patients should be considered for enhanced vaccination strategies and carefully monitored for SARS-CoV-2 infection during cancer treatment.
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Vacina de mRNA-1273 contra 2019-nCoV/administração & dosagem , Anticorpos Antivirais/sangue , Vacina BNT162/administração & dosagem , Imunogenicidade da Vacina , Neoplasias/terapia , Soroconversão , Eficácia de Vacinas , Vacina de mRNA-1273 contra 2019-nCoV/imunologia , Adulto , Idoso , Vacina BNT162/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Neoplasias/imunologia , Estudos Prospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , VacinaçãoRESUMO
How gene expression is controlled to preserve human T cell quiescence is poorly understood. Here we show that non-canonical splicing variants containing long interspersed nuclear element 1 (LINE1) enforce naive CD4+ T cell quiescence. LINE1-containing transcripts are derived from CD4+ T cell-specific genes upregulated during T cell activation. In naive CD4+ T cells, LINE1-containing transcripts are regulated by the transcription factor IRF4 and kept at chromatin by nucleolin; these transcripts act in cis, hampering levels of histone 3 (H3) lysine 36 trimethyl (H3K36me3) and stalling gene expression. T cell activation induces LINE1-containing transcript downregulation by the splicing suppressor PTBP1 and promotes expression of the corresponding protein-coding genes by the elongating factor GTF2F1 through mTORC1. Dysfunctional T cells, exhausted in vitro or tumor-infiltrating lymphocytes (TILs), accumulate LINE1-containing transcripts at chromatin. Remarkably, depletion of LINE1-containing transcripts restores TIL effector function. Our study identifies a role for LINE1 elements in maintaining T cell quiescence and suggests that an abundance of LINE1-containing transcripts is critical for T cell effector function and exhaustion.
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Linfócitos T CD4-Positivos/fisiologia , Cromatina/metabolismo , Regulação da Expressão Gênica , Elementos Nucleotídeos Longos e Dispersos , Splicing de RNA , Linfócitos T CD4-Positivos/imunologia , Cromatina/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Histonas/metabolismo , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfoproteínas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fatores de Transcrição TFII/metabolismo , Transcrição Gênica , NucleolinaRESUMO
To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+ T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+ T lymphocytes, while CD4+ T cells were less expanded and skewed toward TCM and TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+ GZMB+ effector cells were clonally expanded both during the infection and post-infection, while CD8+ GZMK+ lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+ GZMB+ and GZMK+ subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+ T cell population with memory precursor-like features.
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COVID-19/genética , COVID-19/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunofenotipagem , SARS-CoV-2/imunologia , Transcriptoma , Adulto , Idoso , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , COVID-19/virologia , Plasticidade Celular/genética , Plasticidade Celular/imunologia , Evolução Clonal/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Isotipos de Imunoglobulinas/imunologia , Memória Imunológica , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
The Tn antigen is a well-known tumor-associated carbohydrate determinant, often incorporated in glycopeptides to develop cancer vaccines. Herein, four copies of a conformationally constrained mimetic of the antigen TnThr (GalNAc-Thr) were conjugated to the adjuvant CRM197, a protein licensed for human use. The resulting vaccine candidate, mime[4]CRM elicited a robust immune response in a triple-negative breast cancer mouse model, correlated with high frequency of CD4+ T cells and low frequency of M2-type macrophages, which reduces tumor progression and lung metastasis growth. Mime[4]CRM-mediated activation of human dendritic cells is reported, and the proliferation of mime[4]CRM-specific T cells, in cancer tissue and peripheral blood of patients with breast cancer, is demonstrated. The locked conformation of the TnThr mimetic and a proper presentation on the surface of CRM197 may explain the binding of the conjugate to the anti-Tn antibody Tn218 and its efficacy to fight cancer cells in mice.
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BACKGROUND AND AIMS: Primary biliary cholangitis is an autoimmune biliary disease characterized by injury of bile ducts, eventually leading to cirrhosis and death. In most cases, anti-mitochondrial antibodies and persistently elevated serum alkaline phosphatase are the basis for the serological diagnosis. Anti-nuclear antibodies are also useful and may indicate a more aggressive diseases course. In patients in which anti-mitochondrial antibodies are not detected, an accurate diagnosis requires liver histology. This study aims at identifying specific biomarkers for the serological diagnosis of primary biliary cholangitis. METHODS: Sera from patients affected by primary biliary cholangitis, primary sclerosing cholangitis, hepatitis C virus (with and without cryoglobulinemia), hepatocarcinoma and healthy donors were tested on a protein array representing 1658 human proteins. The most reactive autoantigens were confirmed by DELFIA analysis on expanded cohorts of the same mentioned serum classes, and on autoimmune hepatitis sera, using anti-PDC-E2 as reference biomarker. RESULTS: Two autoantigens, SPATA31A3 and GARP, showed high reactivity with primary biliary cholangitis sera, containing or not anti-mitochondrial antibodies. Their combination with PDC-E2 allowed to discriminate primary biliary cholangitis from all tested control classes with high sensitivity and specificity. We found that GARP expression is upregulated upon exposure to biliary salts in human cholangiocytes, an event involving EGFR and insulin pathways. GARP expression was also detected in biliary duct cells of PBC patients. CONCLUSIONS: This study highlighted SPATA31A3 and GARP as new biomarkers for primary biliary cholangitis and unravelled molecular stimuli underlying GARP expression in human cholangiocytes.
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Autoanticorpos/sangue , Autoantígenos/imunologia , Cirrose Hepática Biliar/diagnóstico , Proteínas de Membrana/imunologia , Mitocôndrias/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Feminino , Humanos , Cirrose Hepática Biliar/sangue , Cirrose Hepática Biliar/imunologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Colorectal cancer (CRC) is one of the major causes of cancer-associated mortality worldwide. The currently approved therapeutic agents show a rather limited efficacy. We have recently demonstrated that the atypical cadherin FAT1 is a specific marker of CRC and that the FAT1-specific monoclonal antibody mAb198.3 may offer new therapeutic opportunities for CRC, being efficiently internalized by cancer cells and reducing cancer growth in colon cancer xenograft models. In this study we explored the therapeutic efficacy of mAb198.3 using two drug delivery systems (DDS) for improving the targeted treatment of CRC. The mAb198.3 was either directly bound to super-paramagnetic nanoparticles (spmNPs) or embedded into human erythrocyte-based magnetized carriers, named Erythro-Magneto-Hemagglutinin Virosomes (EMHVs) to produce two different novel mAb198.3 formulations. Both DDS were endowed with magnetic properties and were anchored in the target tumor site by means of an external permanent magnet. The antibody loading efficiency of these two magnetically driven drug delivery systems and the overall therapeutic efficacy of these two formulations were assessed both in vitro and in a proof-of-concept in vivo study. We demonstrated that mAb198.3 bound to spmNPs or embedded into EMHVs was very effective in targeting FAT1-positive colon cancer cells in vitro and accumulating in the tumor mass in vivo. Although both in vivo administered mAb198.3 formulations have approximately 200 lower antibody doses needed, these showed to achieve a relevant therapeutic effect, thus reducing cancer growth more efficiently respect to the naked antibody. These results indicate that the two proposed magnetically driven drug delivery systems have a considerable potential as platforms to improve bioavailability and pharmacodynamics of anti-FAT mAb198.3 and raise new opportunities for a targeted therapy of CRC.
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Anticorpos Monoclonais/química , Caderinas/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas de Magnetita/química , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Composição de Medicamentos/métodos , Eritrócitos/química , Humanos , Imunoterapia/métodos , Magnetismo/métodos , Camundongos Nus , Terapia de Alvo Molecular/métodos , Tamanho da Partícula , Propriedades de Superfície , Distribuição TecidualRESUMO
Antigen immunoreactivity is often determined by surface regions defined by the 3D juxtapositions of amino acids stretches that are not continuous in the linear sequence. As such, mimicking an antigen immunoreactivity by means of putative linear peptide epitopes for diagnostic purposes is not trivial. Here we present a straightforward and robust method to extend the reach of immune-diagnostic probes design by copresenting peptides belonging to the same antigenic surface. In this case study focused on a computationally predicted Zika virus NS1 protein putative antigenic region, we reached a diagnostic confidence by the oriented and spatially controlled coimmobilization of peptide sequences found adjacent within the protein fold, that cooperatively interacted to provide enhanced immunoreactivity with respect to single linear epitopes. Through our method, we were able to differentiate Zika infected individuals from healthy controls. Remarkably, our strategy fits well with the requirements to build high-throughput screening platforms of linear and mixed peptide libraries, and it could possibly facilitate the rapid identification of conformational immunoreactive regions.
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Anticorpos/imunologia , Epitopos/imunologia , Peptídeos/imunologia , Testes Sorológicos/métodos , Sequência de Aminoácidos , Mapeamento de Epitopos/métodos , Epitopos/química , Humanos , Modelos Moleculares , Sondas Moleculares , Peptídeos/química , Conformação Proteica , Curva ROC , Reprodutibilidade dos Testes , Testes Sorológicos/normas , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/imunologia , Zika virus/imunologia , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologiaRESUMO
Tectonic family member 2 (TCTN2) encodes a transmembrane protein that belongs to the tectonic family, which is involved in ciliary functions. Previous studies have demonstrated the role of tectonics in regulating a variety of signaling pathways at the transition zone of cilia. However, the role of tectonics in cancer is still unclear. Here we identify that TCTN2 is overexpressed in colorectal, lung and ovary cancers. We show that different cancer cell lines express the protein that localizes at the plasma membrane, facing the intracellular milieu. TCTN2 over-expression in cancer cells resulted in an increased ability to form colonies in an anchorage independent way. On the other hand, downregulation of TCTN2 using targeted epigenetic editing in cancer cells significantly reduced colony formation, cell invasiveness, increased apoptosis and impaired assembly of primary cilia. Taken together, our results indicate that TCTN2 acts as an oncogene, making it an interesting cancer-associated protein and a potential candidate for therapeutic applications.
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Diagnostic accuracy can be improved considerably by combining multiple markers, whose performance in identifying diseased subjects is usually assessed via receiver operating characteristic (ROC) curves. The selection of multimarker signatures is a complicated process that requires integration of data signatures with sophisticated statistical methods. We developed a user-friendly tool, called CombiROC, to help researchers accurately determine optimal markers combinations from diverse omics methods. With CombiROC data from different domains, such as proteomics and transcriptomics, can be analyzed using sensitivity/specificity filters: the number of candidate marker panels rising from combinatorial analysis is easily optimized bypassing limitations imposed by the nature of different experimental approaches. Leaving to the user full control on initial selection stringency, CombiROC computes sensitivity and specificity for all markers combinations, performances of best combinations and ROC curves for automatic comparisons, all visualized in a graphic interface. CombiROC was designed without hard-coded thresholds, allowing a custom fit to each specific data: this dramatically reduces the computational burden and lowers the false negative rates given by fixed thresholds. The application was validated with published data, confirming the marker combination already originally described or even finding new ones. CombiROC is a novel tool for the scientific community freely available at http://CombiROC.eu.
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Biomarcadores/análise , Interface Usuário-Computador , Área Sob a Curva , Biomarcadores/sangue , Genômica , Hepatite Autoimune/diagnóstico , Hepatite Autoimune/metabolismo , Humanos , Internet , Linfoma/diagnóstico , Linfoma/genética , MicroRNAs/sangue , Distrofia Miotônica/diagnóstico , Distrofia Miotônica/genética , Proteômica , Curva ROC , Sensibilidade e EspecificidadeRESUMO
Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) are highly activated in cancer and involved in tumorigenesis and resistance to anti-cancer therapy. UPR is becoming a promising target of anti-cancer therapies. Thus, the identification of UPR components that are highly expressed in cancer could offer new therapeutic opportunity.In this study, we demonstrate that Endoplasmic Reticulum Metallo Protease 1 (ERMP1) is broadly expressed in a high percentage of breast, colo-rectal, lung, and ovary cancers, regardless of their stage and grade. Moreover, we show that loss of ERMP1 expression significantly hampers proliferation, migration and invasiveness of cancer cells. Furthermore, we show that this protein is an important player in the UPR and defense against oxidative stress. ERMP1 expression is strongly affected by reticular stress induced by thapsigargin and other oxidative stresses. ERMP1 silencing during reticular stress impairs the activation of PERK, a key sensor of the UPR activation. Loss of ERMP1 also prevents the expression of GRP78/BiP, a UPR stress marker involved in the activation of the survival pathway. Finally, ERMP1 silencing in cells exposed to hypoxia leads to inhibition of the Nrf2-mediated anti-oxidant response and to reduction of accumulation of HIF-1, the master transcription factor instructing cells to respond to hypoxic stress. Our results suggest that ERMP1 could act as a molecular starter to the survival response induced by extracellular stresses. Moreover, they provide the rationale for the design of ERMP1-targeting drugs that could act by inhibiting the UPR initial adaptive response of cancer cells and impair cell survival.
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Biomarcadores Tumorais/metabolismo , Estresse do Retículo Endoplasmático , Neoplasias/patologia , Estresse Oxidativo , Peptídeo Hidrolases/metabolismo , Resposta a Proteínas não Dobradas , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Chaperona BiP do Retículo Endoplasmático , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Peptídeo Hidrolases/genética , Prognóstico , Transdução de Sinais , Análise Serial de Tecidos , Células Tumorais CultivadasRESUMO
BACKGROUND: Colorectal cancer (CRC) is one of the major causes of cancer-associated mortality worldwide. The currently approved therapeutic agents have limited efficacy. METHODS: The atypical cadherin FAT1 was discovered as a novel CRC-associated protein by using a monoclonal antibody (mAb198.3). FAT1 expression was assessed in CRC cells by immunohistochemistry (IHC), immunoblots, flow cytometry and confocal microscopy. In addition, in vitro and in vivo tumour models were done to assess FAT1 potential value for therapeutic applications. RESULTS: The study shows that FAT1 is broadly expressed in primary and metastatic CRC stages and detected by mAb198.3, regardless of KRAS and BRAF mutations. FAT1 mainly accumulates at the plasma membrane of cancer cells, whereas it is only marginally detected in normal human samples. Moreover, the study shows that FAT1 has an important role in cell invasiveness while it does not significantly influence apoptosis. mAb198.3 specifically recognises FAT1 on the surface of colon cancer cells and is efficiently internalised. Furthermore, it reduces cancer growth in a colon cancer xenograft model. CONCLUSIONS: This study provides evidence that FAT1 and mAb198.3 may offer new therapeutic opportunities for CRC including the tumours resistant to current EGFR-targeted therapies.
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Anticorpos Monoclonais/farmacologia , Caderinas/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Células HT29 , Humanos , Mutação/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas ras/metabolismoRESUMO
Although nanoparticulate drug delivery systems (NDDSs) can preferentially accumulate in tumors, active targeting by targeting ligands (e.g. monoclonal antibody) is necessary for increasing its targeting efficacy in vivo. We conjugated mAb198.3 on the SiO2@AuNP system surface to make it obtain active targeting efficacy. The FAT1 targeting capability of SiO2@AuNP system is the first issue to be solved. Thus, flow cytometry analysis was attempted to demonstrate that the SiO2@AuNP system could bind to native FAT1 molecules on the surface of Colo205 cells. Also, together with the drug release behavior study of self-decomposable SiO2 NPs, the continuous morphological evolution needed to be clarified. Therefore, to characterize the morphological evolution in vitro, we analyzed the morphology of inner self-decomposable NPs in different time intervals using transmission electron microscopy (TEM). A more comprehensive analysis of this data may be obtained from the article "Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs" in Biomaterials.
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To achieve active tumor targeting and sequential release of 3 drugs to a tumor site in one nanoparticulate system, self-decomposable SiO2 nanoparticles modified by 3-aminopropyltriethoxysilane (APTS) as their inner structure were used to double load HCPT (in the NP core) and Dox (on the NP surface). Meanwhile, monoclonal antibodies (mAb198.3) against the FAT1 antigen and Bcl-2 siRNA were conjugated onto PEGylated Au-PEG-COOH nanoparticles. The obtained drug-loaded SiO2 nanoparticles were coated with the Au-PEG-mAb.198.3/siRNA nanoparticles through electrostatic interaction to form the SiO2@AuNP sequential drug delivery system, which featured the controlled and sequential release of siRNA, Dox and HCPT step by step to maximize its anticancer efficacy. The results revealed that the SiO2@AuNP sequential drug delivery system specifically targeted tumor cells and was internalize rapidly, followed by endosome escape and sequential drug release. Importantly, the sustainable release characteristics of SiO2 made the Tmax difference between HCPT and Dox approximately 8-12 h, and this enhanced the sensitizing efficiency of HCPT on Dox compared with co-administration. The in vivo antitumor results demonstrated that the tumor size after SiO2@AuNP treatment is 1/400 compared with the saline control group and approximately 1/40 of the HCPT/Dox co-treatment group without any noticeable systemic toxicity.
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Antineoplásicos/uso terapêutico , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Antineoplásicos/administração & dosagem , Materiais Biocompatíveis , HumanosRESUMO
BACKGROUND: Herein, we demonstrated the use of a newly generated anti FAT1 antibody (clone mAB198.3) for intracellular delivery of anionic gold NPs, to form active targeting Au nanoparticles with high payload characteristics. METHODS: In vitro characterizations were determined by DLS, confocal microscopy, TEM, western blot, MALDI-TOF MS/MS analysis, MTT, ICP-MS and flow cytometry analysis. In vivo targeting efficacy was investigated by in vivo bio-imaging study and ICP-MS. RESULTS: The specificity of the FAT1 recognition in colon cancer was confirmed by pre-adsorbing mAb198.3, adsorption dramatically abolished the antibody reactivity on colon cancer, thus confirming the binding specificity. The DLS size distribution profile of the AuCOOH, AuCOOH(Cy5)_ mAb198.3, AuCOOH(Cy5)_isotype has showed that the modified gold nanoparticles are well dispersed in water, PBS buffer and cell culture medium with 10 % FBS. By TEM measurement, the size of Au nanoparticles with spherical morphology is about 10-20 nm. AuCOOH_198.3 NPs were stable in an acidic environment, as well as in PBS buffer, cell culture media and media with 10 % serum. MTT results revealed that Au nanoparticles have well biocompatibility. TEM results indicated that conjugation of mAb198.3 on Au nanoparticles can be an effective delivery vehicle for negatively charged gold nanoparticles and increased its intracellular transport. It was also demonstrated by confocal microscopy that AuCOOH(Cy5)_mAb198.3 could attach to the cell membrane in very short time, then gradually delivered into cells. After 4 h incubation, almost all AuCOOH(Cy5)_mAb198.3 have been uptaken into or surrounding the cytoplasm and nucleus. In vivo results showed that only about 20 % of AuCOOH accumulated in tumor site due to EPR effect, while nearly 90 % of AuCOOH_mAb198.3 was found in tumor, providing sufficient evidence for receptor-specific targeting by mAb198.3. CONCLUSION: According to in vitro and in vivo research results, the intracellular uptake of negatively charged AuCOOH_mAB198.3 particles is enhanced to a greater extent. Thus, AuCOOH_mAb198.3 holds significant potential to improve the treatment of cancer.