RESUMO
Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Hematopoiese Clonal/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Pulmonares/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/secundário , Carcinoma Pulmonar de Células não Pequenas/terapia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células/genética , Quimioterapia Adjuvante/métodos , Quitinases/metabolismo , Colectomia , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Conjuntos de Dados como Assunto , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/imunologia , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/imunologia , Granzimas/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Receptor de Morte Celular Programada 1/antagonistas & inibidores , RNA-Seq , Análise de Célula Única , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
Synaptic dysfunction is an early pathogenic event leading to cognitive decline in Huntington's disease (HD). We previously reported that the active ADAM10 level is increased in the HD cortex and striatum, causing excessive proteolysis of the synaptic cell adhesion protein N-Cadherin. Conversely, ADAM10 inhibition is neuroprotective and prevents cognitive decline in HD mice. Although the breakdown of cortico-striatal connection has been historically linked to cognitive deterioration in HD, dendritic spine loss and long-term potentiation (LTP) defects identified in the HD hippocampus are also thought to contribute to the cognitive symptoms of the disease. The aim of this study is to investigate the contribution of ADAM10 to spine pathology and LTP defects of the HD hippocampus. We provide evidence that active ADAM10 is increased in the hippocampus of two mouse models of HD, leading to extensive proteolysis of N-Cadherin, which has a widely recognized role in spine morphology and synaptic plasticity. Importantly, the conditional heterozygous deletion of ADAM10 in the forebrain of HD mice resulted in the recovery of spine loss and ultrastructural synaptic defects in CA1 pyramidal neurons. Meanwhile, normalization of the active ADAM10 level increased the pool of synaptic BDNF protein and activated ERK neuroprotective signaling in the HD hippocampus. We also show that the ADAM10 inhibitor GI254023X restored LTP defects and increased the density of mushroom spines enriched with GluA1-AMPA receptors in HD hippocampal neurons. Notably, we report that administration of the TrkB antagonist ANA12 to HD hippocampal neurons reduced the beneficial effect of GI254023X, indicating that the BDNF receptor TrkB contributes to mediate the neuroprotective activity exerted by ADAM10 inhibition in HD. Collectively, these findings indicate that ADAM10 inhibition coupled with TrkB signaling represents an efficacious strategy to prevent hippocampal synaptic plasticity defects and cognitive dysfunction in HD.
Assuntos
Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide , Hipocampo , Doença de Huntington , Potenciação de Longa Duração , Proteínas de Membrana , Receptor trkB , Transdução de Sinais , Animais , Proteína ADAM10/metabolismo , Proteína ADAM10/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Camundongos , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Hipocampo/metabolismo , Hipocampo/patologia , Receptor trkB/metabolismo , Receptor trkB/antagonistas & inibidores , Potenciação de Longa Duração/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Modelos Animais de Doenças , Caderinas/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Neuroproteção , Masculino , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Camundongos KnockoutRESUMO
Mitofusin-2 (MFN2) is an outer mitochondrial membrane protein essential for mitochondrial networking in most cells. Autosomal dominant mutations in the MFN2 gene cause Charcot-Marie-Tooth type 2A disease (CMT2A), a severe and disabling sensory-motor neuropathy that impacts the entire nervous system. Here, we propose a novel therapeutic strategy tailored to correcting the root genetic defect of CMT2A. Though mutant and wild-type MFN2 mRNA are inhibited by RNA interference (RNAi), the wild-type protein is restored by overexpressing cDNA encoding functional MFN2 modified to be resistant to RNAi. We tested this strategy in CMT2A patient-specific human induced pluripotent stem cell (iPSC)-differentiated motor neurons (MNs), demonstrating the correct silencing of endogenous MFN2 and replacement with an exogenous copy of the functional wild-type gene. This approach significantly rescues the CMT2A MN phenotype in vitro, stabilizing the altered axonal mitochondrial distribution and correcting abnormal mitophagic processes. The MFN2 molecular correction was also properly confirmed in vivo in the MitoCharc1 CMT2A transgenic mouse model after cerebrospinal fluid (CSF) delivery of the constructs into newborn mice using adeno-associated virus 9 (AAV9). Altogether, our data support the feasibility of a combined RNAi and gene therapy strategy for treating the broad spectrum of human diseases associated with MFN2 mutations.
Assuntos
Doença de Charcot-Marie-Tooth , Células-Tronco Pluripotentes Induzidas , Humanos , Camundongos , Animais , Interferência de RNA , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/terapia , Doença de Charcot-Marie-Tooth/metabolismo , Mutação , Hidrolases/genética , Camundongos TransgênicosRESUMO
HIV integrates into the host genome, creating a viral reservoir of latently infected cells that persists despite effective antiretroviral treatment. CD4-positive (CD4+) T cells are the main contributors to the HIV reservoir. CD4+ T cells are a heterogeneous population, and the mechanisms of latency establishment in the different subsets, as well as their contribution to the reservoir, are still unclear. In this study, we analyzed HIV latency establishment in different CD4+ T cell subsets stimulated with interleukin 15 (IL-15), a cytokine that increases both susceptibility to infection and reactivation from latency. Using a dual-reporter virus that allows discrimination between latent and productive infection at the single-cell level, we found that IL-15-treated primary human CD4+ T naive and CD4+ T stem cell memory (TSCM) cells are less susceptible to HIV infection than CD4+ central memory (TCM), effector memory (TEM), and transitional memory (TTM) cells but are also more likely to harbor transcriptionally silent provirus. The propensity of these subsets to harbor latent provirus compared to the more differentiated memory subsets was independent of differential expression of pTEFb components. Microscopy analysis of NF-κB suggested that CD4+ T naive cells express smaller amounts of nuclear NF-κB than the other subsets, partially explaining the inefficient long terminal repeat (LTR)-driven transcription. On the other hand, CD4+ TSCM cells display similar levels of nuclear NF-κB to CD4+ TCM, CD4+ TEM, and CD4+ TTM cells, indicating the availability of transcription initiation and elongation factors is not solely responsible for the inefficient HIV gene expression in the CD4+ TSCM subset. IMPORTANCE The formation of a latent reservoir is the main barrier to HIV cure. Here, we investigated how HIV latency is established in different CD4+ T cell subsets in the presence of IL-15, a cytokine that has been shown to efficiently induce latency reversal. We observed that, even in the presence of IL-15, the less differentiated subsets display lower levels of productive HIV infection than the more differentiated subsets. These differences were not related to different expression of pTEFb, and modest differences in NF-κB were observed for CD4+ T naive cells only, implying the involvement of other mechanisms. Understanding the molecular basis of latency establishment in different CD4+ T cell subsets might be important for tailoring specific strategies to reactivate HIV transcription in all the CD4+ T subsets that compose the latent reservoir.
Assuntos
Linfócitos T CD4-Positivos , Infecções por HIV , Interleucina-15 , Latência Viral , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1 , Humanos , Interleucina-15/farmacologia , NF-kappa B/metabolismo , Provírus , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/virologiaRESUMO
BACKGROUND: von Willebrand factor propeptide (VWFpp) plays an important role in VWF multimerization and storage. VWFpp mutations have been previously associated with types 1, 3 and 2A/IIC von Willebrand disease (VWD). AIMS: To characterize the novel p.Thr274Pro variant identified in two unrelated type 1 VWD patients. METHODS: Phenotype tests were performed to evaluate patients' plasma and platelets following the current ISTH-SSC guidelines. Molecular analysis was performed using next-generation sequencing. The pcDNA3.1-VWF-WT and mutant pcDNA3.1-VWF-Thr274Pro expression vectors were transiently transfected into HEK293 cells to evaluate recombinant (r)VWF constitutive and regulated secretion. For the latter, the transfected cells were stimulated with phorbol-12-myristate-13-acetate. Immunofluorescence staining was performed to assess the localization of WT-rVWF and Thr274Pro-rVWF in endoplasmic reticulum, lysosomes, cis-/trans-Golgi and pseudo-Weibel Palade bodies. RESULTS: Biochemical characterization of patients' plasma samples indicated a type 1 VWD diagnosis. Both patients were heterozygous for the p.Thr274Pro variant. Hybrid Thr274Pro/WT-rVWF showed a secretion reduction of 36±4% according to patients' plasma VWF:Ag levels, whereas Thr274Pro-rVWF secretion was strongly impaired (21±2%). The amount of rVWF in cell lysates was nearly normal for both Thr274P (62±17%) and Thr274Pro/WT-rVWF (72±23%). The regulated secretion was impaired for Thr274Pro/WT-rVWF, whereas Thr274Pro-rVWF was not released at all. Immunofluorescence staining revealed no particular differences between WT and Thr274Pro-rVWF, although Thr274Pro-rVWF showed less pseudo-Weibel Palade bodies with a rounder shape than WT-rVWF. CONCLUSIONS: The novel p.Thr274Pro mutation has a dominant effect and it is responsible of patients' type 1 VWD phenotype through a combined mechanism of reduced synthesis, impaired secretion and multimerization.
Assuntos
Doenças de von Willebrand , Fator de von Willebrand , Células HEK293 , Humanos , Mutação , Fenótipo , Doenças de von Willebrand/diagnóstico , Doenças de von Willebrand/genéticaRESUMO
Brain cholesterol is produced mainly by astrocytes and is important for neuronal function. Its biosynthesis is severely reduced in mouse models of Huntington's disease. One possible mechanism is a diminished nuclear translocation of the transcription factor sterol regulatory element-binding protein 2 (SREBP2) and, consequently, reduced activation of SREBP2-controlled genes in the cholesterol biosynthesis pathway. Here we evaluated the efficacy of a gene therapy based on the unilateral intra-striatal injection of a recombinant adeno-associated virus 2/5 (AAV2/5) targeting astrocytes specifically and carrying the transcriptionally active N-terminal fragment of human SREBP2 (hSREBP2). Robust hSREBP2 expression in striatal glial cells in R6/2 Huntington's disease mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed dopamine receptor D2 (Drd2) transcript levels decline, cleared mutant huntingtin aggregates and attenuated behavioural deficits. We conclude that glial SREBP2 participates in Huntington's disease brain pathogenesis in vivo and that AAV-based delivery of SREBP2 to astrocytes counteracts key features of the disease.
Assuntos
Astrócitos/metabolismo , Corpo Estriado/metabolismo , Técnicas de Transferência de Genes , Terapia Genética/métodos , Doença de Huntington/terapia , Proteína de Ligação a Elemento Regulador de Esterol 2/administração & dosagem , Animais , Astrócitos/patologia , Corpo Estriado/patologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Fenótipo , Proteína de Ligação a Elemento Regulador de Esterol 2/biossíntese , Proteína de Ligação a Elemento Regulador de Esterol 2/genéticaRESUMO
The cellular heterogeneity of the tumor environment of breast cancer (BC) is extremely complex and includes different actors such as neoplastic, stromal, and immunosuppressive cells, which contribute to the chemical and mechanical modification of the environment surrounding the tumor-exasperating immune-escaping mechanisms. In addition to molecular signals that make the tumor microenvironment (TME) unacceptable for the penetrance of the immune system, the physical properties of tumoral extracellular matrix (tECM) also have carved out a fundamental role in the processes of the protection of the tumor niche. Tumor-associated macrophages (TAMs), with an M2 immunosuppressive phenotype, are important determinants for the establishment of a tumor phenotype excluded from T cells. NF-κB transcription factors orchestrate innate immunity and represent the common thread between inflammation and cancer. Many studies have focused on canonical activation of NF-κB; however, activation of non-canonical signaling predicts poor survival and resistance to therapy. In this scenario, we demonstrated the existence of an unusual association of NF-κB components in TAMs that determines the deposition of HSPG2 that affects the stiffness of tECM. These results highlight a new mechanism counterbalanced between physical factors and a new perspective of mechano-pathology to be targeted to counteract immune evasion in BC.
Assuntos
NF-kappa B , Neoplasias , Humanos , Macrófagos , Neoplasias/patologia , Microambiente Tumoral , Macrófagos Associados a TumorRESUMO
Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia.
Assuntos
Anticorpos Monoclonais/imunologia , Ácidos Nucleicos Livres/imunologia , Doenças Fetais/imunologia , Hemofilia A/imunologia , Diagnóstico Pré-Natal/métodos , Proteínas Ribossômicas/imunologia , Especificidade de Anticorpos/imunologia , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/metabolismo , Feminino , Doenças Fetais/sangue , Doenças Fetais/diagnóstico , Células HEK293 , Hemofilia A/sangue , Hemofilia A/diagnóstico , Células Hep G2 , Humanos , Masculino , Gravidez , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Sensibilidade e EspecificidadeRESUMO
The immune system is a fine modulator of the tumor biology supporting or inhibiting its progression, growth, invasion and conveys the pharmacological treatment effect. Tumors, on their side, have developed escaping mechanisms from the immune system action ranging from the direct secretion of biochemical signals to an indirect reaction, in which the cellular actors of the tumor microenvironment (TME) collaborate to mechanically condition the extracellular matrix (ECM) making it inhospitable to immune cells. TME is composed of several cell lines besides cancer cells, including tumor-associated macrophages, cancer-associated fibroblasts, CD4+ and CD8+ lymphocytes, and innate immunity cells. These populations interface with each other to prepare a conservative response, capable of evading the defense mechanisms implemented by the host's immune system. The presence or absence, in particular, of cytotoxic CD8+ cells in the vicinity of the main tumor mass, is able to predict, respectively, the success or failure of drug therapy. Among various mechanisms of immunescaping, in this study, we characterized the modulation of the phenotypic profile of CD4+ and CD8+ cells in resting and activated states, in response to the mechanical pressure exerted by a three-dimensional in vitro system, able to recapitulate the rheological and stiffness properties of the tumor ECM.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Matriz Extracelular/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Evasão Tumoral , Microambiente Tumoral/imunologia , 5'-Nucleotidase/genética , 5'-Nucleotidase/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Fibroblastos Associados a Câncer/imunologia , Fibroblastos Associados a Câncer/patologia , Técnicas de Cultura de Células , Módulo de Elasticidade , Matriz Extracelular/química , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Humanos , Hidrogéis/química , Interferon gama/genética , Interferon gama/imunologia , Ativação Linfocitária , Mecanotransdução Celular , Modelos Biológicos , NF-kappa B/genética , NF-kappa B/imunologia , Fenótipo , Cultura Primária de Células , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Reologia , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/imunologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/genética , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/patologiaRESUMO
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.
Assuntos
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
Multiple Sclerosis (MS) is an inflammatory disease with neurodegenerative alterations, ultimately progressing to neurological handicap. Therapies are effective in counteracting inflammation but not neurodegeneration. Biomarkers predicting disease course or treatment response are lacking. We investigated whether altered gene and protein expression profiles were detectable in the peripheral blood of 78 relapsing remitting MS (RR-MS) patients treated by disease-modifying therapies. A discovery/validation study on RR-MS responsive to glatiramer acetate identified 8 differentially expressed genes: ITGA2B, ITGB3, CD177, IGJ, IL5RA, MMP8, P2RY12, and S100ß. A longitudinal study on glatiramer acetate, Interferon-ß, or Fingolimod treated RR-MS patients confirmed that 7 out of 8 genes were downregulated with reference to the different therapies, whereas S100ß was always upregulated. Thus, we identified a peripheral gene signature associated with positive response in RR-MS which may also explain drug immunomodulatory effects. The usefulness of this signature as a biomarker needs confirmation on larger series of patients.
Assuntos
Fatores Imunológicos/uso terapêutico , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/genética , Transcriptoma/efeitos dos fármacos , Adulto , Feminino , Cloridrato de Fingolimode/uso terapêutico , Perfilação da Expressão Gênica , Acetato de Glatiramer/uso terapêutico , Humanos , Interferon beta/uso terapêutico , Leucócitos Mononucleares/metabolismo , Masculino , Esclerose Múltipla Recidivante-Remitente/sangue , Adulto JovemRESUMO
The pool size ratio measured by quantitative magnetization transfer MRI is hypothesized to closely reflect myelin density, but their relationship has so far been confirmed mostly in ex vivo conditions. We investigate the correspondence between this parameter measured in vivo at 7.0 T, with Black Gold II staining for myelin fibres, and with myelin basic protein and beta-tubulin immunofluorescence in a hybrid longitudinal study of C57BL/6 and SJL/J mice treated with cuprizone, a neurotoxicant causing relatively selective myelin loss followed by spontaneous remyelination upon treatment suspension. Our results confirm that pool size ratio measurements correlate with myelin content, with the correlation coefficient depending on strain and staining method, and demonstrate the in vivo applicability of this MRI technique to experimental mouse models of multiple sclerosis.
Assuntos
Fenômenos Magnéticos , Imageamento por Ressonância Magnética , Bainha de Mielina/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cuprizona , Feminino , Masculino , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismoRESUMO
(19)F-MRI offers unique opportunities to image diseases and track cells and therapeutic agents in vivo. Herein we report a superfluorinated molecular probe, herein called PERFECTA, possessing excellent cellular compatibility, and whose spectral properties, relaxation times, and sensitivity are promising for in vivo (19)F-MRI applications. The molecule, which bears 36 equivalent (19)F atoms and shows a single intense resonance peak, is easily synthesized via a simple one-step reaction and is formulated in water with high stability using trivial reagents and methods.
Assuntos
Radioisótopos de Flúor/farmacocinética , Hidrocarbonetos Fluorados/farmacocinética , Imageamento por Ressonância Magnética , Animais , Radioisótopos de Flúor/administração & dosagem , Radioisótopos de Flúor/química , Hidrocarbonetos Fluorados/administração & dosagem , Hidrocarbonetos Fluorados/química , Injeções Subcutâneas , Modelos Moleculares , Estrutura Molecular , Ratos , Ratos Endogâmicos Lew , Distribuição TecidualRESUMO
The thymus is the main site of immune sensitization to AChR in myasthenia gravis (MG). In our previous studies we demonstrated that Toll-like receptor (TLR) 4 is over-expressed in MG thymuses, suggesting its involvement in altering the thymic microenvironment and favoring autosensitization and autoimmunity maintenance processes, via an effect on local chemokine/cytokine network. Here, we investigated whether TLR4 signaling may favor abnormal cell recruitment in MG thymus via CCL17 and CCL22, two chemokines known to dictate immune cell trafficking in inflamed organs by binding CCR4. We also investigated whether TLR4 activation may contribute to immunodysregulation, via the production of Th17-related cytokines, known to alter effector T cell (Teff)/regulatory T cell (Treg) balance. We found that CCL17, CCL22 and CCR4 were expressed at higher levels in MG compared to normal thymuses. The two chemokines were mainly detected around medullary Hassall's corpuscles (HCs), co-localizing with TLR4(+) thymic epithelial cells (TECs) and CCR4(+) dendritic cells (DCs), that were present in higher number in MG thymuses compared to controls. TLR4 stimulation in MG TECs increased CCL17 and CCL22 expression and induced the production of Th17-related cytokines. Then, to study the effect of TLR4-stimulated TECs on immune cell interactions and Teff activation, we generated an in-vitro imaging model by co-culturing CD4(+) Th1/Th17 AChR-specific T cells, naïve CD4(+)CD25(+) Tregs, DCs and TECs from Lewis rats. We observed that TLR4 stimulation led to a more pronounced Teff activatory status, suggesting that TLR4 signaling in MG thymic milieu may affect cell-to-cell interactions, favoring autoreactive T-cell activation. Altogether our findings suggest a role for TLR4 signaling in driving DC recruitment in MG thymus via CCL17 and CCL22, and in generating an inflammatory response that might compromise Treg function, favoring autoreactive T-cell pathogenic responses.
Assuntos
Células Dendríticas/imunologia , Miastenia Gravis/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Receptor 4 Toll-Like/metabolismo , Adolescente , Adulto , Animais , Autoimunidade , Comunicação Celular , Células Cultivadas , Quimiocina CCL17/genética , Quimiocina CCL17/metabolismo , Quimiocina CCL22/genética , Quimiocina CCL22/metabolismo , Técnicas de Cocultura , Feminino , Humanos , Imunidade Inata , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Endogâmicos Lew , Transdução de Sinais , Timo/imunologia , Timo/patologia , Adulto JovemRESUMO
Malignant mesothelioma is a type of cancer that affects the mesothelium. It is an aggressive and deadly form of cancer that is often caused by exposure to asbestos. At the molecular level, it is characterized by a low number of genetic mutations and high heterogeneity among patients. In this work, we analyzed the plasticity of gene expression of primary mesothelial cancer cells by comparing their properties on 2D versus 3D surfaces. First, we derived from primary human samples four independent primary cancer cells. Then, we used Nichoids, which are micro-engineered 3D substrates, as three-dimensional structures. Nichoids limit the dimension of adhering cells during expansion by counteracting cell migration between adjacent units of a substrate with their microarchitecture. Tumor cells grow effectively on Nichoids, where they show enhanced proliferation. We performed RNAseq analyses on all the samples and compared the gene expression pattern of Nichoid-grown tumor cells to that of cells grown in a 2D culture. The PCA analysis showed that 3D samples were more transcriptionally similar compared to the 2D ones. The 3D Nichoids induced a transcriptional remodeling that affected mainly genes involved in extracellular matrix assembly. Among these genes responsible for collagen formation, COL1A1 and COL5A1 exhibited elevated expression, suggesting changes in matrix stiffness. Overall, our data show that primary mesothelioma cells can be effectively expanded in Nichoids and that 3D growth affects the cells' tensegrity or the mechanical stability of their structure.
Assuntos
Mesotelioma Maligno , Mesotelioma , Humanos , Mesotelioma/genética , Mesotelioma/metabolismo , Mesotelioma/patologia , Colágeno , Movimento Celular/genéticaRESUMO
Huntington's disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact, while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment.
Assuntos
Doença de Huntington , Organoides , Fenótipo , Telencéfalo , Doença de Huntington/patologia , Doença de Huntington/genética , Doença de Huntington/metabolismo , Organoides/patologia , Organoides/metabolismo , Animais , Telencéfalo/patologia , Telencéfalo/citologia , Telencéfalo/metabolismo , Humanos , Camundongos , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/patologia , Análise de Célula Única , Comunicação Celular , Mosaicismo , Neurônios/metabolismo , Neurônios/patologiaRESUMO
Metastatic brain disease (MBD) has seen major advances in clinical management, focal radiation therapy approaches and knowledge of biological factors leading to improved prognosis. Extracellular vesicles (EVs) have been found to play a role in tumor cross-talk with the target organ, contributing to the formation of a premetastatic niche. Human lung and breast cancer cell lines were characterized for adhesion molecule expression and used to evaluate their migration ability in an in vitro model. Conditioned culture media and isolated EVs, characterized by super resolution and electron microscopy, were tested to evaluate their pro-apoptotic properties on human umbilical vein endothelial cells (HUVECs) and human cerebral microvascular endothelial cells (HCMEC/D3) by annexin V binding assay. Our data showed a direct correlation between expression of ICAM1, ICAM2, ß3-integrin and α2-integrin and the ability to firmly adhere to the blood-brain barrier (BBB) model, whereas the same molecules were down-regulated at a later step. Extracellular vesicles released by tumor cell lines were shown to be able to induce apoptosis in HUVEC while brain endothelial cells showed to be more resistant.
RESUMO
Gold nanoparticles (GNPs) are considered promising candidates for healthcare applications, however, their toxicity after long-term exposure to the material remains uncertain. Since the liver is the main filter organ for nanomaterials, this work was aimed at evaluating hepatic accumulation, internalisation and overall safety of well-characterised and endotoxin-free GNPs in healthy mice from 15 minutes to 7 weeks after a single administration. Our data demonstrate that GNPs were rapidly segregated into lysosomes of endothelial cells (LSEC) or Kupffer cells regardless of coating or shape but with different kinetics. Despite the long-lasting accumulation in tissues, the safety of GNPs was confirmed by liver enzymatic levels, as they were rapidly eliminated from the blood circulation and accumulated in the liver without inducing hepatic toxicity. Our results demonstrate that GNPs have a safe and biocompatibile profile despite their long-term accumulation.
Assuntos
Ouro , Nanopartículas Metálicas , Camundongos , Animais , Ouro/toxicidade , Células Endoteliais , Nanopartículas Metálicas/toxicidade , Fígado , Células de KupfferRESUMO
BACKGROUND: The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood. METHODS: Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach. FINDINGS: We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation. INTERPRETATION: We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection. FUNDING: This work was supported by an unrestricted grant from "Fondazione Romeo ed Enrica Invernizzi" to Stefano Biffo and by AIRC under MFAG 2021 - ID. 26178 project - P.I. Manfrini Nicola.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Internalização do Vírus , Pandemias , Receptores Virais/química , Receptores Virais/metabolismo , Ligação Proteica , Pulmão/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Sistema ASC de Transporte de Aminoácidos/metabolismoRESUMO
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.