RESUMO
Neuroligins are synaptic cell adhesion proteins with a role in synaptic function, implicated in neurodevelopmental disorders. The autism spectrum disorder-associated substitution Arg451Cys (R451C) in NLGN3 promotes a partial misfolding of the extracellular domain of the protein leading to retention in the endoplasmic reticulum (ER) and the induction of the unfolded protein response (UPR). The reduced trafficking of R451C NLGN3 to the cell surface leads to altered synaptic function and social behavior. A screening in HEK-293 cells overexpressing NLGN3 of 2662 compounds (FDA-approved small molecule drug library), led to the identification of several glucocorticoids such as alclometasone dipropionate, desonide, prednisolone sodium phosphate, and dexamethasone (DEX), with the ability to favor the exit of full-length R451C NLGN3 from the ER. DEX improved the stability of R451C NLGN3 and trafficking to the cell surface, reduced the activation of the UPR, and increased the formation of artificial synapses between HEK-293 and hippocampal primary neurons. The effect of DEX was validated on a novel model system represented by neural stem progenitor cells and differentiated neurons derived from the R451C NLGN3 knock-in mouse, expressing the endogenous protein. This work shows a potential rescue strategy for an autism-linked mutation affecting cell surface trafficking of a synaptic protein.
Assuntos
Transtorno do Espectro Autista , Animais , Humanos , Camundongos , Transtorno do Espectro Autista/genética , Glucocorticoides , Células HEK293 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Sinapses/metabolismoRESUMO
Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient sensitivity, specificity, and resolution to fully investigate their biodistribution. Our aim was to demonstrate the qualitative and quantitative distribution of unlabeled bepirovirsen, a clinical stage ASO, in livers and kidneys of dosed mice using novel staining and imaging technologies at subcellular resolution. ASOs were detected in formalin-fixed paraffin-embedded (FFPE) and frozen tissues using an automated chromogenic in situ hybridization (ISH) assay: miRNAscope. This was then combined with immunohistochemical detection of cell lineage markers. ASO distribution in hepatocytes versus nonparenchymal cell lineages was quantified using HALO AI image analysis. To complement this, hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) imaging microscopy was used to specifically detect the unique cellular Raman spectral signatures following ASO treatment. Bepirovirsen was localized primarily in nonparenchymal liver cells and proximal renal tubules. Codetection of ASO with distinct cell lineage markers of liver and kidney populations aided target cell identity facilitating quantification. Positive liver signal was quantified using HALO AI, with 12.9% of the ASO localized to the hepatocytes and 87.1% in nonparenchymal cells. HS-CARS imaging specifically detected ASO fingerprints based on the unique vibrational signatures following unlabeled ASO treatment in a totally nonperturbative manner at subcellular resolution. Together, these novel detection and imaging modalities represent a significant increase in our ability to detect unlabeled ASOs in tissues, demonstrating improved levels of specificity and resolution. These methods help us understand their underlying mechanisms of action and ultimately improve the therapeutic potential of these important drugs for treating globally significant human diseases.
Assuntos
Fígado , Oligonucleotídeos Antissenso , Camundongos , Humanos , Animais , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Distribuição Tecidual , Fígado/diagnóstico por imagem , Fígado/metabolismo , Hibridização In Situ , Coloração e RotulagemRESUMO
Carbon dots (CDs) are an emerging class of carbon nanoparticles, which for their characteristics have found applications in many fields such as catalysis, materials and biomedicine. Within this context, the application of CDs as antibacterial agents has received much attention in very recent years, while their use as antifungal nanoparticles has been scarcely investigated. Here we report a systematic investigation of the surface functional groups of CDs to study their influence on these nanoparticles' against Candida albicans. Three classes of CDs have been synthesised and fully characterized. A thorough inâ vitro and inâ vivo biological screening against C. albicans was performed to test their antifungal, antiadhesion and antibiofilm formation activities. Moreover, the interaction with C. albicans cells was investigated by microscopic analysis. Our results evidence how the presence of a positively polarised surface results crucial for the internalization into COS-7 cells. Positively charged nanoparticles were also able to inhibit adhesion and biofilm formation, to interact with the cellular membrane of C. albicans, and to increase the survival of G. mellonella infected larvae after the injection with positive nanoparticles. The antifungal activity of CDs and their extremely low toxicity may represent a new strategy to combat infections sustained by C.albicans.
Assuntos
Antifúngicos , Candida albicans , Animais , Antifúngicos/farmacologia , Carbono , Biofilmes , Larva , Testes de Sensibilidade MicrobianaRESUMO
Lung disease in alpha-1-antitrypsin deficiency (AATD) mainly results from insufficient control of the serine proteases neutrophil elastase (NE) and proteinase-3 due to reduced plasma levels of alpha-1-antitrypsin (AAT) variants. Mutations in the specificity-determining reactive center loop (RCL) of AAT would be predicted to minimally affect protein folding and secretion by hepatocytes but can impair anti-protease activity or alter the target protease. These properly secreted but dysfunctional 'type-2' variants would not be identified by common diagnostic protocols that are predicated on a reduction in circulating AAT. This has potential clinical relevance: in addition to the dysfunctional Pittsburgh and Iners variants reported previously, several uncharacterized RCL variants are present in genome variation databases. To prospectively evaluate the impact of RCL variations on secretion and anti-protease activity, here we performed a systematic screening of amino acid substitutions occurring at the AAT-NE interface. Twenty-three AAT variants that can result from single nucleotide polymorphisms in this region, including 11 present in sequence variation databases, were expressed in a mammalian cell model. All demonstrated unaltered protein folding and secretion. However, when their ability to form stable complexes with NE was evaluated by western blot, enzymatic assays, and a novel ELISA developed to quantify AAT-NE complexes, substrate-like and NE-binding deficient dysfunctional variants were identified. This emphasizes the ability of the RCL to accommodate inactivating substitutions without impacting the integrity of the native molecule and demonstrates that this class of molecule violates a generally accepted paradigm that equates circulating levels with functional protection of lung tissue.
Assuntos
Pneumopatias , Deficiência de alfa 1-Antitripsina , Humanos , Deficiência de alfa 1-Antitripsina/genética , Mutação/genética , Pulmão , Substituição de AminoácidosRESUMO
Several reports have highlighted a potential role of autoreactive B-cells and autoantibodies that correlates with increased disease severity in patients with idiopathic pulmonary fibrosis (IPF). Here we show that patients with IPF have an altered B-cell phenotype and that those subjects who have autoantibodies against the intermediate filament protein periplakin (PPL) have a significantly worse outcome in terms of progression-free survival. Using a mouse model of lung fibrosis, we demonstrate that introducing antibodies targeting the endogenous protein PPL (mimicking naturally occurring autoantibodies seen in patients) directly in the lung increases lung injury, inflammation, collagen and fibronectin expression through direct activation of follicular dendritic cells, which in turn activates and drives proliferation of fibroblasts. This fibrocyte population was also observed in fibrotic foci of patients with IPF and was increased in peripheral blood of IPF patients compared to aged-matched controls. This study reiterates the complex and heterogeneous nature of IPF, identifying new pathways that may prove suitable for therapeutic intervention.
Assuntos
Autoanticorpos , Fibrose Pulmonar Idiopática , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Pulmão/metabolismo , Progressão da Doença , Fibroblastos/metabolismoRESUMO
Background: Cardiovascular disease that is caused by atherosclerosis is the leading cause of death worldwide. Atherosclerosis is primarily triggered by endothelial dysfunction and the accumulation of modified low-density lipoprotein (LDL) particles in the subendothelial space of blood vessels. Early reports have associated oxidized LDL with altered fibrinolysis and atherogenesis. It has been suggested that myeloperoxidase oxidized LDL (Mox-LDL) is involved in atherosclerosis because of its significant pathophysiological role in the modification of LDL in vivo. It has been equally demonstrated that Mox-LDL binds to the lectin-like oxidized low-density lipoprotein receptor-1 (lox-1) scavenger receptor which leads to the upregulation of inflammatory mediators in endothelial cells and the progression of cardiovascular disease. It has been also shown that neuroserpin, a member of the serine proteinase inhibitor (serpin) superfamily, has an important role at the level of fibrinolysis in the nervous tissue. Methods: Since little is known about the effects of Mox-LDL on endothelial cell fibrinolytic activity and the involvement of lox-1 in this process, our study aimed at evaluating the in vitro effects of Mox-LDL on neuroserpin release from human aortic endothelial cells (HAECs) and the role of lox-1 scavenger receptor in this context by relying on lox-1 gene silencing in HAECs, culturing the cells in the presence of Mox-LDL, measuring their neuroserpin expression and release by quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively, and assessing their fibrinolytic activity using the Euglobulin Clot Lysis Time (ECLT) method. Results: Our data show that Mox-LDL decreases endothelial cell fibrinolytic capacity by upregulating neuroserpin in lox-1 knockdown cells. Conclusions: Lox-1 protects the endothelial cells from a Mox-LDL-induced decrease in pro-fibrinolytic capacity, which has important consequences in the context of stroke.
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BACKGROUND: Chimeric antigen receptor (CAR)-T cells can induce powerful immune responses in patients with hematological malignancies but have had limited success against solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) which limits the activity of tumor-infiltrating lymphocytes (TILs) including CAR-T cells. We have developed a next-generation armored CAR (F i-CAR) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is expressed at high levels in a range of aggressive tumors including poorly prognostic triple-negative breast cancer (TNBC). The F i-CAR-T is designed to release an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation within the TME, facilitating activation of CAR-T cells and TILs while limiting toxicity. METHODS: To bolster potency, we developed a F i-CAR construct capable of IL-2-mediated, NFAT-induced secretion of anti-PD-1 single-chain variable fragments (scFv) within the tumor microenvironment, following ROR1-mediated activation. Cytotoxic responses against TNBC cell lines as well as levels and binding functionality of released payload were analyzed in vitro by ELISA and flow cytometry. In vivo assessment of potency of F i-CAR-T cells was performed in a TNBC NSG mouse model. RESULTS: F i-CAR-T cells released measurable levels of anti-PD-1 payload with 5 h of binding to ROR1 on tumor and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Treatment of established PDL1 + TNBC xenograft model with F i-CAR-T cells resulted in significant abrogation in tumor growth and improved survival of mice (71 days), compared to non-armored CAR cells targeting ROR1 (F CAR-T) alone (49 days) or in combination with systemically administered anti-PD-1 antibody (57 days). Crucially, a threefold increase in tumor-infiltrating T cells was observed with F i-CAR-T cells and was associated with increased expression of genes related to cytotoxicity, migration and proliferation. CONCLUSIONS: Our next-generation of ROR1-targeting inducible armored CAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells. This technology provided a significant survival advantage in TNBC xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach in TNBC patients.
Assuntos
Receptores de Antígenos Quiméricos , Anticorpos de Cadeia Única , Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Linfócitos T , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/terapia , Microambiente TumoralRESUMO
Most publications on pediatric COVID-19 in Spain were performed at the beginning of the pandemic when some diagnostic tools were not widely available. This study aims to show the real spectrum of the infection based on wide detection of cases due to symptoms and contact tracing. A descriptive and analytical observational study was performed including pediatric cases (0 to 14 years) from the region of Aragón between May 12 and October 31, 2020. Diagnostics was by PCR detection of viral RNA, rapid antigen detection test, or positive IgG serology. There were 5933 positive children included. Of them, 49.03% were women. The mean age was 7.53 ± 4.28 years. The source of infection could not be determined in 17.8% of cases. As for the rest, was determined to be within the family environment in 67.8%. The percentage of asymptomatic patients was 50.3%. Among symptomatic patients, fever (58.1%) and cough (46.7%) were the most frequent symptoms. Hospitalization was required in 0.52% of infected, intensive care unit admission was on 0.05%, and there was one death (0.02%). Children under the age of one presented some symptoms more frequently (71.6% vs 48.5%; OR 2.68; 95% CI 2.08 to 3.45; p < 0.001) and required more hospitalizations (3.9% vs 0.34%; OR 11.52; 95% CI 5.65 to 23.52; p < 0.001).Conclusion: In our environment, SARS-CoV-2 infection is like other mild respiratory viral infections in the population under the age of 15. The contagion occurs mainly in the family environment, the number of asymptomatic is high, being the symptoms mild and the complications very infrequent. What is Known: ⢠Pediatric infection produced by SARS-CoV-2 has manifested as a mild disease in relation to adult age, although with higher affectation at the youngest ages. ⢠Nearly all studies on epidemiology and clinical spectrum of the disease were conducted with patients diagnosed at the beginning of the pandemic. By then, diagnostic tools were only available in hospitals and in emergency units. What is New: ⢠Once diagnostic means were available in primary care medicine and were used not only for the diagnosis of clinical symptoms of the patient, but for the tracing of case contacts, a much more precise approach to the epidemiology and clinical manifestations of the disease was allowed, as described in this study.
Assuntos
COVID-19 , Adulto , COVID-19/diagnóstico , COVID-19/epidemiologia , Criança , Pré-Escolar , Tosse , Feminino , Febre , Humanos , Pandemias , SARS-CoV-2RESUMO
Neuroserpin is a serine protease inhibitor identified in a search for proteins implicated in neuronal axon growth and synapse formation. Since its discovery over 30 years ago, it has been the focus of active research. Many efforts have concentrated in elucidating its neuroprotective role in brain ischemic lesions, the structural bases of neuroserpin conformational change and the effects of neuroserpin polymers that underlie the neurodegenerative disease FENIB (familial encephalopathy with neuroserpin inclusion bodies), but the investigation of the physiological roles of neuroserpin has increased over the last years. In this review, we present an updated and critical revision of the current literature dealing with neuroserpin, covering all aspects of research including the expression and physiological roles of neuroserpin, both inside and outside the nervous system; its inhibitory and non-inhibitory mechanisms of action; the molecular structure of the monomeric and polymeric conformations of neuroserpin, including a detailed description of the polymerisation mechanism; and the involvement of neuroserpin in human disease, with particular emphasis on FENIB. Finally, we briefly discuss the identification by genome-wide screening of novel neuroserpin variants and their possible pathogenicity.
Assuntos
Neuropeptídeos/metabolismo , Serpinas/metabolismo , Animais , Axônios/metabolismo , Epilepsias Mioclônicas/metabolismo , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Humanos , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Polimerização , NeuroserpinaRESUMO
Formalin is the principal tissue fixative used worldwide for clinical and research purposes. Despite optimal preservation of morphology, its preservation of DNA and RNA is poor. As clinical diagnostics increasingly incorporates molecular-based analysis, the requirement for maintaining nucleic acid quality is of increasing importance. Here we assess an alternative non-formalin-based tissue fixation method, PAXgene Tissue system, with the aim of better preserving nucleic acids, while maintaining the quality of the tissue to be used for vital existing diagnostic techniques. In this study, these criteria are assessed in a clinically representative setting. In total, 203 paired PAXgene Tissue and formalin-fixed samples were obtained. Blind-scored haematoxylin and eosin (H&E) sections showed comparable and acceptable staining. Immunohistochemistry (IHC) staining was suboptimal using existing protocols but improved with minor method adjustment and optimisation. Quality of DNA and RNA was significantly improved by PAXgene tissue fixation [RIN 2.8 versus 3.8 (p < 0.01), DIN 5.68 versus 6.77 (p < 0.001)], which translated into improved performance on qPCR assay. These results demonstrate the potential of PAXgene Tissue to be used routinely in place of formalin, maintaining adequate histological staining and significantly improving the preservation of biological molecules in the genomic era.
Assuntos
DNA/genética , Imuno-Histoquímica , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Fixação de Tecidos , Formaldeído , HumanosRESUMO
BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is considered one of the most common genetic diseases and is characterised by the misfolding and polymerisation of the alpha-1 antitrypsin (AAT) protein within hepatocytes. The relevance of circulating polymers (CP) of AAT in the pathogenesis of lung and liver disease is not completely understood. Therefore, the main objective of our study was to determine whether there is an association between the levels of CP of AAT and the severity of lung and liver disease. METHOD: This was a cross-sectional study in patients with different phenotypes of AATD and controls. To quantify CP, a sandwich ELISA was performed using the 2C1 monoclonal antibody against AAT polymers. Sociodemographic data, clinical characteristics, and liver and lung parameters were collected. RESULTS: A cohort of 70 patients was recruited: 32 Pi*ZZ (11 on augmentation therapy); 29 Z-heterozygous; 9 with other genotypes. CP were compared with a control group of 47 individuals (35 Pi*MM and 12 Pi*MS). ZZ patients had the highest concentrations of CP (p < 0.001) followed by Z heterozygous. The control group and patients with Pi*SS and Pi*SI had the lowest CP concentrations. Pi*ZZ also had higher levels of liver stiffness measurements (LSM) than the remaining AATD patients. Among patients with one or two Z alleles, two patients with lung and liver impairment showed the highest concentrations of CP (47.5 µg/mL), followed by those with only liver abnormality (n = 6, CP = 34 µg/mL), only lung (n = 18, CP = 26.5 µg/mL) and no abnormalities (n = 23, CP = 14.3 µg/mL). Differences were highly significant (p = 0.004). CONCLUSIONS: Non-augmented Pi*ZZ and Z-patients with impaired lung function and increased liver stiffness presented higher levels of CP than other clinical phenotypes. Therefore, CP may help to identify patients more at risk of developing lung and liver disease and may provide some insight into the mechanisms of disease.
Assuntos
Hepatopatias/sangue , Pneumopatias/sangue , Polímeros/metabolismo , Deficiência de alfa 1-Antitripsina/sangue , alfa 1-Antitripsina/sangue , Adulto , Idoso , Biomarcadores/sangue , Estudos Transversais , Feminino , Humanos , Hepatopatias/diagnóstico , Hepatopatias/epidemiologia , Pneumopatias/diagnóstico , Pneumopatias/epidemiologia , Masculino , Pessoa de Meia-Idade , Deficiência de alfa 1-Antitripsina/diagnóstico , Deficiência de alfa 1-Antitripsina/epidemiologiaRESUMO
Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.
Assuntos
Mutação de Sentido Incorreto , Deficiência de alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina , Substituição de Aminoácidos , Células HEK293 , Humanos , Domínios Proteicos , alfa 1-Antitripsina/biossíntese , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/patologiaRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease which presents a grave prognosis for diagnosed patients. Nintedanib (a triple tyrosine kinase inhibitor) and pirfenidone (unclear mechanism of action) are the only approved therapies for IPF, but have limited efficacy. The pathogenic mechanisms of this disease are not fully elucidated; however, a role for mast cells (MCs) has been postulated. OBJECTIVES: The aim of this work was to investigate a role for MCs in IPF and to understand whether nintedanib or pirfenidone could impact MC function. METHODS AND RESULTS: MCs were significantly elevated in human IPF lung and negatively correlated with baseline lung function (FVC). Importantly, MCs were positively associated with the number of fibroblast foci, which has been linked to increased mortality. Furthermore, MCs were increased in the region immediately surrounding the fibroblast foci, and co-culture studies confirmed a role for MC-fibroblast crosstalk in fibrosis. Nintedanib but not pirfenidone inhibited recombinant stem cell factor (SCF)-induced MC survival. Further evaluation of nintedanib determined that it also inhibited human fibroblast-mediated MC survival. This was likely via a direct effect on ckit (SCF receptor) since nintedanib blocked SCF-stimulated ckit phosphorylation, as well as downstream effects on MC proliferation and cytokine release. In addition, nintedanib ablated the increase in lung MCs and impacted high tissue density frequency (HDFm) in a rat bleomycin model of lung fibrosis. CONCLUSION: Nintedanib inhibits MC survival and activation and thus provides a novel additional mechanism by which this drug may exert anti-fibrotic effects in patients with IPF.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/fisiologia , Fibrose Pulmonar Idiopática/patologia , Indóis/farmacologia , Mastócitos/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Idoso , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Bleomicina , Proliferação de Células/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Fibroblastos/patologia , Fibrose , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/fisiopatologia , Pulmão/patologia , Masculino , Mastócitos/patologia , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Piridonas/farmacologia , Ratos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Células-Tronco/farmacologia , Capacidade VitalRESUMO
Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.
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Neuropeptídeos/metabolismo , Serpinas/metabolismo , Linhagem Celular , Glicosilação , Humanos , Neuropeptídeos/química , Dobramento de Proteína , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Serpinas/química , NeuroserpinaRESUMO
A fusion between fibronectin 1 (FN1) and activin receptor 2A (ACVR2A) has been reported previously in isolated cases of the synovial chondromatosis. To analyze further and validate the findings, we performed FISH and demonstrated recurrent FN1-ACVR2A rearrangements in synovial chondromatosis (57%), and chondrosarcoma secondary to synovial chondromatosis (75%), showing that FN1 and/or AVCR2A gene rearrangements do not distinguish between benign and malignant synovial chondromatosis. RNA sequencing revealed the presence of the FN1-ACVR2A fusion in several cases that were negative by FISH suggesting that the true prevalence of this fusion is potentially higher than 57%. In soft tissue chondromas, FN1 alterations were detected by FISH in 50% of cases but no ACVR2A alterations were identified. RNA sequencing identified a fusion involving FN1 and fibroblast growth factor receptor 2 (FGFR2) in the case of soft tissue chondroma and FISH confirmed recurrent involvement of both FGFR1 and FGFR2. These fusions were present in a subset of soft tissue chondromas characterized by grungy calcification, a feature reminiscent of phosphaturic mesenchymal tumor. However, unlike the latter, fibroblast growth factor 23 (FGF23) mRNA expression was not elevated in soft tissue chondromas harboring the FN1-FGFR1 fusion. The mutual exclusivity of ACVR2A rearrangements observed in synovial chondromatosis and FGFR1/2 in soft tissue chondromas suggests these represent separate entities. There have been no reports of malignant soft tissue chondromas, therefore differentiating these lesions will potentially alter clinical management by allowing soft tissue chondromas to be managed more conservatively.
Assuntos
Receptores de Activinas Tipo II/genética , Condroma/genética , Condromatose Sinovial/genética , Fibronectinas/genética , Neoplasias de Tecidos Moles/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Fator de Crescimento de Fibroblastos 23 , Rearranjo Gênico , Humanos , Masculino , Pessoa de Meia-Idade , Fusão Oncogênica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Adulto JovemRESUMO
BACKGROUND: C-reactive protein (CRP) and procalcitonin (PCT) have been described as good predictors of anastomotic leak after colorectal surgery, obtaining the highest diagnostic accuracy on the 5th postoperative day. However, if an enhanced recovery after surgery (ERAS) program is performed, early predictors are needed in order to ensure a safe and early discharge. The aim of this study was to investigate the efficacy of CRP, PCT, and white blood cell (WBC) count determined on first postoperative days, in predicting septic complications, especially anastomotic leak, after laparoscopic colorectal surgery performed within an ERAS program. METHODS: We conducted a prospective study including 134 patients who underwent laparoscopic colorectal surgery within an ERAS program between 2015 and 2017. The primary endpoint investigated was anastomotic leak. CRP, PCT, and WBC count were determined in the blood sample extracted on postoperative day 1 (POD 1), POD 2 and POD 3. RESULTS: Anastomotic leak (AL) was detected in 6 patients (4.5%). Serum levels of CRP and PCT, but not WBC, determined on POD 1, POD 2, and POD 3 were significantly higher in patients who had AL in the postoperative course. Using ROC analysis, the best AUC of the CRP and PCT levels was on POD 3 (0.837 and 0.947, respectively). A CRP cutoff level at 163 mg/l yielded 85% sensitivity, 80% specificity, and 99% negative predictive value (NPV). A PCT cutoff level at 2.5 ng/ml achieved 85% sensitivity, 95% specificity, 44% positive predictive value, and 99% NPV. CONCLUSIONS: CRP and PCT are relevant markers for detecting postoperative AL after laparoscopic colorectal surgery. Furthermore, they can ensure an early discharge with a low probability of AL when an ERAS program is performed.
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Fístula Anastomótica/diagnóstico , Proteína C-Reativa/análise , Neoplasias Colorretais/cirurgia , Laparoscopia/efeitos adversos , Pró-Calcitonina/sangue , Idoso , Biomarcadores/sangue , Feminino , Humanos , Contagem de Leucócitos , Masculino , Assistência Perioperatória , Valor Preditivo dos Testes , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
The serpinopathies are human pathologies caused by mutations that promote polymerisation and intracellular deposition of proteins of the serpin superfamily, leading to a poorly understood cell toxicity. The dementia FENIB is caused by polymerisation of the neuronal serpin neuroserpin (NS) within the endoplasmic reticulum (ER) of neurons. With the aim of understanding the toxicity due to intracellular accumulation of neuroserpin polymers, we have generated transgenic neural progenitor cell (NPC) cultures from mouse foetal cerebral cortex, stably expressing the control protein GFP (green fluorescent protein), or human wild type, G392E or delta NS. We have characterised these cell lines in the proliferative state and after differentiation to neurons. Our results show that G392E NS formed polymers that were mostly retained within the ER, while wild type NS was correctly secreted as a monomeric protein into the culture medium. Delta NS was absent at steady state due to its rapid degradation, but it was easily detected upon proteasomal block. Looking at their intracellular distribution, wild type NS was found in partial co-localisation with ER and Golgi markers, while G392E NS was localised within the ER only. Furthermore, polymers of NS were detected by ELISA and immunofluorescence in neurons expressing the mutant but not the wild type protein. We used control GFP and G392E NPCs differentiated to neurons to investigate which cellular pathways were modulated by intracellular polymers by performing RNA sequencing. We identified 747 genes with a significant upregulation (623) or downregulation (124) in G392E NS-expressing cells, and we focused our attention on several genes involved in the defence against oxidative stress that were up-regulated in cells expressing G392E NS (Aldh1b1, Apoe, Gpx1, Gstm1, Prdx6, Scara3, Sod2). Inhibition of intracellular anti-oxidants by specific pharmacological reagents uncovered the damaging effects of NS polymers. Our results support a role for oxidative stress in the cellular toxicity underlying the neurodegenerative dementia FENIB.
Assuntos
Demência/metabolismo , Epilepsias Mioclônicas/metabolismo , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Neurônios/metabolismo , Neuropeptídeos/toxicidade , Estresse Oxidativo/fisiologia , Polímeros/toxicidade , Serpinas/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Demência/induzido quimicamente , Demência/patologia , Epilepsias Mioclônicas/induzido quimicamente , Epilepsias Mioclônicas/patologia , Transtornos Heredodegenerativos do Sistema Nervoso/induzido quimicamente , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , NeuroserpinaRESUMO
Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.
Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Reparo Gênico Alvo-Dirigido , Deficiência de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Animais , Linhagem Celular , Elementos de DNA Transponíveis/genética , Hepatócitos/metabolismo , Hepatócitos/transplante , Humanos , Fígado/citologia , Camundongos , Albumina Sérica/genética , Albumina Sérica/metabolismo , Albumina Sérica Humana , Fatores de Tempo , alfa 1-Antitripsina/metabolismoRESUMO
Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered ß-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the ß-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation.