RESUMO
BACKGROUND: Guillain-Barré Syndrome (GBS) can lead to significant functional impairments, yet little is understood about the recovery phase and long-term consequences for patients in low- and medium-income countries. OBJECTIVE: To evaluate the functional status and identify factors influencing outcomes among patients with GBS in Colombia. METHODS: Telephone interviews were conducted with GBS patients enrolled in the Neuroviruses Emerging in the Americas Study between 2016 and 2020. The investigation encompassed access to health services and functional status assessments, utilizing the modified Rankin Scale (mRS), GBS Disability Score (GDS), Barthel Index (BI), and International Classification of Functioning (ICF). Univariate analysis, principal component analysis, linear discriminant analysis, and linear regression were employed to explore factors influencing functional status. RESULTS: Forty-five patients (mean age = 50[±22] years) with a median time from diagnosis of 28 months (IQR = 9-34) were included. Notably, 22% and 16% of patients did not receive rehabilitation services during the acute episode and post-discharge, respectively. Most patients demonstrated independence in basic daily activities (median BI = 100, IQR = 77.5-100), improvement in disability as the median mRS at follow-up was lower than at onset (1 [IQR = 0-3] vs. 4.5 [IQR = 4-5], p < 0.001), and most were able to walk without assistance (median GDS = 2, IQR = 0-2). A shorter period from disease onset to interview was associated with worse mRS (p = 0.015) and ICF (p = 0.019). Negative outcomes on GDS and ICF were linked to low socioeconomic status, ICF to the severity of weakness at onset, and BI to an older age. CONCLUSIONS: This study underscores that the functional recovery of GBS patients in Colombia is influenced not only by the natural course of the disease but also by socioeconomic factors, emphasizing the crucial role of social determinants of health.
Assuntos
Síndrome de Guillain-Barré , Infecção por Zika virus , Humanos , Colômbia/epidemiologia , Síndrome de Guillain-Barré/epidemiologia , Síndrome de Guillain-Barré/diagnóstico , Masculino , Feminino , Pessoa de Meia-Idade , Infecção por Zika virus/epidemiologia , Infecção por Zika virus/complicações , Adulto , Idoso , Avaliação da Deficiência , Epidemias , Recuperação de Função Fisiológica , Estado FuncionalRESUMO
Arthropod-borne viruses represent a crucial public health threat. Current arboviral serology assays are either labor intensive or incapable of distinguishing closely related viruses, and many zoonotic arboviruses that may transition to humans lack any serologic assays. In this study, we present a programmable phage display platform, ArboScan, that evaluates antibody binding to overlapping peptides that represent the proteomes of 691 human and zoonotic arboviruses. We confirm that ArboScan provides detailed antibody binding information from animal sera, human sera, and an arthropod blood meal. ArboScan identifies distinguishing features of antibody responses based on exposure history in a Colombian cohort of Zika patients. Finally, ArboScan details epitope level information that rapidly identifies candidate epitopes with potential protective significance. ArboScan thus represents a resource for characterizing human and animal arbovirus antibody responses at cohort scale.
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Anticorpos Antivirais , Arbovírus , Humanos , Arbovírus/imunologia , Arbovírus/isolamento & purificação , Animais , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Peptídeos/imunologia , Peptídeos/química , Infecção por Zika virus/virologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/sangue , Zika virus/imunologia , Epitopos/imunologia , Testes Sorológicos/métodos , Infecções por Arbovirus/virologia , Infecções por Arbovirus/imunologia , Proteoma , Colômbia , Feminino , Biblioteca de Peptídeos , Técnicas de Visualização da Superfície Celular , MasculinoRESUMO
Parkinson's disease with dementia (PDD) is a neurological disorder that clinically and neuropathologically overlaps with Parkinson's disease (PD) and Alzheimer's disease (AD). Although it is assumed that alpha-synuclein ( α -Syn), amyloid beta (A ß ), and the protein Tau might synergistically induce cholinergic neuronal degeneration, presently the pathological mechanism of PDD remains unclear. Therefore, it is essential to delve into the cellular and molecular aspects of this neurological entity to identify potential targets for prevention and treatment strategies. Cholinergic-like neurons (ChLNs) were exposed to rotenone (ROT, 10 µ M) for 24 h. ROT provokes loss of Δ Ψ m , generation of reactive oxygen species (ROS), phosphorylation of leucine-rich repeated kinase 2 (LRRK2 at Ser935) concomitantly with phosphorylation of α -synuclein ( α -Syn, Ser129), induces accumulation of intracellular A ß (iA ß ), oxidized DJ-1 (Cys106), as well as phosphorylation of TAU (Ser202/Thr205), increases the phosphorylation of c-JUN (Ser63/Ser73), and increases expression of proapoptotic proteins TP53, PUMA, and cleaved caspase 3 (CC3) in ChLNs. These neuropathological features resemble those reproduced in presenilin 1 (PSEN1) E280A ChLNs. Interestingly, anti-oxidant and anti-amyloid cannabidiol (CBD), JNK inhibitor SP600125 (SP), TP53 inhibitor pifithrin- α (PFT), and LRRK2 kinase inhibitor PF-06447475 (PF475) significantly diminish ROT-induced oxidative stress (OS), proteinaceous, and cell death markers in ChLNs compared to naïve ChLNs. In conclusion, ROT induces p- α -Syn, iA ß , p-Tau, and cell death in ChLNs, recapitulating the neuropathology findings in PDD. Our report provides an excellent in vitro model to test for potential therapeutic strategies against PDD. Our data suggest that ROT induces a neuropathologic phenotype in ChLNs similar to that caused by the mutation PSEN1 E280A.
Assuntos
Neurônios Colinérgicos , Rotenona , Rotenona/toxicidade , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/patologia , Animais , Doença de Parkinson/patologia , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Demência/patologia , Demência/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Humanos , Células CultivadasRESUMO
Parkinson's disease (PD) is a complex multifactorial progressive neurodegenerative disease characterized by locomotor alteration due to the specific deterioration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc). Mounting evidence shows that human LRRK2 (hLRRK2) kinase activity is involved in oxidative stress (OS)-induced neurodegeneration, suggesting LRRK2 inhibition as a potential therapeutic target. We report that the hLRRK2 inhibitor PF-06447475 (PF-475) prolonged lifespan, increased locomotor activity, maintained DAergic neuronal integrity, and reduced lipid peroxidation (LPO) in female Drosophila melanogaster flies chronically exposed to paraquat (PQ), a redox cycling compound, compared to flies treated with vehicle only. Since LRRK2 is an evolutionary conserved kinase, the present findings reinforce the idea that either reduction or inhibition of the LRRK2 kinase might decrease OS and locomotor alterations associated with PD. Our observations highlight the importance of uncovering the function of the hLRRK2 orthologue dLrrk2 in D. melanogaster as an excellent model for pharmacological screenings.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Locomoção , Longevidade , Estresse Oxidativo , Paraquat , Animais , Estresse Oxidativo/efeitos dos fármacos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Paraquat/toxicidade , Longevidade/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Feminino , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Herbicidas/toxicidadeRESUMO
Background: Familial Alzheimer's disease (FAD) presenilin 1 E280A (PSEN 1 E280A) is characterized by functional impairment and the death of cholinergic neurons as a consequence of amyloid-ß (Aß) accumulation and abnormal phosphorylation of the tau protein. Currently, there are no available therapies that can cure FAD. Therefore, new therapies are urgently needed for treating this disease. Objective: To assess the effect of sildenafil (SIL) on cholinergic-like neurons (ChLNs) harboring the PSEN 1 E280A mutation. Methods: Wild-type (WT) and PSEN 1 E280A ChLNs were cultured in the presence of SIL (25µM) for 24âh. Afterward, proteinopathy, cell signaling, and apoptosis markers were evaluated via flow cytometry and fluorescence microscopy. Results: We found that SIL was innocuous toward WT PSEN 1 ChLNs but reduced the accumulation of intracellular Aß fragments by 87%, decreased the non-physiological phosphorylation of the protein tau at residue Ser202/Thr205 by 35%, reduced the phosphorylation of the proapoptotic transcription factor c-JUN at residue Ser63/Ser73 by 63%, decreased oxidized DJ-1 at Cys106-SO3 by 32%, and downregulated transcription factor TP53 (tumor protein p53), BH-3-only protein PUMA (p53 upregulated modulator of apoptosis), and cleaved caspase 3 (CC3) expression by 20%, 32%, and 22%, respectively, compared with untreated mutant ChLNs. Interestingly, SIL also ameliorated the dysregulation of acetylcholine-induced calcium ion (Ca2+) influx in PSEN 1 E280A ChLNs. Conclusions: Although SIL showed no antioxidant capacity in the oxygen radical absorbance capacity and ferric ion reducing antioxidant power assays, it might function as an anti-amyloid and antiapoptotic agent and functional neuronal enhancer in PSEN 1 E280A ChLNs. Therefore, the SIL has therapeutic potential for treating FAD.
Assuntos
Doença de Alzheimer , Neurônios Colinérgicos , Mutação , Presenilina-1 , Citrato de Sildenafila , Presenilina-1/genética , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/metabolismo , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/patologia , Mutação/genética , Animais , Citrato de Sildenafila/farmacologia , Peptídeos beta-Amiloides/metabolismo , Humanos , Células Cultivadas , Camundongos , Proteínas tau/metabolismo , Proteínas tau/genética , Fosforilação/efeitos dos fármacos , FenótipoRESUMO
Familial Alzheimer's disease (FAD) is a complex and multifactorial neurodegenerative disorder for which no curative therapies are yet available. Indeed, no single medication or intervention has proven fully effective thus far. Therefore, the combination of multitarget agents has been appealing as a potential therapeutic approach against FAD. Here, we investigated the potential of combining tramiprosate (TM), curcumin (CU), and the JNK inhibitor SP600125 (SP) as a treatment for FAD. The study analyzed the individual and combined effects of these two natural agents and this pharmacological inhibitor on the accumulation of intracellular amyloid beta iAß; hyperphosphorylated protein TAU at Ser202/Thr205; mitochondrial membrane potential (ΔΨm); generation of reactive oxygen species (ROS); oxidized protein DJ-1; proapoptosis proteins p-c-JUN at Ser63/Ser73, TP53, and cleaved caspase 3 (CC3); and deficiency in acetylcholine (ACh)-induced transient Ca2+ influx response in cholinergic-like neurons (ChLNs) bearing the mutation I416T in presenilin 1 (PSEN1 I416T). We found that single doses of TM (50 µM), CU (10 µM), or SP (1 µM) were efficient at reducing some, but not all, pathological markers in PSEN 1 I416T ChLNs, whereas a combination of TM, CU, and SP at a high (50, 10, 1 µM) concentration was efficient in diminishing the iAß, p-TAU Ser202/Thr205, DJ-1Cys106-SO3, and CC3 markers by -50%, -75%, -86%, and -100%, respectively, in PSEN1 I417T ChLNs. Although combinations at middle (10, 2, 0.2) and low (5, 1, 0.1) concentrations significantly diminished p-TAU Ser202/Thr205, DJ-1Cys106-SO3, and CC3 by -69% and -38%, -100% and -62%, -100% and -62%, respectively, these combinations did not alter the iAß compared to untreated mutant ChLNs. Moreover, a combination of reagents at H concentration was able to restore the dysfunctional ACh-induced Ca2+ influx response in PSEN 1 I416T. Our data suggest that the use of multitarget agents in combination with anti-amyloid (TM, CU), antioxidant (e.g., CU), and antiapoptotic (TM, CU, SP) actions might be beneficial for reducing iAß-induced ChLN damage in FAD.
Assuntos
Doença de Alzheimer , Antracenos , Curcumina , Presenilina-1 , Taurina/análogos & derivados , Curcumina/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Presenilina-1/genética , Presenilina-1/metabolismo , Antracenos/farmacologia , Animais , Espécies Reativas de Oxigênio/metabolismo , Camundongos , Peptídeos beta-Amiloides/metabolismo , Humanos , Proteínas tau/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacosRESUMO
Several efforts to develop new protocols to differentiate in in vitro human mesenchymal stromal cells (hMSCs) into dopamine (DA) neurons have been reported. We have formulated NeuroForsk 2.0 medium containing fibroblast growth factor type beta (FGFb), brain-derived neurotrophic factor (BDNF), melatonin, purmorphamine, and forskolin. We report for the first time that menstrual stromal cells (MenSCs) cultured in NeuroForsk 2.0 medium for 7 days transdifferentiated into DA-like neurons (DALNs) expressing specific DA lineage markers tyrosine hydroxylase-positive cells (TH+) and DA transporter-positive (DAT+) cells and were responsive to DA-induced transient Ca2+ influx. To test the usefulness of this medium, DALNs were exposed to rotenone (ROT), a naturally occurring organic neurotoxin used extensively to chemically induce an in vitro model of Parkinson's disease (PD), which is a movement disorder characterized by the specific loss of DA neurons. We wanted to determine whether ROT induces apoptotic cell death and autophagy pathway under acute or chronic conditions in DALNs. Here, we report that acute ROT exposure induced several molecular changes in DALNS. ROT induced a loss of mitochondrial membrane potential (ΔΨm), high expression of parkin (PRKN), and high colocalization of dynamin-related protein 1 (DRP1) with the mitochondrial translocase of the outer membrane of mitochondria 20 (TOMM20) protein. Acute ROT also induced the appearance of DJ-1Cys106-SO3, as evidenced by the generation of H2O2 and oxidative stress (OS) damage. Remarkably, ROT triggered the phosphorylation of leucine-rich repeat kinase 2 (LRRK2) at residue Ser935 and phosphorylation of α-Syn at residue Ser129, a pathological indicator. ROT induced the accumulation of lipidated microtubule-associated protein 1B-light chain 3 (LC3B), a highly specific marker of autophagosomes. Finally, ROT induced cleaved caspase 3 (CC3), a marker of activated caspase 3 (CASP3) in apoptotic DALNs compared to untreated DANLs. However, the chronic condition was better at inducing the accumulation of lysosomes than the acute condition. Importantly, the inhibitor of the LRRK2 kinase PF-06447475 (PF-475) almost completely blunted ROT-induced apoptosis and reduced ROT-induced accumulation of lysosomes in both acute and chronic conditions in DALNs. Our data suggest that LRRK2 kinase regulated both apoptotic cell death and autophagy in DALNs under OS. Given that defects in mitochondrial complex I activity are commonly observed in PD, ROT works well as a chemical model of PD in both acute and chronic conditions. Therefore, prevention and treatment therapy should be guided to relieve DALNs from mitochondrial damage and OS, two of the most important triggers in the apoptotic cell death of DALNs.
Assuntos
Doença de Parkinson , Rotenona , Humanos , Rotenona/farmacologia , Rotenona/metabolismo , Dopamina/metabolismo , Caspase 3/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Apoptose , Doença de Parkinson/metabolismo , Neurônios Dopaminérgicos/metabolismo , Autofagia , Doença CrônicaRESUMO
Familial Alzheimer's disease (FAD) is a complex neurodegenerative disorder for which there are no therapeutics to date. Several mutations in presenilin 1 (PSEN 1), which is the catalytic component of γ-secretase complex, are causal of FAD. Recently, the p.Ile416Thr (I416T) PSEN 1 mutation has been reported in large kindred in Colombia. However, cell and molecular information from I416T mutation is scarce. Here, we demonstrate that menstrual stromal cells (MenSCs)-derived planar (2D) PSEN 1 I416T cholinergic-like cells (ChLNS) and (3D) cerebral spheroids (CSs) reproduce the typical neuropathological markers of FAD in 4 post-transdifferentiating or 11 days of transdifferentiating, respectively. The models produce intracellular aggregation of APPß fragments (at day 4 and 11) and phosphorylated protein TAU at residue Ser202/Thr205 (at day 11) suggesting that iAPPß fragments precede p-TAU. Mutant ChLNs and CSs displayed DJ-1 Cys106-SO3 (sulfonic acid), failure of mitochondria membrane potential (ΔΨm), and activation of transcription factor c-JUN and p53, expression of pro-apoptotic protein PUMA, and activation of executer protein caspase 3 (CASP3), all markers of cell death by apoptosis. Moreover, we found that both mutant ChLNs and CSs produced high amounts of extracellular eAß42. The I416T ChLNs and CSs were irresponsive to acetylcholine induced Ca2+ influx compared to WT. The I416T PSEN 1 mutation might work as dominant-negative PSEN1 mutation. These findings might help to understanding the recurring failures of clinical trials of anti-eAß42, and support the view that FAD is triggered by the accumulation of other intracellular AßPP metabolites, rather than eAß42.
Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Neurônios/metabolismo , Colinérgicos , MutaçãoRESUMO
Leucine-rich repeat kinase 2 (LRRK2) has been linked to dopaminergic neuronal vulnerability to oxidative stress (OS), mitochondrial impairment, and increased cell death in idiopathic and familial Parkinson's disease (PD). However, how exactly this kinase participates in the OS-mitochondria-apoptosis connection is still unknown. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 LRRK2 knockout (KO) in the human embryonic kidney cell line 293 (HEK-293) to evaluate the cellular response to the mitochondrial inhibitor complex I rotenone (ROT), a well-known OS and cell death inducer. We report successful knockout of the LRRK2 gene in HEK-293 cells using CRISPR editing (ICE, approximately 60%) and flow cytometry (81%) analyses. We found that HEK-293 LRRK2 WT cells exposed to rotenone (ROT, 50 µM) resulted in a significant increase in intracellular reactive oxygen species (ROS, +7400%); oxidized DJ-1-Cys106-SO3 (+52%); phosphorylation of LRRK2 (+70%) and c-JUN (+171%); enhanced expression of tumor protein (TP53, +2000%), p53 upregulated modulator of apoptosis (PUMA, +1950%), and Parkin (PRKN, +22%); activation of caspase 3 (CASP3, +8000%), DNA fragmentation (+35%) and decreased mitochondrial membrane potential (ΔΨm, -58%) and PTEN induced putative kinase 1 (PINK1, -49%) when compared to untreated cells. The translocation of the cytoplasmic fission protein dynamin-related Protein 1 (DRP1) to mitochondria was also observed by colocalization with translocase of the outer membrane 20 (TOM20). Outstandingly, HEK-293 LRRK2 KO cells treated with ROT showed unaltered OS and apoptosis markers. We conclude that loss of LRRK2 causes HEK-293 to be resistant to ROT-induced OS, mitochondrial damage, and apoptosis in vitro. Our data support the hypothesis that LRRK2 acts as a proapoptotic kinase by regulating mitochondrial proteins (e.g., PRKN, PINK1, DRP1, and PUMA), transcription factors (e.g., c-JUN and TP53), and CASP3 in cells under stress conditions. Taken together, these observations suggest that LRRK2 is an important kinase in the pathogenesis of PD.
Assuntos
Proteínas Reguladoras de Apoptose , Rotenona , Humanos , Rotenona/toxicidade , Caspase 3/metabolismo , Células HEK293 , Proteínas Reguladoras de Apoptose/metabolismo , Estresse Oxidativo , Apoptose/genética , Proteínas Quinases/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismoRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder caused by the progressive loss of dopaminergic (DAergic) neurons in the substantia nigra and the intraneuronal presence of Lewy bodies (LBs), composed of aggregates of phosphorylated alpha-synuclein at residue Ser129 (p-Ser129α-Syn). Unfortunately, no curative treatment is available yet. To aggravate matters further, the etiopathogenesis of the disorder is still unresolved. However, the neurotoxin rotenone (ROT) has been implicated in PD. Therefore, it has been widely used to understand the molecular mechanism of neuronal cell death. In the present investigation, we show that ROT induces two convergent pathways in HEK-293 cells. First, ROT generates H2O2, which, in turn, either oxidizes the stress sensor protein DJ-Cys106-SH into DJ-1Cys106SO3 or induces the phosphorylation of the protein LRRK2 kinase at residue Ser395 (p-Ser395 LRRK2). Once active, the kinase phosphorylates α-Syn (at Ser129), induces the loss of mitochondrial membrane potential (ΔΨm), and triggers the production of cleaved caspase 3 (CC3), resulting in signs of apoptotic cell death. ROT also reduces glucocerebrosidase (GCase) activity concomitant with the accumulation of lysosomes and autophagolysosomes reflected by the increase in LC3-II (microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate II) markers in HEK-293 cells. Second, the exposure of HEK-293 LRRK2 knockout (KO) cells to ROT displays an almost-normal phenotype. Indeed, KO cells showed neither H2O2, DJ-1Cys106SO3, p-Ser395 LRRK2, p-Ser129α-Syn, nor CC3 but displayed high ΔΨm, reduced GCase activity, and the accumulation of lysosomes and autophagolysosomes. Similar observations are obtained when HEK-293 LRRK2 wild-type (WT) cells are exposed to the inhibitor GCase conduritol-ß-epoxide (CBE). Taken together, these observations imply that the combined development of LRRK2 inhibitors and compounds for recovering GCase activity might be promising therapeutic agents for PD.
Assuntos
Glucosilceramidase , Doença de Parkinson , Humanos , Glucosilceramidase/genética , Rotenona/farmacologia , Rotenona/metabolismo , Células HEK293 , Peróxido de Hidrogênio/metabolismo , alfa-Sinucleína/metabolismo , Doença de Parkinson/metabolismo , Lisossomos/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismoRESUMO
Alzheimer's disease (AD) is a chronic neurological condition characterized by the severe loss of cholinergic neurons. Currently, the incomplete understanding of the loss of neurons has prevented curative treatments for familial AD (FAD). Therefore, modeling FAD in vitro is essential for studying cholinergic vulnerability. Moreover, to expedite the discovery of disease-modifying therapies that delay the onset and slow the progression of AD, we depend on trustworthy disease models. Although highly informative, induced pluripotent stem cell (iPSCs)-derived cholinergic neurons (ChNs) are time-consuming, not cost-effective, and labor-intensive. Other sources for AD modeling are urgently needed. Wild-type and presenilin (PSEN)1 p.E280A fibroblast-derived iPSCs, menstrual blood-derived menstrual stromal cells (MenSCs), and umbilical cord-derived Wharton Jelly's mesenchymal stromal cells (WJ-MSCs) were cultured in Cholinergic-N-Run and Fast-N-Spheres V2 medium to obtain WT and PSEN 1 E280A cholinergic-like neurons (ChLNs, 2D) and cerebroid spheroids (CSs, 3D), respectively, and to evaluate whether ChLNs/CSs can reproduce FAD pathology. We found that irrespective of tissue source, ChLNs/CSs successfully recapitulated the AD phenotype. PSEN 1 E280A ChLNs/CSs show accumulation of iAPPß fragments, produce eAß42, present TAU phosphorylation, display OS markers (e.g., oxDJ-1, p-JUN), show loss of ΔΨm, exhibit cell death markers (e.g., TP53, PUMA, CASP3), and demonstrate dysfunctional Ca2+ influx response to ACh stimuli. However, PSEN 1 E280A 2D and 3D cells derived from MenSCs and WJ-MSCs can reproduce FAD neuropathology more efficiently and faster (11 days) than ChLNs derived from mutant iPSCs (35 days). Mechanistically, MenSCs and WJ-MSCs are equivalent cell types to iPSCs for reproducing FAD in vitro.
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Células-Tronco Mesenquimais , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Alzheimer/metabolismo , Neurônios Colinérgicos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Colinérgicos/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismoRESUMO
Parkinson's disease (PD), a progressive neurodegenerative movement disorder, has reached pandemic status worldwide. This neurologic disorder is caused primarily by the specific deterioration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNc). Unfortunately, there are no therapeutic agents that slow or delay the disease progression. Herein, menstrual stromal cell-derived dopamine-like neurons (DALNs) intoxicated with paraquat (PQ2+)/maneb (MB) were used as a model system to elucidate the mechanism by which CBD protects the neural cell from apoptosis in vitro. According to immunofluorescence microscopy, flow cytometry, cell-free assay, and molecular docking analysis, we demonstrate that CBD offers protection to DALNs against PQ2+ (1 mM)/MB (50 µM)-induced oxidative stress (OS) by simultaneously (i) decreasing reactive oxygen species (ROS: O2â¢-, H2O2), (ii) maintaining the mitochondrial membrane potential (ΔΨm), (iii) directly binding to stress sensor protein DJ-1, thereby blunting its oxidation from DJ-1CYS106-SH into DJ-1CYS106-SO3, and (iv) directly binding to pro-apoptotic protease protein caspase 3 (CASP3), thereby disengaging neuronal dismantling. Furthermore, the protective effect of CBD on DJ-1 and CASP3 was independent of CB1 and CB2 receptor signaling. CBD also re-established the Ca2+ influx in DALNs as a response to dopamine (DA) stimuli under PQ2+/MB exposure. Because of its powerful antioxidant and antiapoptotic effects, CBD offers potential therapeutic utility in the treatment of PD.
Assuntos
Canabidiol , Maneb , Doença de Parkinson , Humanos , Paraquat/toxicidade , Paraquat/metabolismo , Maneb/toxicidade , Maneb/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Canabidiol/farmacologia , Canabidiol/metabolismo , Caspase 3/metabolismo , Dopamina/metabolismo , Receptores de Canabinoides/metabolismo , Peróxido de Hidrogênio/farmacologia , Simulação de Acoplamento Molecular , Morte Celular , Neurônios Dopaminérgicos/metabolismo , Estresse OxidativoRESUMO
BACKGROUND: Familial Alzheimer's disease (FAD) is caused by mutations in one or more of 3 genes known as A ß PP, PSEN1, and PSEN2. There are currently no effective therapies for FAD. Hence, novel therapeutics are needed. OBJECTIVE: To analyze the effect of treatment with a combination of epigallocatechin-3-gallate (EGCG) and Melatonin (N-acetyl-5-methoxytryptamine, aMT) in a cerebral spheroid (CS) 3D in vitro model of PSEN 1 E280A FAD. METHODS: We developed a CS in vitro model based on menstrual stromal cells derived from wild-type (WT) and mutant PSEN1 E280A menstrual blood cultured in Fast-N-Spheres V2 medium. RESULTS: Beta-tubulin III, choline acetyltransferase, and GFAP in both WT and mutant CSs spontaneously expressed neuronal and astroglia markers when grown in Fast-N-Spheres V2 medium for 4 or 11 days. Mutant PSEN1 CSs had significantly increased levels of intracellular AßPP fragment peptides and concomitant appearance of oxidized DJ-1 as early as 4 days, and phosphorylated tau, decreased ΔΨm, and increased caspase-3 activity were observed on Day 11. Moreover, mutant CSs were unresponsive to acetylcholine. Treatment with a combination of EGCG and aMT decreased the levels of all typical pathological markers of FAD more efficiently than did EGCG or aMT alone, but aMT failed to restore Ca2 + influx in mutant CSs and decreased the beneficial effect of EGCG on Ca2 + influx in mutant CSs. CONCLUSION: Treatment with a combination of EGCG and aMT can be of high therapeutic value due to the high antioxidant capacity and anti-amyloidogenic effect of both compounds.
RESUMO
OBJECTIVES: To explore the challenges in diagnosing acute flaccid myelitis (AFM) and evaluate clinical features and treatment paradigms associated with under recognition. STUDY DESIGN: This was a retrospective multicenter study of pediatric patients (≤18 years) who were diagnosed with AFM from 2014 to 2018 using the Centers for Disease Control and Prevention's case definition. RESULTS: In 72% of the cases (126 of 175), AFM was not considered in the initial differential diagnosis (n = 108; 61.7%) and/or the patient was not referred for acute care (n = 90; 51.4%) at the initial clinical encounter, and this did not improve over time. Although many features of the presentation were similar in those initially diagnosed with AFM and those who were not; preceding illness, constipation, and reflexes differed significantly between the 2 groups. Patients with a non-AFM initial diagnosis more often required ventilatory support (26.2% vs 12.2%; OR, 0.4; 95% CI, 0.2-1.0; P = .05). These patients received immunomodulatory treatment later (3 days vs 2 days after neurologic symptom onset; 95% CI, -2 to 0; P = .05), particularly intravenous immunoglobulin (5 days vs 2 days; 95% CI, -4 to -2; P < .001). CONCLUSIONS: Delayed recognition of AFM is concerning because of the risk for respiratory decompensation and need for intensive care monitoring. A non-AFM initial diagnosis was associated with delayed treatment that could have a clinical impact, particularly as new treatment options emerge.
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Viroses do Sistema Nervoso Central , Infecções por Enterovirus , Mielite , Doenças Neuromusculares , Criança , Humanos , Mielite/diagnóstico , Mielite/terapia , Doenças Neuromusculares/diagnóstico , Doenças Neuromusculares/terapia , Viroses do Sistema Nervoso Central/diagnóstico , Viroses do Sistema Nervoso Central/terapia , Estudos Retrospectivos , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/terapiaRESUMO
Purpose: Primary central nervous system (CNS) tumors are the second most common cancer in children and adolescents, leading to premature death and disability. Population-based survival estimates aid decision-making in cancer control, however data on survival for primary CNS tumors in Latin America is lacking. We describe survival rates for children with primary CNS tumors treated in ten Colombian cities. Methods: We analyzed data from children and adolescents newly diagnosed with cancer between 2012 and 2021, participating in the Childhood Cancer Clinical Outcomes Surveillance System (VIGICANCER) in ten cities in Colombia. VIGICANCER collects information on clinical outcomes from twenty-seven pediatric oncology units and conducts active follow-up every three months. VIGICANCER does not register craniopharyngiomas; we excluded intracranial germ cell tumors for this report. We used the Kaplan-Meier method to estimate the overall survival probability, stratified by sociodemographic variables, topography, WHO grading, receipt of radiation therapy, and type of surgical resection. We analyzed the prognostic capacity of variables using multivariate proportional Cox's regression, stratified by city and year of diagnosis. Results: During the study period, VIGICANCER included 989 primary CNS tumors in 879 children and 110 adolescents. The cohort median age was 9 years; 53% of patients were males, and 8% were Afro-descendants. Most common tumors were supratentorial astrocytomas (47%), astrocytic tumors (35%), medulloblastomas (20%), ependymomas (11%), and mixed and unspecified gliomas (10%). Five-year overall survival of the entire cohort was 54% (95% CI, 51-58); for supratentorial gliomas, WHO grade I was 77%, II was 62%, III-IV was 27%, respectively, and for medulloblastoma was 61%. The adjusted hazard rate ratio for patients with WHO grade III and IV, for those with subtotal resection, for brainstem location, and for those not receiving radiation therapy was 7.4 (95% CI, 4.7-11.8), 6.4 (95% CI, 4.2-9.8), 2.8 (95% 2.1-3.8), 2.0 (95% CI, 1.3-2.8) and 2.3 (95% CI, 1.7-3.0), respectively. Conclusion: We found that half of Colombia's children and adolescents with primary CNS tumors survive five years, compared to 70% to 80% in high-income countries. In addition to tumor biology and location, gross total resection was crucial for improved survival in this cohort. Systematic monitoring of survival and its determinants provides empirical data for guiding cancer control policies.
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The optimization of resources for research in developing countries forces us to consider strategies in the wet lab that allow the reuse of molecular biology reagents to reduce costs. In this study, we used linear regression as a method for predictive modeling of coverage depth given the number of MinION reads sequenced to define the optimum number of reads necessary to obtain >200X coverage depth with a good lineage-clade assignment of SARS-CoV-2 genomes. The research aimed to create and implement a model based on machine learning algorithms to predict different variables (e.g., coverage depth) given the number of MinION reads produced by Nanopore sequencing to maximize the yield of high-quality SARS-CoV-2 genomes, determine the best sequencing runtime, and to be able to reuse the flow cell with the remaining nanopores available for sequencing in a new run. The best accuracy was -0.98 according to the R squared performance metric of the models. A demo version is available at https://genomicdashboard.herokuapp.com/.
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COVID-19 , SARS-CoV-2 , Humanos , Análise de Sequência de DNA/métodos , SARS-CoV-2/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , GenomaRESUMO
Acute lymphoblastic leukemia (ALL) is hematological neoplasia that affects human beings from early life to adulthood. Although ALL treatment has been effective, an important percentage of ALL patients are resilient to treatment. Therefore, there is an urgent need for testing a new combination of compounds for the treatment of this disease. Recently, combined TPEN and TPGS (T2 combo) have shown selective cytotoxic effects in vitro leukemia cells such as Jurkat, K562, and Ba/F3 cells. In this study, we aimed to test the effect of combined TPEN and TPGS agents (T2 combo) at a fixed dose (TPEN 5 mg/kg: TPGS 100 mg/kg) on leukemic Ba/F3-BCR-ABL P210 BALB-c mice model. We found that 4 successive 2-day apart intravenous injections of T2 combo showed a statistically significant reduction of Ba/F3 BCR-ABL leukemia cells (- 69%) in leukemia BALB/c mice (n = 6) compared to untreated leukemia group (n = 6). Moreover, the T2 combo was innocuous to non-leukemia BALB/c mice (n = 3) compared to untreated non-leukemia mice (control, n = 3). After treatments (day 42), all mice were left to rest until day 50. Outstandingly, the leukemia BALB/c mice treated with the T2 combo showed a lower percentage of Ba/F3-BCR-ABL P210 cells (- 84%) than untreated leukemia BALB/c mice. Furthermore, treatment of leukemia and non-leukemia mice with T2 combo showed no significant tissue alteration/damage according to the histopathological analysis of brain, heart, liver, kidney, and spleen samples; however, T2 combo significantly reduced the number of leukocytes in the bone marrow of treated leukemia mice. We conclude that the T2 combo specifically affects leukemia cells but no other tissue/organs. Therefore, we anticipate that the T2 combo might be a potential pro-oxidant combination for the treatment of leukemia patients.
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Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Camundongos , Animais , Adulto , Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Cromossomo Filadélfia , Camundongos Endogâmicos BALB CRESUMO
Background: Acute lymphoblastic leukemia (ALL) is still incurable hematologic neoplasia in an important percentage of patients. Therefore, new therapeutic approaches need to be developed. Methods: To evaluate the cellular effect of cell-penetrating peptides (C-PP) on leukemia cells, Jurkat cells -a model of ALL were exposed to increasing concentration (50-500 µM) Aß25-35, R7-G-Aß25-35 and Aß25-35-G-R7 peptide for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry (FC), and fluorescent microscopy (FM) analysis were used to assess metabolic viability, cell cycle and proliferation, mitochondria functionality, oxidative stress, and cell death markers. Results: We report for the first time that the R7-G-Aß25-35, but not Aß25-35 peptide, induced selective cell death in Jurkat cells more efficiently than the Aß25-35-G-R7 peptide. Indeed, R7-G-Aß25-35 (200 µM) altered the metabolic activity (-25%), arrested the cell cycle in the G2/M-phase (15%), and induced a significant reduction of cellular proliferation (i.e., -74% reduction of Ki-67 nuclei reactivity). Moreover, R7-G-Aß25-35 induced the dissipation of mitochondrial membrane potential (ΔΨm, 51%) and produced an important amount of reactive oxygen species (ROS, 75% at 8 h) in Jurkat cells. The exposure of cells to antioxidant/cytoprotectant N-acetylcysteine (NAC) did not prevent R7-G-Aß25-35 from a loss of ΔΨm in Jurkat cells. The peptide was also unable to activate the executer CASPASE-3, thereby preserving the integrity of the cellular DNA corroborated by the fact that the caspase-3 inhibitor NSCI was unable to protect cells from R7-G-Aß25-35 -induced cell damage. Further analysis showed that the R7-G-Aß25-35 peptide is specifically localized at the outer mitochondria membrane (OMM) according to colocalization with the protein translocase TOMM20. Additionally, the cytotoxic effect of the poly-R7 peptide resembles the toxic action of the uncoupler FCCP, mitocan oligomycin, and rotenone in Jurkat cells. Importantly, the R7-G-Aß25-35 peptide was innocuous to menstrual mesenchymal stromal cells (MenSC) -normal non-leukemia proliferative cells. Conclusion: Our findings demonstrated that the cationic Aß peptide possesses specific anti-leukemia activity against Jurkat cells through oxidative stress (OS)- and CASPASE-3-independent mechanism but fast mitochondria depolarization.
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Introduction: There is scarce information in Latin America about factors related to stroke patient outcomes in rural areas compared to urban ones. Objective: To evaluate functional outcomes of stroke code patients from rural and urban areas and their relationship with socioeconomic disparity. Methods: Prospective cohort study included patients of urban, semi-urban, and rural origin with stroke code from a high complexity hospital in southwestern Colombia between 2018 and 2019. Demographic, clinical data modified Rankin at discharge, and 3-month follow-up were analyzed. The poverty index, barriers to health access and availability of ambulances by the municipality was assessed at an ecological level. Results: Five hundred and fifty five stroke patients were registered, 21.2% from rural areas, 432 (77.98%) had an ischemic stroke. There were no significant differences in sociodemographic factors and medical background. Urban patients had lower reperfusion therapies rates (23.25%). Favorable mRS at discharge (<3) was higher in urban areas (63.03%) and mortality was superior in rural patients (13.56%). The ambulance rate in semi-urban and rural areas was as low as 0.03 per 100.000 inhabitants, the poverty index was 11.9% in urban areas vs. 23.3% in semi urban and rural areas. Conclusions: Rural patients treated in our center were more likely to present with severe strokes and unfavorable mRS at hospital discharge and 3-month follow-up compared to urban, despite having similar risk factors. There is an inverse relationship, which is not related to the poverty rate or the percentage of people with barriers to access to health. There is a need for further studies that assess barriers inherent in rural patients and establish a regional stroke network.
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TPEN and TPGS have recently shown selective cytotoxic effects in vitro and ex vivo leukemia cells. In this study, we aimed to test the synergistic effect of combined TPEN and TPGS agents (thereafter, T2 combo) on Jurkat (clone-E61), K562, Ba/F3, and non-leukemia peripheral blood lymphocytes (PBL). The ED50 doses (i.e., TPEN ED50: 3.2 µM and TPGS ED50: 34 µM, potency ratio R = 10.62 = TPGS (ED50)/TPEN (ED50)) were identified as dose-effect curve (%DNA fragmentation (sub-G1 phase) versus agent concentration). The most effective synergistic doses were determined according to isobole analysis. The apoptotic and oxidative stress effects of combined doses (TPEN 0.1, 0.5, 1 µM) and TPGS (5, 10, 20 µM)) were evaluated by DNA fragmentation (sub-G1 phase), mitochondrial membrane potential, oxidation of stress sensor protein DJ-1, and activation of executer protein CASPASE-3. They testified to the synergistic effect of the T2 combo (e.g., TPEN 1: TPGS 20, combination index (CI) 0.90 < 1; 1/3.2+ 20/34, > 90% induced apoptosis) in all 3 cell lines. As proof of principle, we challenged complete bone marrow (n = 5) or isolated cells from bone marrow (n = 3) samples from acute pediatric acute B-cell patients and found that T2 combo (1:20; 10:200) dramatically reduced (- 50%) the CD34+/CD19+cell population and increased significantly CD19+/CASP-3+ positive B-ALL cells up to 960%. The T2 combo neither induced DNA fragmentation, altered ΔΨm, nor induced oxidation of stress sensor protein DJ-1, nor activated CASP-3 in PBL cells. We conclude that by using different combinations of TPEN and TPGS, a more efficient treatment strategy can be developed for leukemia patients.