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Intraoperative anti-A/B immunoadsorption (ABO-IA) was recently introduced for ABO-incompatible (ABOi) heart transplantation. Here we report the first case of a patient transplanted with ABO-IA, that was of an age and weight that required two ABO-IA columns run in parallel, to enable the reduction in antibody titres to a sufficiently low level in the time available during implantation of the donor organ.
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INTRODUCTION: Oxygenators for paediatric Extracorporeal Membrane Oxygenation (ECMO) are required to operate over a wide range of flow rates, in a patient group ranging from neonates through to fully grown adolescents. ECMO oxygenators typically have a manufacturer's stated maximum gas: blood flow rate (GBFR) ratio of 2:1, however, many patients require greater ratios than this for adequate CO2 removal. Mismatches in GBFR in theory could result in high gas phase pressures. These increased pressures in theory could cause the formation of gross gaseous microemboli (GME) placing the child at higher risk of neurological injury. METHODS: We evaluated 6 paediatric and 6 adult A.L.ONE™ ECMO oxygenators and assessed their gas phase pressures and GME release, in an ex vivo setting, in GBFR ratios up to greater than 2, across a range of gas flow (1L - 10 L/min) rates with a fraction of inspired oxygen (FiO2) content of 50% and 100%. RESULTS: There were no increases above 10 mmHg observed in gas phase pressures in GBFR >= 2:1 in either adult or paediatric oxygenators. Laboratory examination of GME activity demonstrated a small increase in post-membrane GME release over the study period. GME release was unaffected by FiO2 setting or gas flow rate, with a maximum volume of < 6 µL in both paediatric and adult oxygenators. CONCLUSIONS: In an ex vivo setting, increasing GBFR above 2:1 in a paediatric oxygenator, and to a GBFR of 2:1 in an adult oxygenator did not significantly increase gas phase pressures, and no oxygenator membrane rupture was observed. There were no associations between gas flow rates and GME production.
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The most prominent genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a repeat expansion in the gene C9orf72. Importantly, the transcriptomic consequences of the C9orf72 repeat expansion remain largely unclear. Here, we used short-read RNA sequencing (RNAseq) to profile the cerebellar transcriptome, detecting alterations in patients with a C9orf72 repeat expansion. We focused on the cerebellum, since key C9orf72-related pathologies are abundant in this neuroanatomical region, yet TDP-43 pathology and neuronal loss are minimal. Consistent with previous work, we showed a reduction in the expression of the C9orf72 gene and an elevation in homeobox genes, when comparing patients with the expansion to both patients without the C9orf72 repeat expansion and control subjects. Interestingly, we identified more than 1000 alternative splicing events, including 4 in genes previously associated with ALS and/or FTLD. We also found an increase of cryptic splicing in C9orf72 patients compared to patients without the expansion and controls. Furthermore, we demonstrated that the expression level of select RNA-binding proteins is associated with cryptic splice junction inclusion. Overall, this study explores the presence of widespread transcriptomic changes in the cerebellum, a region not confounded by severe neurodegeneration, in post-mortem tissue from C9orf72 patients.
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Esclerose Lateral Amiotrófica , Proteína C9orf72 , Cerebelo , Degeneração Lobar Frontotemporal , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Cerebelo/patologia , Expansão das Repetições de DNA/genética , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/metabolismo , Degeneração Lobar Frontotemporal/patologia , Perfilação da Expressão Gênica , TranscriptomaRESUMO
BACKGROUND: Anti-human leukocyte antigen (HLA)-antibody production represents a major barrier to heart transplantation, limiting recipient compatibility with potential donors and increasing the risk of complications with poor waiting-list outcomes. Currently there is no consensus to when desensitization should take place, and through what mechanism, meaning that sensitized patients must wait for a compatible donor for many months, if not years. We aimed to determine if intraoperative immunoadsorption could provide a potential desensitization methodology. METHODS: Anti-HLA antibody-containing whole blood was added to a Cardiopulmonary bypass (CPB) circuit set up to mimic a 20 kg patient undergoing heart transplantation. Plasma was separated and diverted to a standalone, secondary immunoadsorption system, with antibody-depleted plasma returned to the CPB circuit. Samples for anti-HLA antibody definition were taken at baseline, when combined with the CPB prime (on bypass), and then every 20 min for the duration of treatment (total 180 min). RESULTS: A reduction in individual allele median fluorescence intensity (MFI) to below clinically relevant levels (<1000 MFI), and in the majority of cases below the lower positive detection limit (<500 MFI), even in alleles with a baseline MFI >4000 was demonstrated. Reduction occurred in all cases within 120 min, demonstrating efficacy in a time period usual for heart transplantation. Flowcytometric crossmatching of suitable pseudo-donor lymphocytes demonstrated a change from T cell and B cell positive channel shifts to negative, demonstrating a reduction in binding capacity. CONCLUSIONS: Intraoperative immunoadsorption in an ex vivo setting demonstrates clinically relevant reductions in anti-HLA antibodies within the normal timeframe for heart transplantation. This method represents a potential desensitization technique that could enable sensitized children to accept a donor organ earlier, even in the presence of donor-specific anti-HLA antibodies.
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Transplante de Coração , Transplante de Rim , Criança , Humanos , Ponte Cardiopulmonar , Doadores de Tecidos , Antígenos HLARESUMO
BACKGROUND: Sensitised patients undergoing Human Leukocyte Antigen-incompatible transplantation are at increased risk of hyperacute rejection and may be predisposed to antibody-mediated rejection, chronic lung allograft dysfunction and higher mortality. CASE: We present a case of primary lung transplantation in the setting of late identification of donor specific antibodies treated with intraoperative target plasma exchange. The patient was treated with fresh human plasma to a final volume of 1.5 times the patient's systemic circulation. From a pre-transplant mean fluorescence intensity of 5002, donor-specific antibodies were undetectable following plasma exchange on single antigen bead assay. CONCLUSIONS: This method represents a potential desensitisation technique for use in the intraoperative period.
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Transplante de Pulmão , Troca Plasmática , Humanos , Lactente , Antígenos HLA , Doadores de Tecidos , Transplante HomólogoRESUMO
BACKGROUND: Intraoperative anti-A/B immunoadsorption (ABO-IA) was recently introduced for ABO-incompatible heart transplantation. Here we report the first case series of patients transplanted with ABO-IA, and compare outcomes with those undergoing plasma exchange facilitated ABO-incompatible heart transplantation (ABO-PE). METHODS: Data were retrospectively analysed on all ABO-incompatible heart transplants undertaken at a single centre between January 1, 2000 and June 1, 2020. Data included all routine laboratory tests, demographics and pre-operative characteristics, intraoperative details and post-operative outcomes. Primary outcome measures were volume of blood product transfusions, maximum post-transplant isohaemagglutinin titres, occurrence of rejection and graft survival. Secondary outcome measures were length of intensive care and hospital stay. Demographic and survival data were also obtained for ABO-compatible transplants during the same time period for comparison. RESULTS: Thirty-seven patients underwent ABO-incompatible heart transplantation, with 27 (73%) using ABO-PE and 10 (27%) using ABO-IA. ABO-IA patients were significantly older than ABO-PE patients (p < 0.001) and the total volume of blood products transfused during the hospital admission was significantly lower (164 [126-212] ml/kg vs 323 [268-379] ml/kg, p < 0.001). No significant differences were noted between methods in either pre or post-transplant maximum isohaemagglutinin titres, incidence of rejection, length of intensive care or total hospital stay. Survival comparison showed no significant difference between antibody reduction methods, or indeed ABO-compatible transplants (p = 0.6). CONCLUSIONS: This novel technique appears to allow a significantly older population than typical to undergo ABO-incompatible heart transplantation, as well as significantly reducing blood product utilization. Furthermore, intraoperative anti-A/B immunoadsorption does not demonstrate increased early post-transplant isohaemagglutinin accumulation or rates of rejection compared to ABO-PE. Early survival is equivalent between ABO-IA, ABO-PE and ABO-compatible heart transplantation.
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Sistema ABO de Grupos Sanguíneos/imunologia , Anticorpos/imunologia , Incompatibilidade de Grupos Sanguíneos/imunologia , Rejeição de Enxerto/prevenção & controle , Transplante de Coração/métodos , Cuidados Intraoperatórios/métodos , Plasmaferese/métodos , Incompatibilidade de Grupos Sanguíneos/complicações , Incompatibilidade de Grupos Sanguíneos/terapia , Criança , Pré-Escolar , Feminino , Seguimentos , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto , Humanos , Lactente , Masculino , Estudos RetrospectivosRESUMO
Traditionally, ABO-incompatible heart transplantation was accomplished using a plasma exchange technique to remove recipient plasma containing donor-incompatible anti-A/B isohaemagglutinins. However, this technique exposed patients to large volumes of allogeneic blood and blood products (up to three times the patient's circulating volume). In 2018, we published the first reported case of an ABO-incompatible heart transplant using an intraoperative immunoadsorption technique which minimises the exposure to blood products by specifically targeting anti-A/B isohaemagglutinins. We have subsequently used this technique in all children undergoing ABO-incompatible heart transplantation and become convinced of its efficacy in this population while observing no adverse effects. This article outlines the practical details required to perform the technique in order to avoid hyperacute rejection.
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Incompatibilidade de Grupos Sanguíneos , Transplante de Coração , Sistema ABO de Grupos Sanguíneos , Criança , Hospitais , Humanos , PlasmafereseRESUMO
Necrotizing pneumonia can lead to respiratory insufficiency in previously healthy children. Extracorporeal membrane oxygenation can be used for hemodynamic salvage and subsequent lung rest awaiting recovery. We present a case of a child initially placed on veno-arterial extracorporeal membrane oxygenation and converted to veno-venous extracorporeal membrane oxygenation. This was done under deep hypothermia in the operating theater.
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Cânula/normas , Oxigenação por Membrana Extracorpórea/métodos , Hipotermia Induzida/métodos , Pneumonia Necrosante/terapia , Pré-Escolar , Feminino , HumanosRESUMO
Congenital hyperinsulinism is a clinical syndrome of pancreatic ß-cell dysfunction characterized by failure to suppress insulin secretion in the presence of hypoglycemia. Here, we describe the concerns, the techniques used to ameliorate these potential problems, and the outcomes for a child with this condition undergoing cardiopulmonary bypass for correction of an atrial septal defect.
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Procedimentos Cirúrgicos Cardíacos/métodos , Ponte Cardiopulmonar/métodos , Cardiopatias Congênitas/cirurgia , Hiperinsulinismo/congênito , Cardiopatias Congênitas/diagnóstico , Humanos , Hiperinsulinismo/diagnóstico , Lactente , MasculinoRESUMO
Sickle cell anaemia results from homozygosity of an A-T point mutation leading to a substitution of glutamic acid for valine at point 6 of the ß-globin gene. A person with sickle cell trait inherits one normal and one mutated allele. Patients with sickle cell anaemia, and to a lesser extent sickle cell trait, have a wide spectrum of haemostatic abnormalities. Here we describe the problems faced in obtaining an accurate activated clotting time measurement, in a patient with sickle cell trait, prior to commencement of cardiopulmonary bypass for decannulation of Berlin Heart.
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Anemia Falciforme/sangue , Coagulação Sanguínea/fisiologia , Traço Falciforme/sangue , Pré-Escolar , Humanos , MasculinoRESUMO
Beta (ß)-thalassemia is a blood disorder with an incidence of 1 in 100,000.1 This case report outlines a patient with ß-thalassemia requiring cardiopulmonary bypass (CPB) and the measures taken to ensure an uneventful procedure.
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Procedimentos Cirúrgicos Cardíacos/métodos , Ponte Cardiopulmonar/métodos , Defeitos dos Septos Cardíacos/complicações , Defeitos dos Septos Cardíacos/cirurgia , Talassemia beta/complicações , Criança , Humanos , Lactente , MasculinoRESUMO
BACKGROUND: Since 1996, ABO-incompatible heart transplantation has been undertaken by performing whole-body plasma exchange to remove isohemagglutinins using the cardiopulmonary bypass (CPB) circuit at the time of transplantation. This requires large volumes of donated blood and blood products, causes hemodynamic instability during the exchange transfusion, and limits practical use to small children. We sought to determine the efficacy of anti-A/B immunoadsorption within the CPB circuit on removal of isohemagglutinins in an ex vivo setting before its use clinically. METHODS: An anti-A/B immunoadsorption column was placed into a CPB circuit mimicking a typical ABO-incompatible transplant patient, which had been primed with type O whole human blood. Samples were taken for determination of isohemagglutinin titers following each plasma volume pass through the anti-A/B immunoadsorption column. RESULTS: There was a linear decrease of at least 1 dilution seen in both anti-A and anti-B IgG and IgM antibodies with each plasma volume pass through the column. This predictable removal allowed the formulation of selection criteria for ABO-incompatible heart transplantation given the reciprocal of titer and patient weight. This degree of predictability allowed us to use it successfully in the clinical setting, reducing antibodies to an undetectable level during ABO-incompatible heart transplantation. CONCLUSIONS: The incorporation of an anti-A/B immunoadsorption column into the extracorporeal circuit reduces allogeneic blood product requirement for ABO-incompatible heart transplantation, while providing efficacious removal of anti-A and anti-B isohemagglutinins. This can be undertaken within the time period of CPB before graft reperfusion and expands the potential recipient pool to larger patients with higher isohemagglutinin titers.
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Sistema ABO de Grupos Sanguíneos , Incompatibilidade de Grupos Sanguíneos , Ponte Cardiopulmonar/métodos , Transplante de Coração/métodos , Técnicas de Imunoadsorção , Estudos de Viabilidade , Hemaglutininas , Humanos , Modelos Cardiovasculares , Modelagem Computacional Específica para o PacienteRESUMO
OBJECTIVES: Colloid osmotic pressure (COP) is a major determinant of fluid shift in paediatric patients undergoing corrective surgery for congenital heart disease (CHD) using cardiopulmonary bypass (CPB). However, very few baseline data are available for those patients requiring surgery within the first few weeks and months of life. Our aim was to determine if our CHD population exhibited COP similar to that of other extremely ill subsets of patients and, if so, whether this related to risk stratification model scores. METHODS: Thirty consecutive patients under 10 kg underwent cardiac surgery with CPB. Data were collected on COP, albumin concentration, prime composition, post-operative length of ventilation and intensive care unit (ICU) stay as well as risk stratification utilising Risk Adjustment for Congenital Heart Surgery (RACHS-1) and Partial Risk Adjustment in Surgery (PRAiS) scoring systems. RESULTS: The patients had a mean pre-bypass COP of 13.9±2.5 mmHg. A significant negative correlation was observed between pre-operative baseline COP and length of ventilation (r=0.7; p<0.001) and a significant negative correlation between PRAiS (r=0.64; p<0.001) and RACHS-1 (r=0.5; p=0.004) scores and baseline COP was seen. CONCLUSIONS: Neonatal and paediatric patients requiring surgical correction of complex congenital cardiac malformations exhibit extremely low baseline COP, comparable to other sick neonatal populations, and are lower than those previously reported. Baseline COP correlates significantly with predicted survival rates and time spent on a ventilator.
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Ponte Cardiopulmonar/métodos , Cardiopatias Congênitas/cirurgia , Pressão Osmótica , Pré-Escolar , Intervalo Livre de Doença , Feminino , Cardiopatias Congênitas/mortalidade , Humanos , Lactente , Masculino , Taxa de SobrevidaRESUMO
Insertion and deletion variants (indels) within poly glycine tracts of fused in sarcoma (FUS) were initially reported as causative of disease in amyotrophic lateral sclerosis (ALS). Subsequent studies identified similar indels in controls and suggested that these indels may confer susceptibility to ALS. We aimed to elucidate the role of previously published and novel exonic indels in FUS in an extensive cohort of 630 ALS patients and 1063 controls. We detected indels in FUS exons 5, 6, 12, and 14 with similar frequencies in patients (0.95%) and controls (0.75%). Exonic indels in poly glycine tracts were also observed with similar frequencies. The largest indel (p.Gly138_Tyr143del) was observed in 1 control. In 1 patient, a 3 base pair deletion in exon 14 (p.Gly475del) was identified, however in vitro studies did not reveal abnormal localization of p.Gly475del mutant FUS. These findings suggest that not all exonic indels in FUS cause disease.
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Esclerose Lateral Amiotrófica/genética , Éxons/genética , Predisposição Genética para Doença/genética , Mutação INDEL/genética , Peptídeos/genética , Proteína FUS de Ligação a RNA/genética , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/epidemiologia , Feminino , Predisposição Genética para Doença/epidemiologia , Humanos , Masculino , Prevalência , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Risco , Sensibilidade e Especificidade , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: Frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) is a heritable form of FTD, but the gene(s) responsible for the majority of autosomal dominant FTD-ALS cases have yet to be found. Previous studies have identified a region on chromosome 9p that is associated with FTD and ALS. METHODS: The authors report the clinical, volumetric MRI, neuropathological and genetic features of a new chromosome 9p-linked FTD-ALS family, VSM-20. RESULTS: Ten members of family VSM-20 displayed heterogeneous clinical phenotypes of isolated behavioural-variant FTD (bvFTD), ALS or a combination of the two. Parkinsonism was common, with one individual presenting with a corticobasal syndrome. Analysis of structural MRI scans from five affected family members revealed grey- and white-matter loss that was most prominent in the frontal lobes, with mild parietal and occipital lobe atrophy, but less temporal lobe atrophy than in 10 severity-matched sporadic bvFTD cases. Autopsy in three family members showed a consistent and unique subtype of FTLD-TDP pathology. Genome-wide linkage analysis conclusively linked family VSM-20 to a 28.3 cM region between D9S1808 and D9S251 on chromosome 9p, reducing the published minimal linked region to a 3.7 Mb interval. Genomic sequencing and expression analysis failed to identify mutations in the 10 known and predicted genes within this candidate region, suggesting that next-generation sequencing may be needed to determine the mutational mechanism associated with chromosome 9p-linked FTD-ALS. CONCLUSIONS: Family VSM-20 significantly reduces the region linked to FTD-ALS on chromosome 9p. A distinct pattern of brain atrophy and neuropathological findings may help to identify other families with FTD-ALS caused by this genetic abnormality.
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Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Cromossomos Humanos Par 9/genética , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Adulto , Esclerose Lateral Amiotrófica/complicações , Autopsia , Encéfalo/patologia , Análise Mutacional de DNA , Família , Feminino , Demência Frontotemporal/complicações , Ligação Genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Imuno-Histoquímica , Escore Lod , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/genética , LinhagemRESUMO
Mutations in the gene encoding fused in sarcoma (FUS) were recently identified as a novel cause of amyotrophic lateral sclerosis (ALS), emphasizing the genetic heterogeneity of ALS. We sequenced the genes encoding superoxide dismutase (SOD1), TAR DNA-binding protein 43 (TARDBP) and FUS in 99 sporadic and 17 familial ALS patients ascertained at Mayo Clinic. We identified two novel mutations in FUS in two out of 99 (2.0%) sporadic ALS patients and established the de novo occurrence of one FUS mutation. In familial patients, we identified three (17.6%) SOD1 mutations, while FUS and TARDBP mutations were excluded. The de novo FUS mutation (g.10747A>G; IVS13-2A>G) affects the splice-acceptor site of FUS intron 13 and was shown to induce skipping of FUS exon 14 leading to the C-terminal truncation of FUS (p.G466VfsX14). Subcellular localization studies showed a dramatic increase in the cytoplasmic localization of FUS and a reduction of normal nuclear expression in cells transfected with truncated compared to wild-type FUS. We further identified a novel in-frame insertion/deletion mutation in FUS exon 12 (p.S402_P411delinsGGGG) which is predicted to expand a conserved poly-glycine motif. Our findings extend the mutation spectrum in FUS leading to ALS and describe the first de novo mutation in FUS.
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Esclerose Lateral Amiotrófica/genética , Proteína FUS de Ligação a RNA/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Proteína FUS de Ligação a RNA/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Adulto JovemRESUMO
Mutations in three genes (PSEN1, PSEN2, and APP) have been identified in patients with early-onset (<65 years) Alzheimer's disease (AD). We performed a screening for mutations in the coding regions of presenilins, as well as exons 16 and 17 of the APP gene in a total of 231 patients from the Iberian peninsular with a clinical diagnosis of early-onset AD (mean age at onset of 52.9 years; range 31-64). We found three novel mutations in PSEN1, one novel mutation in PSEN2, and a novel mutation in the APP gene. Four previously described mutations in PSEN1 were also found. The same analysis was carried in 121 elderly healthy controls from the Iberian peninsular, and a set of 130 individuals from seven African populations belonging to the Centre d'Etude du Polymorphisme Humain-Human Genome Diversity Panel (CEPH-HGDP), in order to determine the extent of normal variability in these genes. Interestingly, in the latter series, we found five new non-synonymous changes in all three genes and a presenilin 2 variant (R62H) that has been previously related to AD. In some of these mutations, the pathologic consequence is uncertain and needs further investigation. To address this question we propose and use a systematic algorithm to classify the putative pathology of AD mutations.
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Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Presenilina-1/genética , Presenilina-2/genética , Adulto , África , Idoso , Éxons/genética , Predisposição Genética para Doença/genética , Testes Genéticos , Humanos , Pessoa de Meia-Idade , Mutação/genética , Portugal , EspanhaRESUMO
Here, we describe the generation and characterization of a novel tau transgenic mouse model (mTau) that overexpresses wild-type murine tau protein by twofold compared with endogenous levels. Transgenic tau expression was driven by a BAC transgene containing the entire wild-type mouse tau locus, including the endogenous promoter and the regulatory elements associated with the tau gene. The mTau model therefore differs from other tau models in that regulation of the genomic mouse transgene mimics that of the endogenous gene, including normal exon splicing regulation. Biochemical data from the mTau mice demonstrated that modest elevation of mouse tau leads to tau hyperphosphorylation at multiple pathologically relevant epitopes and accumulation of sarkosyl-insoluble tau. The mTau mice show a progressive increase in hyperphosphorylated tau pathology with age up to 15 to 18 months, which is accompanied by gliosis and vacuolization. In contrast, older mice show a decrease in tau pathology levels, which may represent hippocampal neuronal loss occurring in this wild-type model. Collectively, these results describe a novel model of tauopathy that develops pathological changes reminiscent of early stage Alzheimer's disease and other related neurodegenerative diseases, achieved without overexpression of a mutant human tau transgene. This model will provide an important tool for understanding the early events leading to the development of tau pathology and a model for analysis of potential therapeutic targets for sporadic tauopathies.
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Degeneração Neural/complicações , Degeneração Neural/patologia , Tauopatias/complicações , Tauopatias/patologia , Proteínas tau/metabolismo , Envelhecimento/patologia , Animais , Axônios/patologia , Axônios/ultraestrutura , Cruzamento , Cromossomos Artificiais Bacterianos/genética , Regulação da Expressão Gênica , Vetores Genéticos/genética , Genoma/genética , Camundongos , Camundongos Transgênicos , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Fosforilação , SolubilidadeRESUMO
Mutations in the progranulin gene (GRN) are an important cause of frontotemporal lobar degeneration (FTLD) with ubiquitin and TAR DNA-binding protein 43 (TDP43)-positive pathology. The clinical presentation associated with GRN mutations is heterogeneous and may include clinical probable Alzheimer's disease. All GRN mutations identified thus far cause disease through a uniform disease mechanism, i.e. the loss of functional GRN or haploinsufficiency. To determine if expression of GRN in plasma could predict GRN mutation status and could be used as a biological marker, we optimized a GRN ELISA and studied plasma samples of a consecutive clinical FTLD series of 219 patients, 70 control individuals, 72 early-onset probable Alzheimer's disease patients and nine symptomatic and 18 asymptomatic relatives of GRN mutation families. All FTLD patients with GRN loss-of-function mutations showed significantly reduced levels of GRN in plasma to about one third of the levels observed in non-GRN carriers and control individuals (P < 0.001). No overlap in distributions of GRN levels was observed between the eight GRN loss-of-function mutation carriers (range: 53-94 ng/ml) and 191 non-GRN mutation carriers (range: 115-386 ng/ml). Similar low levels of GRN were identified in asymptomatic GRN mutation carriers. Importantly, ELISA analyses also identified one probable Alzheimer's disease patient (1.4%) carrying a loss-of-function mutation in GRN. Biochemical analyses further showed that the GRN ELISA only detects full-length GRN, no intermediate granulin fragments. This study demonstrates that using a GRN ELISA in plasma, pathogenic GRN mutations can be accurately detected in symptomatic and asymptomatic carriers. The approximately 75% reduction in full-length GRN, suggests an unbalanced GRN metabolism in loss-of-function mutation carriers whereby more GRN is processed into granulins. We propose that plasma GRN levels could be used as a reliable and inexpensive tool to identify all GRN mutation carriers in early-onset dementia populations and asymptomatic at-risk individuals.
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Demência/diagnóstico , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mutação , Adulto , Idoso , Doença de Alzheimer/sangue , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Biomarcadores/sangue , Estudos de Coortes , Demência/sangue , Demência/genética , Ensaio de Imunoadsorção Enzimática/métodos , Saúde da Família , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Progranulinas , Sensibilidade e EspecificidadeRESUMO
The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.