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Alelos , Heterocigoto , Fenotipo , Globinas alfa , Talasemia beta , Humanos , Talasemia beta/genética , Globinas alfa/genética , Masculino , Femenino , AdultoRESUMEN
BACKGROUND: Cerebral amyloid angiopathy (CAA) is a disease caused by the accumulation of the amyloid-beta protein and is a major cause of intracerebral hemorrhage (ICH) and vascular dementia in the elderly. The presence of the amyloid-beta protein in the vessel wall may induce a chronic state of cerebral inflammation by activating astrocytes, microglia, and pro-inflammatory substances. Minocycline, an antibiotic of the tetracycline family, is known to modulate inflammation, gelatinase activity, and angiogenesis. These processes are suggested to be key mechanisms in CAA pathology. Our aim is to show the target engagement of minocycline and investigate in a double-blind placebo-controlled randomized clinical trial whether treatment with minocycline for 3 months can decrease markers of neuroinflammation and of the gelatinase pathway in cerebrospinal fluid (CSF) in CAA patients. METHODS: The BATMAN study population consists of 60 persons: 30 persons with hereditary Dutch type CAA (D-CAA) and 30 persons with sporadic CAA. They will be randomized for either placebo or minocycline (15 sporadic CAA/15 D-CAA minocycline, 15 sporadic CAA/15 D-CAA placebo). At t = 0 and t = 3 months, we will collect CSF and blood samples, perform a 7-T MRI, and collect demographic characteristics. DISCUSSION: The results of this proof-of-principle study will be used to assess the potential of target engagement of minocycline for CAA. Therefore, our primary outcome measures are markers of neuroinflammation (IL-6, MCP-1, and IBA-1) and of the gelatinase pathway (MMP2/9 and VEGF) in CSF. Secondly, we will look at the progression of hemorrhagic markers on 7-T MRI before and after treatment and investigate serum biomarkers. TRIAL REGISTRATION: ClinicalTrials.gov NCT05680389. Registered on January 11, 2023.
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Angiopatía Amiloide Cerebral Familiar , Angiopatía Amiloide Cerebral , Anciano , Humanos , Péptidos beta-Amiloides , Antibacterianos/farmacología , Angiopatía Amiloide Cerebral/diagnóstico por imagen , Angiopatía Amiloide Cerebral/tratamiento farmacológico , Angiopatía Amiloide Cerebral/complicaciones , Angiopatía Amiloide Cerebral Familiar/complicaciones , Angiopatía Amiloide Cerebral Familiar/patología , Hemorragia Cerebral/etiología , Gelatinasas , Inflamación , Minociclina , Enfermedades Neuroinflamatorias , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
BACKGROUND: Familial Mediterranean Fever (FMF) is the earliest described and most prevalent hereditary auto-inflammatory disease. Its clinical presentation is diverse, leading to possible delay in diagnosis and treatment. Due to immigration, FMF became common in non-Mediterranean European regions. In the present single centre retrospective study, the clinical, demographic, and genetic characteristics of patients with FMF of different ancestry in Amsterdam are described. METHODS: Case records of patients with FMF, who met the Tel-Hashomer diagnostic criteria, were retrospectively analysed. The international disease severity score was used. RESULTS: Between 1990-2012, 53 patients were identified, 28 were female. Main country of origin was Turkey. The mean age at the time of analysis was 29.1 years; 13.8 years at onset of symptoms; and at time of diagnosis, 22.0 years. Most frequent symptoms were peritonitis (91%) and fever (81%). The mean C-reactive protein and erythrocyte sedimentation rate during acute attacks were 133 mg/l and 37 mm/first hour, respectively. One patient developed amyloidosis as a complication. Seventeen patients underwent abdominal surgery before diagnosis. Most patients (92%) received colchicine treatment and were responsive (81%). Most patients classified their disease as a mild disease (42%). MEFV gene mutation analysis was performed in 46 patients; most patients were compound heterozygotes (n = 17), and the most frequent mutation was M694V (n = 18). CONCLUSION: FMF in Amsterdam is diagnosed in relatively young patients and the delay to diagnosis is 8.2 years. Disease manifestations and genetic distribution of our FMF patients are comparable to those in Mediterranean regions, suggesting that ancestry is more important than environment.
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Colchicina/uso terapéutico , Fiebre Mediterránea Familiar , Peritonitis , Pirina/genética , Adolescente , Adulto , Edad de Inicio , Demografía , Intervención Médica Temprana , Fiebre Mediterránea Familiar/epidemiología , Fiebre Mediterránea Familiar/genética , Fiebre Mediterránea Familiar/fisiopatología , Fiebre Mediterránea Familiar/terapia , Femenino , Humanos , Masculino , Mutación , Países Bajos/epidemiología , Peritonitis/diagnóstico , Peritonitis/etiología , Prevalencia , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Moduladores de Tubulina/uso terapéuticoRESUMEN
OBJECTIVE: To study whether genetic variation in coagulation and fibrinolysis genes contributes to cerebrovascular complications in bacterial meningitis. METHODS: We performed a nationwide prospective genetic association study in adult community-acquired bacterial meningitis patients. The exons and flanking regions of 16 candidate genes involved in coagulation and fibrinolysis pathways were sequenced. We analyzed whether genetic variation in these genes resulted in a higher risk of cerebrovascular complications, unfavorable outcome and differences in thrombocyte count on admission. RESULTS: From 2006 to 2011, a total of 1101 bacterial meningitis patients were identified of whom 622 supplied DNA for genotyping and passed genetic quality control steps. In 139 patients (22%) the episode of bacterial meningitis was complicated by cerebral infarction, and 188 (30%) had an unfavorable outcome. We identified the functional variant rs494860 in the protein Z (PROZ) gene as our strongest association with occurrence of cerebral infarction (odds ratio (OR) 0.49 (95% confidence interval 0.33-0.73), pâ¯=â¯5.2â¯×â¯10-4). After Bonferroni correction for multiple testing no genetic variant was significantly associated (p-value threshold 2.7â¯×â¯10-4). CONCLUSION: Our study suggests a functional genetic variation in the PROZ gene, rs494860, may be of importance in bacterial meningitis pathogenesis and cerebral infarction risk. Replication of this finding in other cohort studies populations is needed.
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Coagulación Sanguínea/genética , Trastornos Cerebrovasculares/etiología , Fibrinólisis/genética , Estudios de Asociación Genética , Meningitis Bacterianas/complicaciones , Meningitis Neumocócica/complicaciones , Adulto , Anciano , Infarto Cerebral/epidemiología , Infarto Cerebral/etiología , Trastornos Cerebrovasculares/microbiología , Estudios de Cohortes , Infecciones Comunitarias Adquiridas/epidemiología , Infecciones Comunitarias Adquiridas/microbiología , Femenino , Variación Genética , Humanos , Masculino , Meningitis Bacterianas/epidemiología , Persona de Mediana Edad , Países Bajos/epidemiología , Oportunidad Relativa , Estudios Prospectivos , Análisis de Secuencia de ADNRESUMEN
AIM: The NET, encoded by SLC6A2, is responsible for presynaptic NE-reuptake. 123I-mIBG is clinically used to evaluate cardiac sympathetic function. However, it is unknown if polymorphism of SLC6A2 influences cardiac sympathetic activity as assessed with 123I-mIBG. Therefore we studied the influence of SLC6A2 SNPs on myocardial 123I-mIBG parameters in CHF. MATERIALS AND METHODS: Forty-nine adults with stable CHF (age 66.5 ± 8.1 years, LVEF 22.3 ± 6.4) were enrolled. Fifteen minutes (early) and 4 hours (late) after administration of 123I-mIBG planar images were acquired. The H/M ratio was calculated from the manually drawn ROI over the left ventricle and a fixed mediastinal ROI. Fourteen exons of the SLC6A2 gene were analyzed from whole blood samples. RESULTS: We found 6 different SLC6A2 SNPs, although none were functional. LVEF was the only independent predictor for early (adjusted R 2 = 0.063, p = 0.045) and late H/M ratio (adjusted R 2 = 0.116, p = 0.010). NT-proBNP was the only independent predictor for 123I-mIBG WO (adjusted R 2 = 0.074, p = 0.032). SLC6A2 SNPs were not associated with any myocardial 123I-mIBG-derived parameter. CONCLUSION: In this specific CHF population polymorphism of SLC6A2 gene was not associated with any 123I-mIBG derived parameters.
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3-Yodobencilguanidina , Insuficiencia Cardíaca/diagnóstico por imagen , Insuficiencia Cardíaca/genética , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/genética , Polimorfismo Genético/genética , Cintigrafía , Anciano , Enfermedad Crónica , Femenino , Humanos , Radioisótopos de Yodo , Masculino , Persona de Mediana Edad , Radiofármacos , Volumen SistólicoRESUMEN
BACKGROUND AND AIM: T cells are key players in the chronic intestinal inflammation that characterises Crohn's disease. Here we aim to map the intestinal T-cell receptor [TCR] repertoire in patients with Crohn's disease, using next-generation sequencing technology to examine the clonality of the T-cell compartment in relation to mucosal inflammation and response to therapy. METHODS: Biopsies were taken from endoscopically inflamed and uninflamed ileum and colon of 19 patients with Crohn's disease. From this cohort, additional biopsies were taken after 8 weeks of remission induction therapy from eight responders and eight non-responders. Control biopsies from 11 patients without inflammatory bowel disease [IBD] were included. The TCRß repertoire was analysed by next-generation sequencing of biopsy RNA. RESULTS: Both in Crohn's disease patients and in non-IBD controls, a broad intestinal T-cell repertoire was found, with a considerable part consisting of expanded clones. Clones in Crohn's disease were more expanded [p = 0.008], with the largest clones representing up to as much as 58% of the total repertoire. There was a substantial overlap of the repertoire between inflamed and uninflamed tissue and between ileum and colon. Following therapy, responders showed larger changes in the T-cell repertoire than non-responders, although a considerable part of the repertoire remained unchanged in both groups. CONCLUSIONS: The intestinal T-cell repertoire distribution in Crohn's disease is different from that in the normal gut, containing profoundly expanded T-cell clones that take up a large part of the repertoire. The T-cell repertoire is fairly stable regardless of endoscopic mucosal inflammation or response to therapy.
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Enfermedad de Crohn/inmunología , Enfermedad de Crohn/patología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Linfocitos T/patología , Adalimumab/uso terapéutico , Adulto , Antiinflamatorios/uso terapéutico , Biopsia , Budesonida/uso terapéutico , Proteína C-Reactiva/metabolismo , Estudios de Casos y Controles , Células Clonales/efectos de los fármacos , Colon/patología , Enfermedad de Crohn/tratamiento farmacológico , Femenino , Fármacos Gastrointestinales/uso terapéutico , Humanos , Íleon/patología , Inflamación/inmunología , Inflamación/patología , Infliximab/uso terapéutico , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Linfocitos T/efectos de los fármacos , Adulto JovenRESUMEN
Mycobacterium leprae infection gives rise to the immunologically and histopathologically classified spectrum of leprosy. At present, several tools for the stratification of patients are based on acquired immunity markers. However, the role of innate immunity, particularly the complement system, is largely unexplored. The present retrospective study was undertaken to explore whether the systemic levels of complement activation components and regulators can stratify leprosy patients, particularly in reference to the reactional state of the disease. Serum samples from two cohorts were analysed. The cohort from Bangladesh included multi-bacillary (MB) patients with (n = 12) or without (n = 46) reaction (R) at intake and endemic controls (n = 20). The cohort from Ethiopia included pauci-bacillary (PB) (n = 7) and MB (n = 23) patients without reaction and MB (n = 15) patients with reaction. The results showed that the activation products terminal complement complex (TCC) (P ≤ 0·01), C4d (P ≤ 0·05) and iC3b (P ≤ 0·05) were specifically elevated in Bangladeshi patients with reaction at intake compared to endemic controls. In addition, levels of the regulator clusterin (P ≤ 0·001 without R; P < 0·05 with R) were also elevated in MB patients, irrespective of a reaction. Similar analysis of the Ethiopian cohort confirmed that, irrespective of a reaction, serum TCC levels were increased significantly in patients with reactions compared to patients without reactions (P ≤ 0·05). Our findings suggests that serum TCC levels may prove to be a valuable tool in diagnosing patients at risk of developing reactions.
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Clusterina/sangre , Activación de Complemento , Complemento C3b/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Inmunidad Innata , Lepra/inmunología , Adolescente , Adulto , Bangladesh , Biomarcadores/sangre , Etiopía , Femenino , Interacciones Huésped-Patógeno , Humanos , Lepra/sangre , Lepra/diagnóstico , Lepra/microbiología , Masculino , Persona de Mediana Edad , Mycobacterium leprae/inmunología , Mycobacterium leprae/patogenicidad , Estudios RetrospectivosRESUMEN
AIM: The current literature shows no consensus on the localization and number of characteristic neuronal inclusions [p62 and dipeptide repeat proteins (DRPs) positive, TDP-43-negative and TDP-43 positive] in the brain and spinal cord of patients with the hexanucleotide repeat expansion on chromosome 9 (C9ORF72-positive patients). This may be due to small sample sizes. A valid brain map of the inclusions in C9ORF72-positive patients may improve clinicopathological correlations and may serve as a reference for neuropathologists. METHODS: We performed a systematic review on 42 pathological studies to assess the pooled prevalence rates and density (a measure of the number of inclusions per brain region) of (phosphorylated)-TDP-43, p62 and DRP neuronal inclusions in seven brain regions and the spinal cord of 261 C9ORF72-positive patients with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and ALS-FTD. RESULTS: In the cerebellum and hippocampus, the pooled prevalence rates of TDP-43 neuronal cytoplasmic inclusions (NCIs; cerebellum: 3.9%; hippocampus: 68.3%) were lower than those of DRP (cerebellum: 97.2%; hippocampus 97.1%). Moreover, TDP-43 inclusion density was lower compared with p62 inclusion density in these regions. The pooled prevalence rate of TDP-43 NCI in the substantia nigra was high (94.4%). DISCUSSION: The findings of this systematic review largely confirm findings of previous smaller studies on the localization and prevalence of inclusions in the central nervous system of C9ORF72-positive patients. The high prevalence of TDP-43 inclusions in the substantia nigra is a relatively new finding and is probably related to the relatively high prevalence of parkinsonism in C9ORF72-positive patients.
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Esclerosis Amiotrófica Lateral/patología , Encéfalo/patología , Demencia Frontotemporal/patología , Cuerpos de Inclusión/patología , Médula Espinal/patología , Adulto , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Expansión de las Repeticiones de ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Demencia Frontotemporal/genética , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Unión al ARN/metabolismoRESUMEN
BACKGROUND: Mortality and morbidity in patients with bacterial meningitis result from the proinflammatory response and dysregulation of coagulation and fibrinolysis. Thrombin-activatable fibrinolysis inhibitor (TAFI) is activated by free thrombin or thrombin in complex with thrombomodulin, and plays an antifibrinolytic role during fibrin clot degradation, but also has an anti-inflammatory role by inactivating proinflammatory mediators, such as complement activation products. OBJECTIVE: To assess the role of TAFI in pneumococcal meningitis. METHODS: We performed a prospective nationwide genetic association study in patients with bacterial meningitis, determined TAFI and complement levels in cerebrospinal fluid (CSF), and assessed the function of TAFI in a pneumococcal meningitis mouse model by using Cpb2 (TAFI) knockout mice. RESULTS: Polymorphisms (reference sequences: rs1926447 and rs3742264) in the CPB2 gene, coding for TAFI, were related to the development of systemic complications in patients with pneumococcal meningitis. Higher protein levels of TAFI in CSF were significantly associated with CSF complement levels (C3a, iC3b, and C5b-9) and with more systemic complications in patients with bacterial meningitis. The risk allele of rs1926447 (TT) was associated with higher levels of TAFI in CSF. In the murine model, consistent with the human data, Cpb2-deficient mice had decreased disease severity, as reflected by lower mortality, and attenuated cytokine levels and bacterial outgrowth in the systemic compartment during disease, without differences in the brain compartment, as compared with wild-type mice. CONCLUSIONS: These findings suggest that TAFI plays an important role during pneumococcal meningitis, which is likely to be mediated through inhibition of the complement system, and influences the occurrence of systemic complications and inflammation.
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Carboxipeptidasa B2/fisiología , Meningitis Meningocócica/líquido cefalorraquídeo , Meningitis Neumocócica/líquido cefalorraquídeo , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Animales , Daño Encefálico Crónico/etiología , Carboxipeptidasa B2/líquido cefalorraquídeo , Carboxipeptidasa B2/deficiencia , Carboxipeptidasa B2/genética , Hemorragia Cerebral/etiología , Infecciones Comunitarias Adquiridas/sangre , Infecciones Comunitarias Adquiridas/líquido cefalorraquídeo , Infecciones Comunitarias Adquiridas/complicaciones , Infecciones Comunitarias Adquiridas/genética , Complemento C3a/líquido cefalorraquídeo , Complemento C3b/líquido cefalorraquídeo , Complejo de Ataque a Membrana del Sistema Complemento/líquido cefalorraquídeo , Citocinas/sangre , Femenino , Fibrinólisis , Humanos , Masculino , Meningitis Meningocócica/sangre , Meningitis Meningocócica/complicaciones , Meningitis Meningocócica/genética , Meningitis Neumocócica/sangre , Meningitis Neumocócica/complicaciones , Meningitis Neumocócica/genética , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Insuficiencia Respiratoria/etiología , Choque Séptico/etiología , Resultado del TratamientoRESUMEN
Genetic factors are important in all forms of dementia, especially in early onset dementia. The frequency of major gene defects in dementia has not been investigated in the Netherlands. Furthermore, whether the recently in a FTD family identified PRKAR1B gene is associated with an Alzheimer's disease (AD) like phenotype, has not been studied. With this study, we aimed to investigate the mutation frequency of the major AD and FTD genes and the PRKAR1B gene in a well-defined Dutch cohort of patients with early onset dementia. Mutation analysis of the genes PSEN1, APP, MAPT, GRN, C9orf72 and PRKAR1B was performed on DNA of 229 patients with the clinical diagnosis AD and 74 patients with the clinical diagnosis FTD below the age of 70 years. PSEN1 and APP mutations were found in, respectively 3.5 and 0.4 % of AD patients, and none in FTD patients. C9orf72 repeat expansions were present in 0.4 % of AD and in 9.9 % of FTD patients, whereas MAPT and GRN mutations both were present in 0.4 % in AD patients, and in 1.4 % resp. 2.7 % in FTD patients. We did not find any pathogenic mutations in the PRKAR1B gene. PSEN1 mutations are the most common genetic cause in Dutch AD patients, whereas MAPT and GRN mutations were found in less than 5 percent. C9orf72 repeat expansions were the most common genetic defect in FTD patients. No pathogenic PRKAR1B mutations were found in the early onset AD and FTD patients of our study.
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Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Demencia/diagnóstico , Demencia/genética , Mutación/genética , Adulto , Edad de Inicio , Anciano , Estudios de Cohortes , Demencia/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Países Bajos/epidemiologíaRESUMEN
Peripheral nerve injury leads to Wallerian degeneration, followed by regeneration, in which functionality and morphology of the nerve are restored. We previously described that deficiency for complement component C6, which prevents formation of the membrane attack complex, slows down degeneration and results in an earlier recovery of sensory function after sciatic nerve injury compared to WT animals. In this study, we determine whether C6(-/-) rats have an intrinsic trait that affects sciatic nerve regeneration after injury. To study the contribution of complement activation on degeneration and regeneration with only minimal effect of complement activation, a crush injury model with only modest complement deposition was used. We compared the morphological and functional aspects of crushed nerves during degeneration and regeneration in C6(-/-) and WT animals. Morphological changes of myelin and axons showed similar degeneration and regeneration patterns in WT and C6(-/-) injured nerves. Functional degeneration and regeneration, recorded by ex vivo electrophysiology and in vivo foot flick test, showed that the timeline of the restoration of nerve conduction and sensory recovery also followed similar patterns in WT and C6(-/-) animals. Our findings suggest that C6 deficiency by itself does not alter the regrowth capacity of the peripheral nerve after crush injury.
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Complemento C6/deficiencia , Regeneración Nerviosa , Traumatismos de los Nervios Periféricos/fisiopatología , Nervio Ciático/lesiones , Nervio Ciático/fisiología , Animales , Axones/fisiología , Complemento C6/fisiología , Masculino , Vaina de Mielina/fisiología , Ratas , Degeneración Walleriana/fisiopatologíaRESUMEN
BACKGROUND: Asthma and atopy share common characteristics including type 2 helper-T-cell-mediated inflammation. However, only asthma is associated with variable airways obstruction. The complex cellular and molecular pathways distinguishing asthma and atopy can now be captured by transcriptomic analysis (RNA-Seq). We hypothesized that the transcriptomic profile of airway smooth muscle (ASM) distinguishes atopic asthma from atopic healthy controls. First, we compared the ASM transcriptomic profiles of endobronchial biopsies between glucocorticoid-free, atopic asthma patients, and atopic and nonatopic healthy controls. Second, we investigated the association between ASM transcriptomic profiles and airway function. METHODS: Twelve asthma patients and 12 control subjects (six atopic, six nonatopic) underwent bronchoscopy. RNA of laser-dissected ASM from 96 bronchial biopsy specimens was sequenced with Roche GS FLX. Gene networks were identified using Ingenuity Pathway Analysis. RNA-Seq reads were assumed to follow a negative binomial distribution. With the current sample size, the estimated false discovery rate was approximately 1%. RESULTS: One hundred and seventy four ASM genes were differentially expressed between asthma patients and atopic controls, 108 between asthma patients and nonatopic controls, and 135 between atopic and nonatopic controls. A set of eight genes discriminated asthma patients from nonasthmatic controls, irrespective of atopy. Four of these genes (RPTOR, VANGL1, FAM129A, LEPREL1) were associated with airway hyper-responsiveness (P < 0.05). CONCLUSION: Airway smooth muscle from asthma patients can be distinguished from that of atopic and nonatopic control subjects by a specific gene expression profile, which is associated with airway hyper-responsiveness.
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Asma/genética , Hipersensibilidad/genética , Músculo Liso , Transcriptoma/genética , Adulto , Femenino , Perfilación de la Expresión Génica , Humanos , Captura por Microdisección con Láser , Masculino , Adulto JovenRESUMEN
BACKGROUND: Myoclonus dystonia syndrome (MDS) is a hyperkinetic movement disorder caused, in a proportion of cases, by mutations of the maternally imprinted epsilon-sarcoglycan gene (SGCE). SGCE mutation rates vary between cohorts, suggesting genetic heterogeneity. E- and ζ-sarcoglycan are both expressed in brain tissue. In this study we tested whether zeta-sarcoglycan gene (SGCZ) mutations also contribute to this disorder. METHODS: Patients with clinically suspected MDS and no SGCE mutation were recruited and classified, according to previously published criteria, as to their likelihood of the movement disorder. All SGCZ exons and intron/exon boundaries were screened by direct sequencing. RESULTS: Fifty-four SGCE mutation-negative patients were recruited from the UK and the Netherlands. Subdivided according to the likelihood of the movement disorder resulted in 17 'definite', 16 'probable' and 21 'possible' cases. No pathogenic SGCZ mutations were identified. CONCLUSIONS: SGCZ mutations are unlikely to contribute to the genetic heterogeneity in MDS.
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Trastornos Distónicos/genética , Mutación/genética , Mioclonía/genética , Sarcoglicanos/genética , Adolescente , Adulto , Niño , Exones/genética , Femenino , Humanos , Intrones/genética , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
OBJECTIVE: To identify potential autoreactive B-cell and plasma-cell clones by quantitatively analysing the complete human B-cell receptor (BCR) repertoire in synovium and peripheral blood in early and established rheumatoid arthritis (RA). METHODS: The BCR repertoire was screened in synovium and blood of six patients with early RA (ERA) (<6 months) and six with established RA (ESRA) (>20 months). In two patients, the repertoires in different joints were compared. Repertoires were analysed by next-generation sequencing from mRNA, generating >10 000 BCR heavy-chain sequence reads per sample. For each clone, the degree of expansion was calculated as the percentage of the total number of reads encoding the specific clonal sequence. Clones with a frequency ≥ 0.5% were considered dominant. RESULTS: Multiple dominant clones were found in inflamed synovium but hardly any in blood. Within an individual patient, the same dominant clones were detected in different joints. The majority of the synovial clones were class-switched; however, the fraction of clones that expressed IgM was higher in ESRA than ERA patients. Dominant synovial clones showed autoreactive features: in ERA in particular the clones were enriched for immunoglobulin heavy chain gene segment V4-34 (IGHV4-34) and showed longer CDR3 lengths. Dominant synovial clones that did not encode IGHV4-34 also had longer CDR3s than peripheral blood. CONCLUSIONS: In RA, the synovium forms a niche where expanded--potentially autoreactive--B cells and plasma cells reside. The inflamed target tissue, especially in the earliest phase of disease, seems to be the most promising compartment for studying autoreactive cells.
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Artritis Reumatoide/inmunología , Autoinmunidad/inmunología , Linfocitos B/inmunología , Membrana Sinovial/inmunología , Secuencia de Aminoácidos , Artritis Reumatoide/genética , Células Clonales/inmunología , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Femenino , Humanos , Cambio de Clase de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Activación de Linfocitos/inmunología , Masculino , Datos de Secuencia Molecular , Células Plasmáticas/inmunología , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Peripheral nerve damage induces a sequence of degeneration and regeneration events with a specific time course that leads to (partial) functional recovery. Quantitative electrophysiological analysis of degeneration and recovery over time is essential to understand the process. NEW METHOD: The presented ex vivo neurophysiological method evaluates functional recovery of the propagation of the compound action potential after crush injury of the rat sciatic nerve. A 32 channel electrode array was used to monitor compound action potential propagation at time points between 1h and 35 days after semi-quantitative crush injury of the rat sciatic nerve. RESULTS: The compound action potential was characterized by four measures: the latency, the duration, the amplitude and a measure that combined time and location. These four parameters reflected the subsequent steps in early axonal degradation, the transition to rapid degeneration followed by sprouting and the long period of remyelination that accompanied regeneration. COMPARISON WITH EXISTING METHODS: The neurophysiology measures of the compound action potential were compared with the morphology of the nerve at representative time points and analysis of functional recovery of action potential propagation was compared with a behavioral test: the foot flick test. CONCLUSIONS: Our data suggests that the ex vivo electrophysiological method is complementary to the classical behavioral foot flick test in that it allows a detailed time analysis of the degeneration and early regeneration phases at a high spatial and temporal sensitivity. The results were well-matched with observations made with immunohistochemical and morphological methods.
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Degeneración Nerviosa , Regeneración Nerviosa , Nervio Ciático/lesiones , Neuropatía Ciática/patología , Neuropatía Ciática/fisiopatología , Potenciales de Acción , Animales , Axones/patología , Axones/fisiología , Estimulación Eléctrica , Electrodos , Electrofisiología/métodos , Técnica del Anticuerpo Fluorescente , Técnicas In Vitro , Masculino , Compresión Nerviosa , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Conducción Nerviosa , Neurofisiología/métodos , Ratas , Recuperación de la Función , Nervio Ciático/patología , Nervio Ciático/fisiopatología , Neuropatía Ciática/etiología , Sensación/fisiología , Nervio Tibial/patología , Nervio Tibial/fisiopatología , Factores de TiempoRESUMEN
CD8(+) T-cell responses against latent viruses can cover considerable portions of the CD8(+) T-cell compartment for many decades, yet their initiation and maintenance remains poorly characterized in humans. A key question is whether the clonal repertoire that is raised during the initial antiviral response can be maintained over these long periods. To investigate this we combined next-generation sequencing of the T-cell receptor repertoire with tetramer-sorting to identify, quantify and longitudinally follow virus-specific clones within the CD8(+) T-cell compartment. Using this approach we studied primary infections of human cytomegalovirus (hCMV) and Epstein Barr virus (EBV) in renal transplant recipients. For both viruses we found that nearly all virus-specific CD8(+) T-cell clones that appeared during the early phase of infection were maintained at high frequencies during the 5-year follow-up and hardly any new anti-viral clones appeared. Both in transplant recipients and in healthy carriers the clones specific for these latent viruses were highly dominant within the CD8(+) T-cell receptor Vß repertoire. These findings suggest that the initial antiviral response in humans is maintained in a stable fashion without signs of contraction or changes of the clonal repertoire.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Receptores de Antígenos de Linfocitos T/genética , Antígenos Virales/inmunología , Citomegalovirus/genética , Herpesvirus Humano 4/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Trasplante de Riñón/inmunología , Persona de Mediana Edad , Factores de Tiempo , Latencia del Virus , Adulto JovenRESUMEN
OBJECTIVE: To profile quantitatively the T-cell repertoire in multiple joints and peripheral blood of patients with recent onset (early) or established rheumatoid arthritis (RA) using a novel next-generation sequencing protocol to identify potential autoreactive clones. METHODS: Synovium of patients with recent onset (early) RA (<6 months) (n=6) or established RA (>18 months) (n=6) was screened for T-cell clones by sequencing over 10 000 T-cell receptors (TCR) per sample. T cells from paired blood samples were analysed for comparison. From two patients synovial T cells were obtained from multiple inflamed joints. The degree of expansion of each individual clone was based on its unique CDR3 sequence frequency within a sample. Clones with a frequency of over 0.5% were considered to be highly expanded clones (HEC). RESULTS: In early RA synovium, the T-cell repertoire was dominated by 35 HEC (median, range 2-70) accounting for 56% of the TCR sequenced. The clonal dominance in the synovium was patient specific and significantly greater than in established RA (median of 11 HEC (range 5-24) in established RA synovium accounting for 9.8% of T cells; p<0.01). 34% (range 28-40%) of the most expanded T-cell clones were shared between different joints in the same patients, compared with only 4% (range 0-8%) between synovium and blood (p=0.01). CONCLUSIONS: In RA, a systemic autoimmune disease, the inflamed synovium forms a niche for specific expanded T-cell clones, especially in early disease. This suggests that, at least in RA, autoreactive T cells should be addressed specifically in the inflamed tissue, preferably in the early phase of the disease.
Asunto(s)
Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Autoinmunidad/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Biopsia , Microambiente Celular/inmunología , Células Clonales/citología , Células Clonales/inmunología , Progresión de la Enfermedad , Humanos , Membrana Sinovial/inmunología , Membrana Sinovial/patologíaRESUMEN
Since the membrane attack complex (MAC), an end product of the activated complement cascade, has been shown to play a role in neurodegeneration, we investigated to which extent MAC contributes to structural reorganization, neuronal cell death, and seizure development in two rat models for temporal lobe epilepsy. We used the electrically-induced status epilepticus (SE) model and the kindling model in C6-deficient rats (that are unable to form MAC) and wild-type (WT) PVG/c rats. Structural reorganization was investigated using hilar cell counts and mossy fiber sprouting. Seizure development was monitored using electroencephalographic (EEG) recordings. 4 weeks after electrically stimulated SE, hilar cell counts in C6-deficient and WT post-SE rats were significantly decreased compared to an unstimulated control group, but not different between C6-deficient and WT post-SE. Since seizure development was unexpectedly absent in most post-SE rats we assessed epileptogenesis using the kindling rate as main parameter. Kindling development was slightly delayed in C6-deficient rats compared to WT rats. The lack of effect of C6 deficiency on hilar cell death and mossy fiber sprouting after electrically-induced SE or kindling argues against a role of the terminal complement complex in neuronal cell death induced by SE or seizures. A small but significant delay of kindling epileptogenesis suggests a subtle role of MAC in seizure spread. Whether complement components upstream of MAC play a crucial role in neuronal death and/or epileptogenesis needs to be further investigated.
Asunto(s)
Encéfalo/fisiopatología , Complemento C6/deficiencia , Fibras Musgosas del Hipocampo/patología , Convulsiones/fisiopatología , Animales , Encéfalo/patología , Muerte Celular , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Modelos Animales de Enfermedad , Estimulación Eléctrica , Electroencefalografía , Epilepsia del Lóbulo Temporal/patología , Epilepsia del Lóbulo Temporal/fisiopatología , Femenino , Excitación Neurológica , Fibras Musgosas del Hipocampo/fisiopatología , Ratas , Convulsiones/patologíaRESUMEN
Neisseria meningitidis is an obligate human pathogen. While it is a frequent commensal of the upper respiratory tract, in some individuals the bacterium spreads to the bloodstream, causing meningitis and/or sepsis, which are serious conditions with high morbidity and mortality. Here we report the availability of the genome sequence of the widely used serogroup B laboratory strain H44/76.