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PCR-based screening assays targeting strain-specific genetic markers allow the timely detection and specific differentiation of bacterial strains. Especially in situations where an infection cluster occurs, fast assay development is crucial for supporting targeted control measures. However, the turnaround times (TATs) for assay setup may be high due to insufficient knowledge about screening assay methods, workflows, and software tools. Here, two blind-coded and quality-controlled ring trials were performed in which five German laboratories established PCR-based screening assays from genomic data that specifically target selected bacterial clusters within two bacterial monospecies sample panels. While the first ring trial was conducted without a time limit to train the participants and assess assay feasibility, in the second ring trial, a challenging time limit of 2 weeks was set to force fast assay development as soon as genomic data were available. During both ring trials, we detected high interlaboratory variability regarding the screening assay methods and targets, the TATs for assay setup, and the number of screening assays. The participants designed between one and four assays per cluster that targeted cluster-specific unique genetic sequences, genes, or single nucleotide variants using conventional PCRs, high-resolution melting assays, or TaqMan PCRs. Assays were established within the 2-week time limit, with TATs ranging from 4 to 13 days. TaqMan probe delivery times strongly influenced TATs. In summary, we demonstrate that a specific exercise improved the preparedness to develop functional cluster-specific PCR-based screening assays from bacterial genomic data. Furthermore, the parallel development of several assays enhances assay availability.
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Bactérias , Genoma Bacteriano , Humanos , Reação em Cadeia da Polimerase/métodos , Genoma Bacteriano/genética , GenômicaRESUMO
INTRODUCTION: In order to improve patient care and to increase food safety within the framework of One Health, the project "Integrated Genomic Surveillance of Zoonotic Agents (IGS-Zoo)" aims to develop concepts for a genomic surveillance of Shiga toxin(Stx)-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany. METHODS: An online survey was conducted to assess the currently available and applied STEC/EHEC typing methods in the federal laboratories of veterinary regulation, food control, and public health service. RESULTS: Twenty-six questionnaires from 33 participants were evaluated with regard to STEC/EHEC. The number of STEC/EHEC-suspected samples that the laboratories process per year ranges between 10 and 3500, and out of these they obtain between 3 and 1000 pathogenic isolates. Currently the most frequently used typing method is the determination of Stx- and intimin-coding genes using polymerase chain reaction (PCR). Whole genome sequencing (WGS) is currently used by eight federal state laboratories, and nine are planning to implement it in the future. The most common obstacle for further typing of STEC/EHEC is that isolation from sample material is often unsuccessful despite apparent PCR detection of the stx genes. DISCUSSION: The results of the survey should facilitate the integration of the analysis methods developed in the project and emphasize the target groups' individual needs for corresponding training concepts.
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Escherichia coli Êntero-Hemorrágica , Infecções por Escherichia coli , Escherichia coli Shiga Toxigênica , Humanos , Toxina Shiga/genética , Alemanha , Escherichia coli Shiga Toxigênica/genética , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/veterináriaRESUMO
High levels of antibodies against glutamic acid decarboxylase (GAD) are observed in patients with different neurological disorders, but cells producing these autoantibodies are largely unexplored. We detect circulating GAD-reactive B cells in peripheral blood that readily differentiate into antibody-producing cells. These cells are highly elevated in most patients with GAD-antibody-associated disorders (n = 15) compared to controls (n = 19). They mainly produce GAD65 antibodies of the IgG1 and IgG4 subclasses and are as abundant as B cells reactive for common recall antigens. Bone marrow cells represent an additional source of GAD antibodies. The identification of GAD-antibody-producing cells has implications for the selection of cell-specific biologics. ANN NEUROL 2019;85:448-454.
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Autoanticorpos/imunologia , Linfócitos B/imunologia , Ataxia Cerebelar/imunologia , Glutamato Descarboxilase/imunologia , Encefalite Límbica/imunologia , Plasmócitos/imunologia , Rigidez Muscular Espasmódica/imunologia , Adolescente , Adulto , Idoso , Células da Medula Óssea/imunologia , Estudos de Casos e Controles , Feminino , Humanos , Imunoglobulina G/imunologia , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVE: Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patients with inflammatory demyelinating diseases of the central nervous system (CNS). We analyzed their pathogenic activity by affinity-purifying these antibodies (Abs) from patients and transferring them to experimental animals. METHODS: Patients with Abs to MOG were identified by cell-based assay. We determined the cross-reactivity to rodent MOG and the recognized MOG epitopes. We produced the correctly folded extracellular domain of MOG and affinity-purified MOG-specific Abs from the blood of patients. These purified Abs were used to stain CNS tissue and transferred in 2 models of experimental autoimmune encephalomyelitis. Animals were analyzed histopathologically. RESULTS: We identified 17 patients with MOG Abs from our outpatient clinic and selected 2 with a cross-reactivity to rodent MOG; both had recurrent optic neuritis. Affinity-purified Abs recognized MOG on transfected cells and stained myelin in tissue sections. The Abs from the 2 patients recognized different epitopes on MOG, the CC' and the FG loop. In both patients, these Abs persisted during our observation period of 2 to 3 years. The anti-MOG Abs from both patients were pathogenic upon intrathecal injection in 2 different rat models. Together with cognate MOG-specific T cells, these Abs enhanced T-cell infiltration; together with myelin basic protein-specific T cells, they induced demyelination associated with deposition of C9neo, resembling a multiple sclerosis type II pathology. INTERPRETATION: MOG-specific Abs affinity purified from patients with inflammatory demyelinating disease induce pathological changes in vivo upon cotransfer with myelin-reactive T cells, suggesting that these Abs are similarly pathogenic in patients. Ann Neurol 2018;84:315-328.
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Autoanticorpos/sangue , Encéfalo/metabolismo , Encéfalo/patologia , Glicoproteína Mielina-Oligodendrócito/sangue , Adulto , Idoso , Animais , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Cobaias , Humanos , Inflamação/sangue , Inflamação/diagnóstico , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Endogâmicos Lew , Adulto JovemRESUMO
The BAFF-APRIL system is best known for its control of B cell homeostasis, and it is a target of therapeutic intervention in autoimmune diseases and lymphoma. By analyzing the expression of the three receptors of this system, B cell maturation Ag (BCMA), transmembrane activator and CAML interactor, and BAFF receptor, in sorted human immune cell subsets, we found that BCMA was transcribed in plasmacytoid dendritic cells (pDCs) in both blood and lymphoid tissue. Circulating human pDCs contained BCMA protein without displaying it on the cell surface. After engagement of TLR7/8 or TLR9, BCMA was detected also on the cell surface of pDCs. The display of BCMA on the surface of human pDCs was accompanied by release of soluble BCMA (sBCMA); inhibition of γ-secretase enhanced surface expression of BCMA and reduced the release of sBCMA by pDCs. In contrast with human pDCs, murine pDCs did not express BCMA, not even after TLR9 activation. In this study, we extend the spectrum of BCMA expression to human pDCs. sBCMA derived from pDCs might determine local availability of its high-affinity ligand APRIL, because sBCMA has been shown to function as an APRIL-specific decoy. Further, therapeutic trials targeting BCMA in patients with multiple myeloma should consider possible effects on pDCs.
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Antígeno de Maturação de Linfócitos B/imunologia , Células Dendríticas/imunologia , Transdução de Sinais/imunologia , Animais , Receptor do Fator Ativador de Células B/imunologia , Antígeno de Maturação de Linfócitos B/biossíntese , Separação Celular , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Camundongos , Reação em Cadeia da PolimeraseRESUMO
Monoclonal Abs against CD20 reduce the number of relapses in multiple sclerosis (MS); commonly this effect is solely attributed to depletion of B cells. Recently, however, a subset of CD3(+)CD20(+) T cells has been described that is also targeted by the anti-CD20 mAb rituximab. Because the existence of cells coexpressing CD3 and CD20 is controversial and features of this subpopulation are poorly understood, we studied this issue in detail. In this study, we confirm that 3-5% of circulating human T cells display CD20 on their surface and transcribe both CD3 and CD20. We report that these CD3(+)CD20(+) T cells pervade thymus, bone marrow, and secondary lymphatic organs. They are found in the cerebrospinal fluid even in the absence of inflammation; in the cerebrospinal fluid of MS patients they occur at a frequency similar to B cells. Phenotypically, these T cells are enriched in CD8(+) and CD45RO(+) memory cells and in CCR7(-) cells. Functionally, they show a higher frequency of IL-4-, IL-17-, IFN-γ-, and TNF-α-producing cells compared with T cells lacking CD20. CD20-expressing T cells respond variably to immunomodulatory treatments given to MS patients: they are reduced by fingolimod, alemtuzumab, and dimethyl fumarate, whereas natalizumab disproportionally increases them in the blood. After depletion by rituximab, they show earlier and higher repopulation than CD20(+) B cells. Taken together, human CD3(+)CD20(+) T cells pervade lymphatic organs and the cerebrospinal fluid, have a strong ability to produce different cytokines, and respond to MS disease modifying drugs.
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Antígenos CD20/biossíntese , Complexo CD3/biossíntese , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Alemtuzumab , Anticorpos Monoclonais Humanizados/farmacologia , Separação Celular , Citocinas/biossíntese , Fumarato de Dimetilo/farmacologia , Cloridrato de Fingolimode/farmacologia , Citometria de Fluxo , Humanos , Fatores Imunológicos/farmacologia , Natalizumab/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens. Here we report sequence data of the STEC strain BfR-EC-18960, which has integrated IS elements in the B-subunit of the Shiga toxin Stx2b gene. The strain was isolated from deer meat at a local butchery in Germany in 2021.
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The characterization of Shiga toxin-producing Escherichia coli (STEC) is necessary to assess their pathogenic potential, but isolation of the strain from complex matrices such as milk remains challenging. In previous work, we have shown the potential of long-read metagenomics to characterize eae-positive STEC from artificially contaminated raw milk without isolating the strain. The presence of multiple E. coli strains in the sample was shown to potentially hinder the correct characterization of the STEC strain. Here, we aimed at determining the STEC:commensal ratio that would prevent the characterization of the STEC. We artificially contaminated pasteurized milk with different ratios of an eae-positive STEC and a commensal E. coli and applied the method previously developed. Results showed that the STEC strain growth was better than the commensal E. coli after enrichment in acriflavine-supplemented BPW. The STEC was successfully characterized in all samples with at least 10 times more STEC post-enrichment compared to the commensal E. coli. However, the presence of equivalent proportions of STEC and commensal E. coli prevented the full characterization of the STEC strain. This study confirms the potential of long-read metagenomics for STEC characterization in an isolation-free manner while refining its limit regarding the presence of background E. coli strains.
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Thermotolerant Campylobacter spp. are fecal contaminants of chicken meat with serious implications for human health. E. coli is considered as hygiene indicator since, in contrast to Campylobacter. spp., the bacterium is generally present in the avian gut. Stress exposure may transiently cease bacterial division. Therefore, colony forming units (CFU) may underestimate the infection risk of pathogens. We developed a viability real-time PCR (v-qPCR) for the quantification of viable E. coli targeting the uidA gene, encoding ß-glucuronidase, which is usually detected for phenotypic species identification. The short- and long-term effects of decontaminating chicken skin on the survival of both C. jejuni and an ESBL-producing E. coli were evaluated by CFU and v-qPCR. The results showed that freezing and storage in cool conditions are potentially underestimated by CFU but not by v-qPCR. The effect of treatment with peroxyacetic acid on survival was consistently detected by CFU and v-qPCR. v-qPCR analysis detected bacterial survival upon the application of lactic acid, which awaits further analysis. Interestingly, both bacteria showed similar kinetics of inactivation upon the application of reduction strategies, suggesting that E. coli might be a complementary hygiene indicator. We conclude that v-qPCR can improve food safety under the consideration of some limitations.
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Shiga toxin-producing Escherichia coli (STEC) are a cause of severe human illness and are frequently associated with haemolytic uraemic syndrome (HUS) in children. It remains difficult to identify virulence factors for STEC that absolutely predict the potential to cause human disease. In addition to the Shiga-toxin (stx genes), many additional factors have been reported, such as intimin (eae gene), which is clearly an aggravating factor for developing HUS. Current STEC detection methods classically rely on real-time PCR (qPCR) to detect the presence of the key virulence markers (stx and eae). Although qPCR gives an insight into the presence of these virulence markers, it is not appropriate for confirming their presence in the same strain. Therefore, isolation steps are necessary to confirm STEC viability and characterize STEC genomes. While STEC isolation is laborious and time-consuming, metagenomics has the potential to accelerate the STEC characterization process in an isolation-free manner. Recently, short-read sequencing metagenomics have been applied for this purpose, but assembly quality and contiguity suffer from the high proportion of mobile genetic elements occurring in STEC strains. To circumvent this problem, we used long-read sequencing metagenomics for identifying eae-positive STEC strains using raw cow's milk as a causative matrix for STEC food-borne outbreaks. By comparing enrichment conditions, optimizing library preparation for MinION sequencing and generating an easy-to-use STEC characterization pipeline, the direct identification of an eae-positive STEC strain was successful after enrichment of artificially contaminated raw cow's milk samples at a contamination level as low as 5 c.f.u. ml-1. Our newly developed method combines optimized enrichment conditions of STEC in raw milk in combination with a complete STEC analysis pipeline from long-read sequencing metagenomics data. This study shows the potential of the innovative methodology for characterizing STEC strains from complex matrices. Further developments will nonetheless be necessary for this method to be applied in STEC surveillance.
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Leite , Escherichia coli Shiga Toxigênica , Animais , Microbiologia de Alimentos , Leite/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Toxina Shiga/genética , Escherichia coli Shiga Toxigênica/isolamento & purificaçãoRESUMO
Shiga toxin-producing Escherichia coli (STEC) can cause severe human illness, which are frequently linked to the consumption of contaminated beef or dairy products. However, recent outbreaks associated with contaminated flour and undercooked dough in the United States and Canada, highlight the potential of plant based food as transmission routes for STEC. In Germany STEC has been isolated from flour, but no cases of illness have been linked to flour. In this study, we characterized 123 STEC strains isolated from flour and flour products collected between 2015 and 2019 across Germany. In addition to determination of serotype and Shiga toxin subtype, whole genome sequencing (WGS) was used for isolates collected in 2018 to determine phylogenetic relationships, sequence type (ST), and virulence-associated genes (VAGs). We found a high diversity of serotypes including those frequently associated with human illness and outbreaks, such as O157:H7 (stx2c/d, eae), O145:H28 (stx2a, eae), O146:H28 (stx2b), and O103:H2 (stx1a, eae). Serotypes O187:H28 (ST200, stx2g) and O154:H31 (ST1892, stx1d) were most prevalent, but are rarely linked to human cases. However, WGS analysis revealed that these strains, as well as, O156:H25 (ST300, stx1a) harbour high numbers of VAGs, including eae, nleB and est1a/sta1. Although STEC-contaminated flour products have yet not been epidemiologically linked to human clinical cases in Germany, this study revealed that flour can serve as a vector for STEC strains with a high pathogenic potential. Further investigation is needed to determine the sources of STEC contamination in flour and flour products particularly in regards to these rare serotypes.
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Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Farinha/microbiologia , Contaminação de Alimentos/análise , Toxina Shiga/genética , Animais , Canadá , Bovinos , Surtos de Doenças , Infecções por Escherichia coli/transmissão , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/genética , Microbiologia de Alimentos , Variação Genética/genética , Genoma Bacteriano/genética , Alemanha , Humanos , Filogenia , Virulência/genética , Fatores de Virulência/genética , Sequenciamento Completo do GenomaRESUMO
Shiga toxin-producing Escherichia coli (STEC) in sprouts have caused large scale outbreaks in the past involving severe illness. The combination of this very diverse pathogen and a food matrix with high numbers of background microbiota poses a particular challenge for detection and isolation. An acid treatment of the enrichment before plating on agar has been shown to improve the recovery of STEC from sprouts. After enrichment in buffered peptone water (BPW) at 37 °C we applied an acid treatment, followed by plating on tryptone bile x-glucuronide (TBX) agar (acid bile method). An inter-laboratory study was organized with 21 laboratories taking part to evaluate the performance parameters and applicability of the acid bile method. A sample set of six sprout samples was prepared consisting of two uninoculated samples and four spiked samples, each containing one of two STEC strains at one of two concentrations (low and high). Analyzing a set of six samples at the National Reference Laboratory (NRL E. coli), we determined the relative abundance of STEC without, after acid-, after bile- and after acid-bile treatment using real-time PCR. The participating laboratories successfully applied the acid bile method and were better able to detect (sensitivity 92.9% vs. 70.0%) and isolate (sensitivity 87.5% vs. 31.3%) STEC from positive samples using the acid bile method compared to non-acid methods. The relative limit of detection (RLOD) after isolation using the acid bile method (vs. non-acid method) was <1 for both STEC strains used, BfR-EC-14434 O133:H25 (0.146) and BfR-EC-16015 O26:H11 (0.073). A collection of STEC (n = 71) of diverse type and characteristics was assessed for their resistance towards the acid bile treatment selection. The majority (n = 65) of STEC strains could be recovered after acid treatment on TBX plates. However, a few strains (n = 6), among them clinical isolates were (partly) sensitive. These results suggest that an acid bile method is a rapid and reasonable approach to improve the recovery of STEC from sprouts when used in combination with methods targeting other selection markers.
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Ácidos e Sais Biliares/metabolismo , Microbiologia de Alimentos/métodos , Ácido Clorídrico/metabolismo , Escherichia coli Shiga Toxigênica/isolamento & purificação , Verduras/microbiologia , Ágar , Animais , Meios de Cultura/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Plântula/microbiologia , Escherichia coli Shiga Toxigênica/metabolismoRESUMO
The presence of bacteria carrying antimicrobial resistance (AMR) genes in wildlife is an indicator that resistant bacteria of human or livestock origin are widespread in the environment. In addition, it could represent an additional challenge for human health, since wild animals could act as efficient AMR reservoirs and epidemiological links between human, livestock and natural environments. The aim of this study was to investigate the occurrence and the antibiotic resistance patterns of several bacterial species in certain wild animals in Germany, including wild boars (Sus scrofa), roe deer (Capreolus capreolus) and wild ducks (family Anatidae, subfamily Anatinae) and geese (family Anatidae, subfamily Anserinae). In the framework of the German National Zoonoses Monitoring Program, samples from hunted wild boars, roe deer and wild ducks and geese were collected nationwide in 2016, 2017, and 2019, respectively. Fecal samples were tested for the presence of Salmonella spp. (in wild boars and wild ducks and geese), Campylobacter spp. (in roe deer and wild ducks and geese), Shiga toxin-producing Escherichia (E.) coli (STEC), commensal E. coli and extended-spectrum beta-lactamase- (ESBL) or ampicillinase class C (AmpC) beta-lactamase-producing E. coli (in wild boars, roe deer and wild ducks and geese). In addition, the presence of methicillin-resistant Staphylococcus aureus (MRSA) was investigated in nasal swabs from wild boars. Isolates obtained in the accredited regional state laboratories were submitted to the National Reference Laboratories (NRLs) for confirmation, characterization and phenotypic resistance testing using broth microdilution according to CLSI. AMR was assessed according to epidemiological cut-offs provided by EUCAST. Salmonella spp. were isolated from 13 of 552 (2.4%) tested wild boar fecal samples, but absent in all 101 samples from wild ducks and geese. Nine of the 11 isolates that were submitted to the NRL Salmonella were susceptible to all tested antimicrobial substances. Campylobacter spp. were isolated from four out of 504 (0.8%) roe deer fecal samples, but not from any of the samples from wild ducks and geese. Of the two isolates received in the NRL Campylobacter, neither showed resistance to any of the substances tested. From roe deer, 40.2% of the fecal samples (144 of 358) yielded STEC compared to 6.9% (37 of 536) from wild boars. In wild ducks and geese, no STEC isolates were found. Of 150 STEC isolates received in the NRL (24 from wild boars and 126 from roe deer), only one from each animal species showed resistance. Of the 219 isolates of commensal E. coli from wild boars tested for AMR, 210 were susceptible to all 14 tested substances (95.9%). In roe deer this proportion was even higher (263 of 269, 97.8%), whereas in wild ducks and geese this proportion was lower (41 of 49, 83.7%). Nevertheless, selective isolation of ESBL-/AmpC-producing E. coli yielded 6.5% (36 of 551) positive samples from wild boars, 2.3% (13 of 573) from roe deer and 9.8% (10 of 102) from wild ducks and geese. Among the 25 confirmed ESBL-/AmpC-producing isolates from wild boars, 14 (56.0%) showed resistance up to five classes of substances. This proportion was lower in roe deer (3 of 12, 25%) and higher in wild ducks and geese (7 of 10, 70%). None of the 577 nasal swabs from wild boars yielded MRSA. Results indicate that overall, the prevalence of resistant bacteria from certain wild animals in Germany is low, which may reflect not only the low level of exposure to antimicrobials but also the low level of resistant bacteria in the areas where these animals live and feed. However, despite this low prevalence, the patterns observed in bacteria from the wild animals included in this study are an indicator for specific resistance traits in the environment, including those to highest priority substances such as 3rd generation cephalosporins, fluoroquinolones and colistin. Therefore, also continuous monitoring of the occurrence of such bacteria in wildlife by selective isolation is advisable. Furthermore, the possible role of wildlife as reservoir and disperser of resistant bacteria would need to be assessed, as wild animals, and in particular wild ducks and geese could become spreaders of resistant bacteria given their capacity for long-range movements.
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Dynorphin neuropeptides are believed to act as endogenous anticonvulsants, though direct evidence for such a role in humans is sparse. We now report pronounced increases of prodynorphin mRNA expression in the dentate gyrus of patients with temporal lobe epilepsy in comparison to controls. We detected a conspicuously right skewed, bimodal distribution of mRNA levels among patients, suggestive of a dynamic up-regulation of prodynorphin expression in epilepsy. Highest transcript levels were seen postictally. Our data argue for an essential role of dynorphin in the termination of seizures.
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Giro Denteado/metabolismo , Encefalinas/genética , Encefalinas/metabolismo , Epilepsia do Lobo Temporal/patologia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Regulação para Cima/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Isótopos de Carbono , Criança , Pré-Escolar , Giro Denteado/fisiopatologia , Diprenorfina , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/fisiopatologia , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Mudanças Depois da Morte , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
BACKGROUND: Early diagnosis of CNS lymphoma (CNSL) is essential for successful therapy of this rapidly progressing brain tumor. However, in patients presenting with focal brain lesions, fast and reliable diagnosis of PCNSL remains a challenge. A proliferation-inducing ligand (APRIL) and B cell activating factor (BAFF) are important factors in the pathophysiology, diagnosis, and prognosis of systemic B cell malignancies. However, their utility as biomarkers for the diagnosis of CNSL and their effects on CNSL cells remain unclear. METHODS: In this prospective study, we analyzed the levels of APRIL and BAFF in the cerebrospinal fluid (CSF) of 116 patients with suspected focal brain lesions, including 53 CNSL patients. Additionally, we serially measured their levels during chemotherapy and relapse. Furthermore, we analyzed the effect of APRIL and BAFF on two B cell lymphoma cell lines using proliferation, viability, and chemotaxis assays. RESULTS: CSF levels of APRIL and BAFF reliably differentiated CNSL from other focal brain lesions (including primary and metastatic brain tumors, autoimmune-inflammatory lesions, and neuroinfectious lesions) with a specificity of 93.7% (APRIL, BAFF) and a sensitivity of 62.3% (APRIL) and 47.1% (BAFF). Serial CSF analysis of CNSL patients during chemotherapy and relapse demonstrates a close correlation of APRIL CSF levels and the course of this disease. In vitro, APRIL and BAFF showed anti-apoptotic effects during MTX treatment and mediated chemotaxis of malignant B cells. CONCLUSION: This study extends the spectrum of valuable diagnostic biomarkers in CNSL. In patients with focal brain lesions, measurement of APRIL in CSF could help accelerating the diagnosis of CNSL. Moreover, our results highlight an important role of APRIL and BAFF in the pathophysiology of CNSL.
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Fator Ativador de Células B/metabolismo , Biomarcadores Tumorais/sangue , Neoplasias do Sistema Nervoso Central/metabolismo , Linfoma/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Fator Ativador de Células B/genética , Quimiotaxia , Regulação Neoplásica da Expressão Gênica , Humanos , Estudos Prospectivos , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
Neurodegenerative parkinsonian syndromes comprise a number of disorders that are characterized by similar clinical features but are separated on the basis of different pathologies, i.e., aggregates of α-synuclein or tau protein. Due to the overlap of signs and symptoms a precise differentiation is often difficult, especially early in the disease course. Enormous efforts have been taken to develop tau-selective PET imaging agents, but strong off-target binding to monoamine oxidase B (MAO-B) has been observed across first generation ligands. Nonetheless, astrogliosis-related MAO-B elevation is a common histopathological known feature of all parkinsonian syndromes and might be itself an interesting imaging target. Therefore, this study aimed to investigate the performance of [18F]-THK5351, a combined MAO-B and tau tracer for differential diagnosis of parkinsonian syndromes. [18F]-THK5351 PET was performed in 34 patients: six with Parkinson's disease (PD), nine with multiple system atrophy with predominant parkinsonism (MSA-P), six with MSA with predominant cerebellar ataxia (MSA-C), and 13 with progressive supranuclear palsy (PSP) Richardson's syndrome. Volume-of-interest-based quantification of standardized-uptake-values was conducted in different parkinsonian syndrome-related target regions. PET results were subjected to multinomial logistic regression to create a prediction model discriminating among groups. Furthermore, we correlated tracer uptake with clinical findings. Elevated [18F]-THK5351 uptake in midbrain and diencephalon differentiated PSP patients from PD and MSA-C. MSA-C patients were distinguishable by high tracer uptake in the pons and cerebellar deep white matter when compared to PSP and PD patients, whereas MSA-P patients tended to show higher tracer uptake in the lentiform nucleus. A multinomial logistic regression classified 33/34 patients into the correct clinical diagnosis group. Tracer uptake in the pons, cerebellar deep white matter, and striatum was closely associated with the presence of cerebellar and parkinsonian symptoms of MSA patients. The current study demonstrates that combined MAO-B and tau binding of THK5351 facilitates differential diagnosis of parkinsonian syndromes. Furthermore, our data indicate a correlation of MSA phenotype with [18F]-THK5351 uptake in certain brain regions, illustrating their relevance for the emergence of clinical symptoms and underlining the potential of THK5351 PET as a biomarker that correlates with pathological changes as well as with disease stage.
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BACKGROUND: In the current study, nine foodborne "Locus of Enterocyte Effacement" (LEE)-negative Shiga toxin-producing Escherichia coli (STEC) strains were selected for whole genome sequencing and analysis for yet unknown genetic elements within the already known LEE integration sites selC, pheU and pheV. Foreign DNA ranging in size from 3.4 to 57 kbp was detected and further analyzed. Five STEC strains contained an insertion of foreign DNA adjacent to the selC tRNA gene and five and seven strains contained foreign DNA adjacent to the pheU and pheV tRNA genes, respectively. We characterized the foreign DNA insertion associated with selC (STEC O91:H21 strain 17584/1), pheU (STEC O8:H4 strain RF1a and O55:Hnt strain K30) and pheV (STEC O91:H21 strain 17584/1 and O113:H21 strain TS18/08) as examples. RESULTS: In total, 293 open reading frames partially encoding putative virulence factors such as TonB-dependent receptors, DNA helicases, a hemolysin activator protein precursor, antigen 43, anti-restriction protein KlcA, ShiA, and phosphoethanolamine transferases were detected. A virulence type IV toxin-antitoxin system was detected in three strains. Additionally, the ato system was found in one strain. In strain 17584/1 we were able to define a new genomic island which we designated GIselC 17584/1. The island contained integrases and mobile elements in addition to genes for increased fitness and those playing a putative role in pathogenicity. CONCLUSION: The data presented highlight the important role of the three tRNAs selC, pheU, and pheV for the genomic flexibility of E. coli.
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OBJECTIVES: To evaluate the presence of antibodies to conformation-intact myelin oligodendrocyte glycoprotein (MOG) in a subgroup of adult patients with clinically definite multiple sclerosis (MS) preselected for a specific clinical phenotype including severe spinal cord, optic nerve, and brainstem involvement. METHODS: Antibodies to MOG were investigated using a cell-based assay in 3 groups of patients: 104 preselected patients with MS (group 1), 55 age- and sex-matched, otherwise unselected patients with MS (group 2), and in 22 brain-biopsied patients with demyelinating diseases of the CNS (n = 19 with MS), 4 of whom classified as MS type II (group 3). Recognized epitopes were identified with mutated variants of MOG. RESULTS: Antibodies to MOG were found in about 5% (5/104) of preselected adult patients with MS. In contrast, in groups 2 and 3, none of the patients tested positive for MOG antibodies. Patients with MS with antibodies to MOG predominantly manifested with concomitant severe brainstem and spinal cord involvement and had a severe disease course with high relapse rates and failure to several disease-modifying therapies. Three of them had been treated with plasma exchange with a favorable response. All anti-MOG-positive patients with MS showed typical MS lesions on brain MRI. Longitudinal analysis up to 9 years revealed fluctuations and reappearance of anti-MOG reactivity. Epitope mapping indicated interindividual heterogeneity, yet intraindividual stability of the antibody response. CONCLUSIONS: Antibodies to MOG can be found in a distinct subgroup of adult MS with a specific clinical phenotype and may indicate disease heterogeneity.
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OBJECTIVE: To evaluate the frequency of the cryoporin/NLRP3 low-penetrance mutations V198M and Q703K in patients who reported at least 2 symptoms compatible with cryopyrin-associated periodic syndromes (CAPS) and to characterize the phenotype in mutation-positive patients. METHODS: The frequency of the V198M and Q703K mutations was investigated in a selected cohort of 108 patients from our neuroimmunology department. We describe the clinical, neurologic, immunologic, and neuroradiologic features of the mutation carriers. RESULTS: Seventeen patients (16%) tested positive for either of the 2 mutations (V198M: n = 2; Q703K: n = 15). Eleven patients (65%) had severe headache syndromes. Six of these 11 patients were diagnosed with migraine. Nine patients (53%) had a concomitant diagnosis of multiple sclerosis (MS). In 3 patients, we identified additional family members with the respective mutation as well as the diagnosis of MS. Severe recurrent cranial nerve (CN) affection was the hallmark feature in 7 of the 8 (88%) non-MS mutation carriers. Brain MRI showed abnormalities in all but 2 patients (88%) and detected CN inflammation in 4 patients. Interleukin-6 was elevated in the CSF of 2 patients in the non-MS cohort during acute CAPS episodes with severe CNS inflammation. 5 of 9 treated patients (56%) responded to anti-interleukin-1 therapy. CONCLUSION: CAPS constitute rare but treatable and commonly misdiagnosed autoinflammatory syndromes. Our data expand the spectrum of CAPS-associated neurologic manifestations. They also broaden our concept of autoimmunity and autoinflammation by linking CAPS and MS.
RESUMO
Survival of plasma cells is regulated by B-cell maturation antigen (BCMA), a membrane-bound receptor activated by its agonist ligands BAFF and APRIL. Here we report that γ-secretase directly cleaves BCMA, without prior truncation by another protease. This direct shedding is facilitated by the short length of BCMA's extracellular domain. In vitro, γ-secretase reduces BCMA-mediated NF-κB activation. In addition, γ-secretase releases soluble BCMA (sBCMA) that acts as a decoy neutralizing APRIL. In vivo, inhibition of γ-secretase enhances BCMA surface expression in plasma cells and increases their number in the bone marrow. Furthermore, in multiple sclerosis, sBCMA levels in spinal fluid are elevated and associated with intracerebral IgG production; in systemic lupus erythematosus, sBCMA levels in serum are elevated and correlate with disease activity. Together, shedding of BCMA by γ-secretase controls plasma cells in the bone marrow and yields a potential biomarker for B-cell involvement in human autoimmune diseases.