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BACKGROUND: The search for intrinsic factors, which account for a protein's capability to act as an allergen, is ongoing. Fold stability has been identified as a molecular feature that affects processing and presentation, thereby influencing an antigen's immunologic properties. OBJECTIVE: We assessed how changes in fold stability modulate the immunogenicity and sensitization capacity of the major birch pollen allergen Bet v 1. METHODS: By exploiting an exhaustive virtual mutation screening, we generated mutants of the prototype allergen Bet v 1 with enhanced thermal and chemical stability and rigidity. Structural changes were analyzed by means of x-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulations. Stability was monitored by using differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy. Endolysosomal degradation was simulated in vitro by using the microsomal fraction of JAWS II cells, followed by liquid chromatography coupled to mass spectrometry. Immunologic properties were characterized in vitro by using a human T-cell line specific for the immunodominant epitope of Bet v 1 and in vivo in an adjuvant-free BALB/c mouse model. RESULTS: Fold stabilization of Bet v 1 was pH dependent and resulted in resistance to endosomal degradation at a pH of 5 or greater, affecting presentation of the immunodominant T-cell epitope in vitro. These properties translated in vivo into a strong allergy-promoting TH2-type immune response. Efficient TH2 cell activation required both an increased stability at the pH of the early endosome and efficient degradation at lower pH in the late endosomal/lysosomal compartment. CONCLUSIONS: Our data indicate that differential pH-dependent fold stability along endosomal maturation is an essential protein-inherent determinant of allergenicity.
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Alérgenos/química , Antígenos de Plantas/química , Alérgenos/genética , Alérgenos/imunologia , Animais , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Endossomos , Feminino , Concentração de Íons de Hidrogênio , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Camundongos Endogâmicos BALB C , Mutação , Pólen/imunologia , Dobramento de Proteína , Estabilidade ProteicaRESUMO
BACKGROUND: The Rhesus (Rh) complex consists of a core comprising the Rh proteins (RhD/RhCE) and the Rh-associated glycoprotein (RhAG) with accessory chains (GPB, LW, CD47). Molecular defects of the RHAG gene may cause a regulator Rhnull phenotype without Rh antigen expression or a Rhmod phenotype with decreased Rh antigen expression. STUDY DESIGN AND METHODS: Blood samples of a donor with strongly diminished Rh antigens and five family members were analyzed by serological phenotyping, flow cytometry, molecular testing, and gene expression analysis of Rh complex candidate genes. RESULTS: RHAG sequencing identified a missense mutation, c.241G>C (p.Gly81Arg) and a splice site mutation, c.640 + 3del14, among the cohort. Compound heterozygosity of these novel alleles identified in the propositus and two siblings gave rise to a strongly diminished expression of RhAG, Rh, and CD47 antigens on the RBC surface. CONCLUSION: The Rhmod phenotype was caused by a novel RHAG splice site mutation in association with a non-functional allele. The primary depression of RhAG is most likely due to posttranslational events that affect the interaction and processing of the RhAG glycoprotein and gave rise to a secondary depression of RhD, RhCE, and CD47, the major members of the Rh complex.
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Proteínas Sanguíneas/genética , Glicoproteínas de Membrana/genética , Sistema do Grupo Sanguíneo Rh-Hr/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Proteínas Sanguíneas/metabolismo , Membrana Eritrocítica/metabolismo , Heterozigoto , Humanos , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Sistema do Grupo Sanguíneo Rh-Hr/metabolismo , IrmãosRESUMO
BACKGROUND: Today's modern research of B and T cell antigen receptors (the immunoglobulins (IG) or antibodies and T cell receptors (TR)) forms the basis for detailed analyses of the human adaptive immune system. For instance, insights in the state of the adaptive immune system provide information that is essentially important in monitoring transplantation processes and the regulation of immune suppressiva. In this context, algorithms and tools are necessary for analyzing the IG and TR diversity on nucleotide as well as on amino acid sequence level, identifying highly proliferated clonotypes, determining the diversity of the cell repertoire found in a sample, comparing different states of the human immune system, and visualizing all relevant information. RESULTS: We here present IMEX, a software framework for the detailed characterization and visualization of the state of human IG and TR repertoires. IMEX offers a broad range of algorithms for statistical analysis of IG and TR data, CDR and V-(D)-J analysis, diversity analysis by calculating the distribution of IG and TR, calculating primer efficiency, and comparing multiple data sets. We use a mathematical model that is able to describe the number of unique clonotypes in a sample taking into account the true number of unique sequences and read errors; we heuristically optimize the parameters of this model. IMEX uses IMGT/HighV-QUEST analysis outputs and includes methods for splitting and merging to enable the submission to this portal and to combine the outputs results, respectively. All calculation results can be visualized and exported. CONCLUSION: IMEX is an user-friendly and flexible framework for performing clonality experiments based on CDR and V-(D)-J rearranged regions, diversity analysis, primer efficiency, and various different visualization experiments. Using IMEX, various immunological reactions and alterations can be investigated in detail. IMEX is freely available for Windows and Unix platforms at http://bioinformatics.fh-hagenberg.at/immunexplorer/.
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Algoritmos , DNA/análise , Rearranjo Gênico , Imunoglobulinas/genética , Receptores de Antígenos de Linfócitos T/genética , Software , DNA/sangue , Primers do DNA/genética , Humanos , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Rapid and reliable Rhesus D typing is crucial for blood donation centers. In instances of massive blood transfusion or reduced antigen expression, DNA-based phenotype prediction becomes mandatory. Our molecular RHD typing approach involves an initial real-time PCR for the most common aberrant RHD types in our region, RHD*01W.1 (weak D type 1), RHD*01W.2 (weak D type 2), RHD*01W.3 (weak D type 3), and RHD*07.01 (DVII). For comprehensive coverage, Sanger sequencing of RHD coding regions is performed in the case of PCR target-negative results. We evaluated the specificity and accuracy of these methods using the recently launched LightCycler® PRO real-time platform. All findings demonstrated remarkable accuracy. Notably, the LightCycler® PRO instrument offers a distinct advantage in data interpretation and integration via the HL7 interface. This study underlines the importance of including advanced molecular techniques in blood typing protocols, especially in scenarios where conventional serological methods may be insufficient.
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BACKGROUND: Next-generation sequencing (NGS) has changed genomics significantly. More and more applications strive for sequencing with different platforms. Now, in 2012, after a decade of development and evolution, NGS has been accepted for a variety of research fields. Determination of sequencing errors is essential in order to follow next-generation sequencing beyond research use only. This study describes the overall 454 system performance of using multiple GS Junior runs with an in-house established and validated diagnostic assay for human leukocyte antigen (HLA) exon sequencing. Based on this data, we extracted, evaluated and characterized errors and variants of 60 HLA loci per run with respect to their adjacencies. RESULTS: We determined an overall error rate of 0.18% in a total of 118,484,408 bases. 31.3% of all reads analyzed (n=349,503) contain one or more errors. The largest group are deletions that account for 50% of the errors. Incorrect bases are not distributed equally along sequences and tend to be more frequent at sequence ends. Certain sequence positions in the middle or at the beginning of the read accumulate errors. Typically, the corresponding quality score at the actual error position is lower than the adjacent scores. CONCLUSIONS: Here we present the first error assessment in a human next-generation sequencing diagnostics assay in an amplicon sequencing approach. Improvements of sequence quality and error rate that have been made over the years are evident and it is shown that both have now reached a level where diagnostic applications become feasible. Our presented data are better than previously published error rates and we can confirm and quantify the often described relation of homopolymers and errors. Nevertheless, a certain depth of coverage is needed, in particular with challenging areas of the sequencing target. Furthermore, the usage of error correcting tools is not essential but might contribute towards the capacity and efficiency of a sequencing run.
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Éxons , Antígenos HLA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade , Análise de Sequência de DNA/métodos , HumanosRESUMO
BACKGROUND: Human leukocyte antigen matching at allelic resolution is proven clinically significant in hematopoietic stem cell transplantation, lowering the risk of graft-versus-host disease and mortality. However, due to the ever growing HLA allele database, tissue typing laboratories face substantial challenges. In light of the complexity and the high degree of allelic diversity, it has become increasingly difficult to define the classical transplantation antigens at high-resolution by using well-tried methods. Thus, next-generation sequencing is entering into diagnostic laboratories at the perfect time and serving as a promising tool to overcome intrinsic HLA typing problems. Therefore, we have developed and validated a scalable automated HLA class I and class II typing approach suitable for diagnostic use. RESULTS: A validation panel of 173 clinical and proficiency testing samples was analysed, demonstrating 100% concordance to the reference method. From a total of 1,273 loci we were able to generate 1,241 (97.3%) initial successful typings. The mean ambiguity reduction for the analysed loci was 93.5%. Allele assignment including intronic sequences showed an improved resolution (99.2%) of non-expressed HLA alleles. CONCLUSION: We provide a powerful HLA typing protocol offering a short turnaround time of only two days, a fully integrated workflow and most importantly a high degree of typing reliability. The presented automated assay is flexible and can be scaled by specific primer compilations and the use of different 454 sequencing systems. The workflow was successfully validated according to the policies of the European Federation for Immunogenetics. Next-generation sequencing seems to become one of the new methods in the field of Histocompatibility.
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Antígenos HLA/análise , Teste de Histocompatibilidade/métodos , Automação Laboratorial/métodos , Humanos , Tipagem de Sequências Multilocus/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reprodutibilidade dos TestesAssuntos
Polimorfismo de Nucleotídeo Único , Sistema do Grupo Sanguíneo Rh-Hr/genética , Alelos , Substituição de Aminoácidos , Expressão Gênica , Humanos , Fenótipo , Domínios Proteicos , Sistema do Grupo Sanguíneo Rh-Hr/biossíntese , Sistema do Grupo Sanguíneo Rh-Hr/química , Análise de Sequência de DNAAssuntos
Alérgenos/imunologia , Phleum/imunologia , Proteínas de Plantas/imunologia , Poaceae/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Adulto , Citocinas/imunologia , Fazendeiros , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linfócitos T/imunologia , População UrbanaRESUMO
BACKGROUND: Knowledge of allergen-specific T cell epitopes is a prerequisite not only for therapeutic approaches but also for elucidating immunological mechanisms of type I allergy. Ex vivo detection of allergen-specific T cells using class II tetramer technology has become an important tool for investigating immune responses in atopic and healthy individuals. METHODS: Using (3)H-thymidine incorporation assays, T cell epitopes specific for the major timothy grass pollen allergen Phl p 5.0101 were mapped in 11 allergic donors and two different mouse strains. Different protocols for expansion/restimulation of T cells from the blood of allergic donors and detection of allergen-specific T cells by Class II Ultimer staining were evaluated. RESULTS: We identified several new Phl p 5.0101 class II T cell epitopes in allergic patients and confirmed previously published ones. Additionally, we discovered the major T cell epitopes in BALB/c and C57BL/6 mice. Using a novel Class II Ultimer, we detected epitope-specific T cells expanded from the blood of an allergic donor. CONCLUSIONS: Epitope mapping of Phl p 5.0101 revealed an immunodominant epitope in BALB/c and C57BL/6 mice and an immunodominant region in humans (amino acids 259-282), which was recognized by 8 out of 11 allergic donors. Detection of Phl p 5-specific T cells was demonstrated using a Class II Ultimer specific for epitope 196-210. Successful detection of ultimer-positive T cells was strongly dependent on a resting phase after in vitro expansion.
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Epitopos de Linfócito T/imunologia , Proteínas de Plantas/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Mapeamento de Epitopos , Feminino , Humanos , Epitopos Imunodominantes/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Coloração e RotulagemRESUMO
The human genome project triggered the introduction of next generation sequencing (NGS) systems. Although originally developed for total genome sequencing, metagenomics and plant genetics, the ultra-deep sequencing feature of NGS was utilized for diagnostic purposes in HIV resistance and tropism as well in detecting new mutations and tumor clones in oncology. Recent publications exploited the feature of clonal sequencing for immunogenetics to dissolve the growing number of ambiguities. This concept is quite reliable if all exons of interest are tested and the amplification region includes flanking introns. Challenging questions on quality control, cost effectiveness, workflow, and management of enormous loads of data remain if NGS is considered as routine method in the immunogenetics laboratory. If solved, NGS has big potential to have a major impact on immunogenetics by way of providing ambiguity-free HLA-typing results faster, but will also have a great influence on how immunogenetics testing and workflows are organized.
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Background: There is limited data evaluating the prescription practices for antithrombotic therapy in patients with atrial fibrillation (AF) following elective percutaneous coronary intervention (PCI). Objective: This single-center, single-operator, retrospective cohort study aimed to evaluate trends of antithrombotic treatment strategies in patients with AF undergoing elective PCI. Methods: Patients with AF who electively underwent PCI performed by a single interventionalist between April 2013 and May 2018 were identified. The primary outcome was the antithrombotic therapy at discharge assessed by chart review: triple (TAT, triple antithrombotic therapy) or dual (DAT, dual antithrombotic therapy) antithrombotic therapy and vitamin K antagonist (VKA) or non-vitamin K antagonist oral anticoagulant (NOAC), respectively. Results: Of 6,135 screened patients, 259 met the inclusion criteria. Among these, 133 (51%) patients received NOAC- and 126 (49%) VKA-therapy. Compared with patients on NOAC therapy, patients treated with VKA had higher bleeding risk (mean HAS-BLED-Score; 2.3 vs. 2.0; p = 0.02) and more co-morbidities (estimated glomerular filtration rate <30 ml/min, 11 vs. 4%; p = 0.04; diabetes mellitus, 33 vs. 20%; p = 0.03; history of previous PCI, 37 vs. 21%; p < 0.01). TAT was prescribed more frequently if the prescription included VKA compared with NOAC (61 vs. 41%; p < 0.01). Prescription of TAT and VKA decreased throughout the observed period (2013: 100% vs. 2018: 6%; p < 0.01 and 2013: 91% vs. 2018: 28%; p < 0.01). Conclusion: These observational data from a single center registry show a decrease of TAT- and VKA- prescription in favor of DAT with NOAC. Whether these observations are consistent with national or global trends should to be evaluated in further studies.
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Alelos , Substituição de Aminoácidos , Sistema do Grupo Sanguíneo de Kell/genética , Glicoproteínas de Membrana/genética , Metaloendopeptidases/genética , Mutação de Sentido Incorreto , Mutação Puntual , Membrana Eritrocítica/metabolismo , Humanos , Glicoproteínas de Membrana/química , Metaloendopeptidases/química , Dados de Sequência Molecular , Transporte Proteico , Estudos RetrospectivosRESUMO
BACKGROUND: Aberrant RHD alleles leading to a reduced expression of D antigen on the red blood cell (RBC) surface may be mistyped as D- by serology. To quantify the occurrence of weak D, DEL, and D+/- chimera among apparent D- first-time blood donors, polymerase chain reaction (PCR) screening was implemented as a routine service. STUDY DESIGN AND METHODS: A total of 23,330 pretyped D- samples were tested for RHD markers in Exons 4, 7, and 10 in pools of 20 by PCR. Samples with positive results in PCR were reevaluated by exon-specific PCRs, DNA sequencing, and serologic methods. RESULTS: Among 94 PCR-positive samples, 74 exhibited a weak D or DEL phenotype, dubbed weak D type 1, weak D type 2, weak D type 5, weak D type 32, weak D type 4.3, RHD(M295I), RHD(del147), and RHD(1227G>A). The most prevalent alleles were weak D type 4.3 (n = 31) and RHD(IVS3+1G>A) (n = 24). CONCLUSIONS: As a clinical consequence, 74 blood donor samples carrying weak D and DEL phenotypes with the potential of causing secondary immunizations in recipients were reclassified as D+. Those samples were reliably amplified by RHD Exon 7 PCR; therefore, its usage in the Upper Austrian population is recommended. The association of the weak D type 4.3 samples with a ce leads to the policy that all apparently D- donors should be tested with genotyping methods; otherwise, potentially immunogenic RHD alleles may be overseen.
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Doadores de Sangue , Sistema do Grupo Sanguíneo Rh-Hr/genética , Imunoglobulina rho(D)/sangue , Alelos , Áustria , Tipagem e Reações Cruzadas Sanguíneas/métodos , Estudos de Coortes , Análise Mutacional de DNA , Frequência do Gene , Humanos , Polimorfismo Genético , Valor Preditivo dos Testes , Sistema do Grupo Sanguíneo Rh-Hr/análise , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Imunoglobulina rho(D)/análise , Testes SorológicosRESUMO
DNA sequencing is the gold standard for high-resolution genotyping of the highly polymorphic human leukocyte antigen (HLA) loci. In the case of hematopoietic stem cell transplantation, four-digit typing of HLA class I and II genes is indicated. We developed a group-specific, real-time polymerase chain reaction (PCR) strategy for amplification of DRB1 applying TaqMan chemistry on different real-time machines. For evaluation of genotyping accuracy and ambiguity resolution, 115 well-characterized samples were adapted. Separate analysis of each allele was possible in 100 samples (87%). Of the samples, 13% (n = 15) showed amplification in one of the eight group-specific PCR mixes: one sample according to the group DRB1*15, 16, along with 14 samples according to the group DRB1*03, *11, *13, *14. Further testing of the 14 (12.2%) samples associated with group DRB1*03, *11, *13, *14 was performed with specific intron primers. In conclusion this typing scheme generates results within 1 day, thereby making sequencing for different requirements attractive.
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Antígenos HLA-DR/genética , Reação em Cadeia da Polimerase/métodos , Alelos , Sequência de Bases , Cadeias HLA-DRB1 , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The Human Major Histocompatibility Complex (MHC) is a highly polymorphic region full of immunoregulatory genes. The MHC codes for the human leukocyte antigens (HLA), proteins that present on the cellular surface and that are involved in self-non-self recognition. For matching donors and recipients for organ and stem-cell transplants it is important to know an individual's HLA haplotype determinable in this region. Now, as next-generation sequencing (NGS) platforms mature and become more and more accepted as a standard method, NGS applications have spread from research laboratories to the clinic, where they provide valid genetic insights. Here, we describe a cost-effective microarray-based sequence capture, enrichment, and NGS sequencing approach to characterize MHC haplotypes. Using this approach, ~4 MB of MHC sequence for four DNA samples (donor, recipient and the parents of the recipient) were sequenced in parallel in one NGS instrument run. We complemented this approach using microarray-based genome-wide SNP analysis. Taken together, the use of recently developed tools and protocols for sequence capture and massively parallel sequencing allows for detailed MHC analysis and donor-recipient matching.
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Complexo Principal de Histocompatibilidade/genética , Genótipo , Antígenos HLA/genética , Haplótipos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/métodosRESUMO
Rabbit anti-thymocyte globulins (ATGs) are widely used for the prevention of acute and chronic graft versus host disease (aGVHD, cGVHD) following allogeneic hematopoietic stem cell transplantation (HSCT). However, most prospective and retrospective studies did not reveal an overall survival (OS) benefit associated with ATG. Homozygosity for human leukocyte antigen (HLA)-C group 1 killer-cell immunoglobulin-like receptor ligands (KIR-L), i.e. C1/1 KIR-L status, was recently shown to be a risk factor for severe aGVHD. Congruously, we have previously reported favorable outcomes in C1/1 recipients after ATG-based transplants in a monocentric analysis. Here, within an extended cohort, we test the hypothesis that incorporation of ATG for GVHD prophylaxis may improve survival particularly in HSCT recipients with at least one C1 KIR-ligand. Retrospectively, 775 consecutive allogeneic (excluding haploidentical) HSCTs were analyzed, including peripheral blood and bone marrow grafts for adults with hematological diseases at two Austrian HSCT centers. ATG-Fresenius/Grafalon, Thymoglobuline, and alemtuzumab were applied in 256, 87, and 7 transplants, respectively (subsequently summarized as "ATG"), while 425 HSCT were performed without ATG. Median follow-up of surviving patients is 48 months. Adjusted for age, disease-risk, HLA-match, donor and graft type, sex match, cytomegalovirus serostatus, conditioning intensity, and type of post-grafting GVHD prophylaxis, Cox regression analysis of the entire cohort (n = 775) revealed a significant association of ATG with decreased non-relapse mortality (NRM) (risk ratio (RR), 0.57; p = 0.001), and overall mortality (RR, 0.71; p = 0.014). Upon stratification for HLA-C KIR-L, the greatest benefit for ATG emerged in C1/1 recipients (n = 291), by reduction of non-relapse (RR, 0.34; p = 0.0002) and overall mortality (RR, 0.50; p = 0.003). Less pronounced, ATG decreased NRM (RR, 0.60; p = 0.036) in HLA-C group 1/2 recipients (n = 364), without significantly influencing overall mortality (RR, 0.70; p = 0.065). After exclusion of higher-dose ATG-based transplants, serotherapy significantly improved both NRM (RR, 0.54; p = 0.019; n = 322) and overall mortality (RR, 0.60; p = 0.018) in C1/2 recipients as well. In both, C1/1 (RR, 1.70; p = 0.10) and particularly in C1/2 recipients (RR, 0.94; p = 0.81), there was no statistically significant impact of ATG on relapse incidence. By contrast, in C2/2 recipients (n = 121), ATG neither reduced NRM (RR, 1.10; p = 0.82) nor overall mortality (RR, 1.50; p = 0.17), but increased the risk for relapse (RR, 4.38; p = 0.02). These retrospective findings suggest ATG may provide a survival benefit in recipients with at least one C1 group KIR-L, by reducing NRM without significantly increasing the relapse risk.
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Chondrogenic differentiation of adipose-derived stem cells (ASC) is challenging but highly promising for cartilage repair. Large donor variability of chondrogenic differentiation potential raises the risk for transplantation of cells with reduced efficacy and a low chondrogenic potential. Therefore, quick potency assays are required to control the potency of the isolated cells before cell transplantation. Current in vitro methods to analyze the differentiation capacity are time-consuming, and thus, a novel enhancer and tissue-specific promoter combination was used for the detection of chondrogenic differentiation of ASC in a novel quick potency bioassay. Human primary ASC were cotransfected with the Metridia luciferase-based collagen type II reporter gene pCMVE_ACDCII-MetLuc together with a Renilla control plasmid and analyzed for their chondrogenic potential. On day 3 after chondrogenic induction, the luciferase activity was induced in all tested donors under three-dimensional culture conditions and, in a second approach, also under two-dimensional (2D) culture conditions. With our newly developed quick potency bioassay, we can determine chondrogenic potential already after 3 days of chondrogenic induction and under 2D culture conditions. This will enhance the efficiency of testing cell functionality, which should allow in the future to predict the suitability of cells derived from individual patients for cell therapies in a very short time and at low costs.
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Tecido Adiposo/citologia , Bioensaio/métodos , Diferenciação Celular , Condrócitos/citologia , Condrogênese/fisiologia , Luciferases/metabolismo , Tecido Adiposo/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Humanos , Luciferases/genética , Reação em Cadeia da Polimerase/métodos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Engenharia Tecidual/métodosRESUMO
BACKGROUND: In recent years various studies showed, that hepatitis E virus (HEV) is a growing public health problem in many developed countries. Therefore, HEV infections might bear a transmission risk by blood transfusions. The clinical relevance still requires further investigations. The aim of this study was to provide an overview of acute HEV infections in Upper Austrian blood donors as well as a risk estimation of this transfusion-related infection. METHODS AND FINDINGS: A total of 58,915 blood donors were tested for HEV RNA using a commercial HEV RT-PCR Kit. 7 of these donors (0.01%) were PCR-positive with normal laboratory parameters in absence of clinical signs of hepatitis. Viral load determined by quantitative real-time PCR showed a HEV nucleic acid concentration of 2,217 293,635 IU/ml. At follow-up testing (2-11 weeks after donation) all blood donors had negative HEV RNA results. Additionally, genotyping was performed by amplification and sequencing of the ORF1 or ORF2 region of the HEV genome. All HEV RNA positive donor samples revealed a genotype 3 isolate. For the antibody screening, anti-HEV IgM and IgG were detected by ELISA. Follow up serological testing revealed that no donor was seropositive for HEV IgM or IgG antibodies at time of donation. Moreover, we verified the prevalence of anti-HEV IgG in 1,203 of the HEV RNA negative tested blood donors. Overall 13.55% showed positive results for anti-HEV IgG. CONCLUSIONS: In the presented study, we investigated HEV infections in blood donations of Upper Austria over 1 year. We concluded that 1 out of 8,416 blood donations is HEV RNA positive. Seroprevalence of anti HEV IgG results in an age-related increase of 13.55%. Therefore, based on this data, we recommend HEV-PCR screening to prevent transmission of hepatitis E virus by transfusion.