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The in vitro diagnostic medical devices regulation (IVDR) will take effect in May 2022. This regulation has a large impact on both the manufacturers of in vitro diagnostic medical devices (IVD) and clinical laboratories. For clinical laboratories, the IVDR poses restrictions on the use of laboratory developed tests (LDTs). To provide a uniform interpretation of the IVDR for colleagues in clinical practice, the IVDR Task Force was created by the scientific societies of laboratory specialties in the Netherlands. A guidance document with explanations and interpretations of relevant passages of the IVDR was drafted to help laboratories prepare for the impact of this new legislation. Feedback from interested parties and stakeholders was collected and used to further improve the document. Here we would like to present our approach to our European colleagues and inform them about the impact of the IVDR and, importantly we would like to present potentially useful approaches to fulfill the requirements of the IVDR for LDTs.

Next-generation sequencing and next-generation metabolic screening are, independently, increasingly applied in clinical diagnostics of inborn errors of metabolism (IEM). Integrated into a single bioinformatic method, these two &ndash, omics technologies can potentially further improve the diagnostic yield for IEM. Here, we present cross-omics: a method that uses untargeted metabolomics results of patient&rsquo, s dried blood spots (DBSs), indicated by Z-scores and mapped onto human metabolic pathways, to prioritize potentially affected genes. We demonstrate the optimization of three parameters: (1) maximum distance to the primary reaction of the affected protein, (2) an extension stringency threshold reflecting in how many reactions a metabolite can participate, to be able to extend the metabolite set associated with a certain gene, and (3) a biochemical stringency threshold reflecting paired Z-score thresholds for untargeted metabolomics results. Patients with known IEMs were included. We performed untargeted metabolomics on 168 DBSs of 97 patients with 46 different disease-causing genes, and we simulated their whole-exome sequencing results in silico. We showed that for accurate prioritization of disease-causing genes in IEM, it is essential to take into account not only the primary reaction of the affected protein but a larger network of potentially affected metabolites, multiple steps away from the primary reaction.

Aim Mutations in the RS-domain of RNA-binding motif protein 20 (RBM20) have recently been identified to segregate with aggressive forms of familial dilated cardiomyopathy (DCM). Loss of RBM20 in rats results in missplicing of the sarcomeric gene titin (TTN). The functional and physiological consequences of RBM20 mutations outside the mutational hotspot of RBM20 have not been explored to date. In this study, we investigated the pathomechanism of DCM caused by a novel RBM20 mutation in human cardiomyocytes. Methods and results We identified a family with DCM carrying a mutation (RBM20 E913K/+) in a glutamate-rich region of RBM20. Western blot analysis of endogenous RBM20 protein revealed strongly reduced protein levels in the heart of an RBM20 E913K/+ carrier. RNA deep-sequencing demonstrated massive inclusion of exons coding for the spring region of titin in the RBM20 E913K/+ carrier. Titin isoform analysis revealed a dramatic shift from the less compliant N2B towards the highly compliant N2BA isoforms in RBM20 E913K/+ heart. Moreover, an increased sarcomere resting-length was observed in single cardiomyocytes and isometric force measurements revealed an attenuated Frank-Starling mechanism (FSM), which was rescued by protein kinase A treatment. Conclusion A mutation outside the mutational hotspot of RBM20 results in haploinsufficiency of RBM20. This leads to disturbed alternative splicing of TTN, resulting in a dramatic shift to highly compliant titin isoforms and an impaired FSM. These effects may contribute to the early onset, and malignant course of DCM caused by RBM20 mutations. Altogether, our results demonstrate that heterozygous loss of RBM20 suffices to profoundly impair myocyte biomechanics by its disturbance of TTN splicing.

Background— Atrial fibrillation (AF) is associated with sudden cardiac death. We aimed to study whether AF is associated with ventricular fibrillation (VF), the most common cause of sudden cardiac death and whether this association is independent of confounders, ie, concomitant disease, use of antiarrhythmic or QT-prolonging drugs, and acute myocardial infarction. Methods and Results— We performed a community-based case-control study. Cases were patients with out-of-hospital cardiac arrest because of ECG-documented VF. Controls were age-/sex-matched non-VF subjects from the community. VF risk in AF patients was studied by means of (conditional) logistic regression, adjusting for all available confounders. We studied 1397 VF cases and 3474 controls. AF occurred in 215 cases (15.4%) and 90 controls (2.6%). AF was associated with a 3-fold increased risk of VF (adjusted odds ratio, 3.1 [2.1–4.5]). VF risk in AF cases was increased to the same extent across all age/sex groups and in AF cases who had no comorbidity (adjusted odds ratio 3.0 [1.6–5.5]) or used no confounding drugs (antiarrhythmics, 2.4 [1.4–4.3]; QT-prolonging drugs, 3.1 [1.8–5.4]). VF risk was similarly increased in AF cases with acute myocardial infarction–related VF (adjusted odds ratio 2.6 [1.4–4.8]), and those with non-acute myocardial infarction–related VF (adjusted odds ratio 4.3 [1.9–10.1]). Conclusions— AF is independently associated with a 3-fold increased risk of VF. Comorbidity, use of antiarrhythmic or QT-prolonging drugs, or acute myocardial infarction does not fully account for this increased risk.

Background Genetic variation is an important determinant of RNA transcription and splicing, which in turn contributes to variation in human traits, including cardiovascular diseases. Results Here we report the first in-depth survey of heart transcriptome variation using RNA-sequencing in 97 patients with dilated cardiomyopathy and 108 non-diseased controls. We reveal extensive differences of gene expression and splicing between dilated cardiomyopathy patients and controls, affecting known as well as novel dilated cardiomyopathy genes. Moreover, we show a widespread effect of genetic variation on the regulation of transcription, isoform usage, and allele-specific expression. Systematic annotation of genome-wide association SNPs identifies 60 functional candidate genes for heart phenotypes, representing 20% of all published heart genome-wide association loci. Focusing on the dilated cardiomyopathy phenotype we found that eQTL variants are also enriched for dilated cardiomyopathy genome-wide association signals in two independent cohorts. Conclusions RNA transcription, splicing, and allele-specific expression are each important determinants of the dilated cardiomyopathy phenotype and are controlled by genetic factors. Our results represent a powerful resource for the field of cardiovascular genetics. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1286-z) contains supplementary material, which is available to authorized users.

Hematopoietic stem cell transplantation with HLA-DPB1–mismatched donors leads to an increased risk of acute graft-versus-host disease (GVHD). Studies have indicated a prognostic value for classifying HLA-DPB1 mismatches based on T cell–epitope (TCE) groups. The aim of this study was to determine the contribution of indirect recognition of HLA-DP–derived epitopes, as determined with the Predicted Indirectly ReCognizable HLA Epitopes (PIRCHE) method. We therefore conducted a retrospective single-center analysis on 80 patients transplanted with a 10/10 matched unrelated donor that was HLA-DPB1 mismatched. HLA-DPB1 mismatches that were classified as GVH nonpermissive by the TCE algorithm correlated to higher numbers of HLA class I as well as HLA class II presented PIRCHE (PIRCHE-I and -II) compared with permissive or host-versus-graft nonpermissive mismatches. Patients with acute GVHD grades II to IV presented significantly higher numbers of PIRCHE-I compared with patients without acute GVHD (P < .05). Patients were divided into 2 groups based on the presence or absence of PIRCHE. Patients with PIRCHE-I or -II have an increased hazard of acute GVHD when compared with patients without PIRCHE-I or -II (hazard ratio [HR], 3.19; 95% confidence interval [CI], 1.10 to 9.19; P < .05; and HR, 4.07; 95% CI, .97 to 17.19; P = .06, respectively). Patients classified as having an HLA-DPB1 permissive mismatch by the TCE model had an increased risk of acute GVHD when comparing presence of PIRCHE-I with absence of PIRCHE-I (HR, 2.96; 95% CI, .84 to 10.39; P = .09). We therefore conclude that the data presented in this study describe an attractive and feasible possibility to better select permissible HLA-DPB1 mismatches by including both a direct and an indirect recognition model.

Inherited paternal HLA antigens from the semi-allogeneic fetus may trigger maternal immune responses during pregnancy, leading to the production of child-specific HLA antibodies. The prevalence of these HLA antibodies increases with the number of successful pregnancies. In the present study, we investigated the effect of a single prior miscarriage on HLA antibody formation during a subsequent successful pregnancy. Women with a successful pregnancy with one or more prior miscarriages (n = 229) and women with a successful pregnancy without a prior miscarriage (n = 58), and their children were HLA typed. HLA antibody analyses were performed in these women to identify whether HLA antibodies were formed against mismatched HLA class-I antigens of the last child. The percentage of immunogenic antigens was significantly lower after a single successful pregnancy that was preceded by a single miscarriage (n = 18 women) compared to a successful pregnancy that was preceded by a first successful pregnancy (n = 62 women). Thus, our data suggest that a previous miscarriage has a different impact on child-specific HLA antibody formation during a subsequent successful pregnancy than a previous successful pregnancy. The lower immunogenicity in these women cannot be explained by reduced numbers of immunogenic B-cell and T-cell epitopes. In conclusion, our observations indicate that increasing gravidity is not related to an increased prevalence of HLA antibodies in a single successful pregnancy that was preceded by a single prior miscarriage.

Gene expression is commonly used to study the activation of dendritic cells (DCs) to identify proteins that determine whether these cells induce an immunostimulatory or tolerogenic immune response. RNA expression, however, does not necessarily predict protein abundance and often requires large numbers of experiments for statistical significance. Proteomics provides a direct view on protein expression but is costly and time consuming. Here, we combined a comprehensive quantitative proteome and transcriptome analysis on a single batch of immature and cytokine cocktail matured human DCs and integrated resulting data sets at the pathway level. Although overall correlation between differential mRNA and protein expression was low, correlation between components of DC relevant pathways was significantly higher. Differentially expressed proteins and genes partly mapped to identical but also to different pathway components demonstrating that RNA and protein data not only supported but also complemented each other. We identified 5 dominant pathways, which confirmed the importance of cytokines, cell adhesion, and migration in DC maturation and also indicated a fundamental role for lipid metabolism. From these pathways we extracted novel maturation markers that might improve DC vaccine design. For several of the candidate markers we confirmed widespread significance examining DCs from multiple individuals, underscoring the validity of our approach. We conclude that integration of different but related data sets at the pathway level can significantly increase the predictive power of multi "omics" analyses.

Inhibitor of DNA binding (Id) proteins function as inhibitors of members of the basic helix-loop-helix family of transcription factors and have been demonstrated to play an important role in regulating lymphopoiesis. However, the role of these proteins in regulation of myelopoiesis is currently unclear. In this study, we have investigated the role of Id1 and Id2 in the regulation of granulopoiesis. Id1 expression was initially up-regulated during early granulopoiesis, which was then followed by a decrease in expression during final maturation. In contrast, Id2 expression was up-regulated in terminally differentiated granulocytes. In order to determine whether Id expression plays a critical role in regulating granulopoiesis, Id1 and Id2 were ectopically expressed in CD34+ cells by retroviral transduction. Our experiments demonstrate that constitutive expression of Id1 inhibits eosinophil development, whereas in contrast neutrophil differentiation was modestly enhanced. Constitutive Id2 expression accelerates final maturation of both eosinophils and neutrophils, whereas inhibition of Id2 expression blocks differentiation of both lineages. Transplantation of β2-microglobulin-/- nonobese diabetic severe combined immunodeficient (NOD/SCID) mice with CD34+ cells ectopically expressing Id1 resulted in enhanced neutrophil development, whereas ectopic expression of Id2 induced both eosinophil and neutrophil development. These data demonstrate that both Id1 and Id2 play a critical, although differential role in granulopoiesis.

In human neutrophils, both changes in intracellular Ca(2+) concentrations, [Ca(2+)]i, and activation of phosphatidylinositol-3 kinase (PtdIns3K) have been proposed to play a role in regulating cellular function induced by chemoattractants. In this study we have investigated the role of [Ca(2+)]i and its effector molecule calmodulin in human neutrophils. Increased [Ca(2+)]i alone was sufficient to induce phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2), p38 mitogen activated kinase (p38 MAPK), protein kinase B (PKB) and glycogen synthase kinase-3alpha (GSK-3alpha). Inhibition of calmodulin using a calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), did not effect N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced ERK, p38 MAPK or GSK-3alpha phosphorylation, but attenuated fMLP induced PKB phosphorylation. PCR analysis of human neutrophil cDNA demonstrated variable expression of members of the Ca(2+)/calmodulin-dependent kinase family. The roles of calmodulin and PtdIns3K in regulating neutrophil effector functions were further compared. Neutrophil migration was abrogated by inhibition of calmodulin, while no effect was observed when PtdIns3K was inhibited. In contrast, production of reactive oxygen species was sensitive to inhibition of both calmodulin and PtdIns3K. Finally, we demonstrated that chemoattractants are unable to modulate neutrophil survival, despite activation of PtdIns3K and elevation [Ca(2+)]i. Taken together, our data indicate critical roles for changes in [Ca(2+)]i and calmodulin activity in regulating neutrophil migration and respiratory burst and suggest that chemoattractant induced PKB phosphorylation may be mediated by a Ca(2+)/calmodulin sensitive pathway in human neutrophils.

During the first months of HIV infection, the virus typically evolves several immune escape mutations. These mutations are found in epitopes in viral proteins and reduce the impact of the CD8+ T cells specific for these epitopes. Recent data show that only a subset of the epitopes escapes, that most of these escapes evolve early, and that the rate of immune escape slows down considerably. To investigate why the evolution of immune escape slows down over the time of infection, we have extended a consensus mathematical model to allow several immune responses to control the virus together. In the extended model, most escapes also occur early, and the immune escape rate becomes small later, and typically only a minority of the epitopes escape. We show that escaping one of the many immune responses provides little advantage after viral setpoint has been approached because the total killing rate hardly depends on the breadth of the immune response. If the breadth of the immune response slowly wanes during disease progression, the model predicts an increase in the rate of immune escape at late stages of infection. Overall, the most striking prediction of the model is that HIV evolves a small number of immune escapes, in both relative and absolute terms, when the CTL immune response is broad.

Objectives This study aimed to evaluate HIV sequence evolution in whole genes and in CD8 T-cell epitope regions following immunotherapy and subsequent analytical treatment interruption (ATI). A second objective of this study was to analyze associations between vaccine-specific immune responses and epitope mutation rates. Design HIV-1-infected patients on combined antiretroviral therapy (cART) were subjected to immunotherapy by the administration of an autologous dendritic cell-based therapeutic vaccine expressing Tat, Rev, and Nef and subsequent ATI. Methods HIV-1 genes were amplified and sequenced from plasma RNA obtained before initiation of cART as well as during ATI. Control sequences for virus evolution in untreated HIV-1-infected individuals were obtained from the HIV Sequence Database (Los Alamos). CD8 T-cell epitope regions were defined based on literature data and prediction models. HIV-1-specific immune responses were evaluated to analyze their impact on sequence evolution. Results Viral sequence evolution in the tat, rev, and nef genes of vaccinated patients was similar to that of controls. The number of mutations observed inside and outside CD8 T-cell epitopes was comparable for vaccine-targeted and nontargeted proteins. We found no evidence for an impact of vaccine-induced or enhanced immune responses on the number of mutations inside or outside epitopes. Conclusion Therapeutic vaccination of HIV-1-infected patients with a dendritic cell-based vaccine targeting Tat, Rev, and Nef did not affect virus evolution at the whole gene level nor at the CD8 T-cell epitope level.

Genetic factors such as the MHC influence the immunocompetence of an individual. MHC genes are the most polymorphic genes in primates, which is often interpreted as an adaptation to establish good T cell responses to a wide range of (evolving) pathogens. Chimpanzee MHC (Patr) genes are less polymorphic than human MHC (HLA) genes, which is surprising because chimpanzee is the older species of the two and is therefore expected to display more variation. To quantify the effect of the reduced polymorphism, we compared the peptide binding repertoire of human and chimpanzee MHC molecules. Using a peptide-MHC binding predictor and proteomes of >900 mammalian viruses, we show that, at the population level, the total peptide binding repertoire of Patr-A molecules is ∼36% lower than that of their human counterparts, whereas the reduction of the peptide binding repertoire of the Patr-B locus is only 15%. In line with these results, different Patr-A molecules turn out to have largely overlapping peptide binding repertoires, whereas the Patr-B molecules are more distinct from each other. This difference is somewhat less apparent at the individual level, where we found that only 25% of the viruses are significantly better presented by “simulated” humans with heterozygous HLA-A and -B loci. Taken together, our results indicate that the Patr-B molecules recovered more after the selective sweep, whereas the Patr-A locus shows the most signs of the selective sweep with regard to its peptide binding repertoire.

Objective and design: HIV-1 is known to adapt to the human immune system, leading to accumulation of escape mutations during the course of infection within an individual. Cross-sectional studies have shown an inverse correlation between the prevalence of human leukocyte antigen (HLA) alleles in a population and the number of cytotoxic T lymphocyte (CTL) escape mutations in epitopes restricted by those HLA alleles. Recently, it was demonstrated that at a population level HIV-1 is adapting to the humoral immune response, which is reflected in an increase in resistance to neutralizing antibodies over time. Here we investigated whether adaptations to cellular immunity have also accumulated during the epidemic. Methods: We compared the number of CTL epitopes in HIV-1 strains isolated from individuals who seroconverted at the beginning of the HIV-1 epidemic and from individuals who seroconverted in recent calendar time. Results: The number of CTL epitopes in HIV-1 variants restricted by the most common HLA alleles in the population did not change significantly during the epidemic. In contrast, we found a significant loss of CTL epitopes restricted by HLA-B alleles associated with a low relative hazard of HIV-1 disease progression during the epidemic. Such a loss was not observed for CTL epitopes restricted by HLA-A alleles. Conclusion: Despite the large degree of HLA polymorphism, HIV-1 has accumulated adaptations to CTL responses within 20 years of the epidemic. The fact that such adaptations are driven by the HLA-B molecules that provide best protection against HIV-1 disease progression has important implications for our understanding of HIV evolution.

Analyses of biological processes would benefit from accurate definitions of protein complexes. High-throughput mass spectrometry data offer the possibility of systematically defining protein complexes; however, the predicted compositions vary substantially depending on the algorithm applied. We determine consensus compositions for 409 core protein complexes from Saccharomyces cerevisiae by merging previous predictions with a new approach. Various analyses indicate that the consensus is comprehensive and of high quality. For 85 out of 259 complexes not recorded in GO, literature search revealed strong support in the form of coprecipitation. New complexes were verified by an independent interaction assay and by gene expression profiling of strains with deleted subunits, often revealing which cellular processes are affected. The consensus complexes are available in various formats, including a merge with GO, resulting in 518 protein complex compositions. The utility is further demonstrated by comparison with binary interaction data to reveal interactions between core complexes.

Hematopoiesis is a highly regulated process resulting in the formation of all blood lineages. Aberrant regulation of phosphatidylinositol-3-kinase (PI3K) signaling has been observed in hematopoietic malignancies, suggesting that regulated PI3K signaling is critical for regulation of blood cell production. An ex vivo differentiation system was used to investigate the role of PI3K and its downstream effector, protein kinase B (PKB/c-akt) in myelopolesis. PI3K activity was essential for hematopoietic progenitor survival. High PKB activity was found to promote neutrophil and monocyte development, while, conversely, reduction of PKB activity was required to induce optimal eosinophil differentiation. In addition, transplantation of beta 2-microglobulin(-/-) NOD/SCID mice with CD34(+) cells ectopically expressing constitutively active PKB resulted in enhanced neutrophil and monocyte development, whereas ectopic expression of dominant-negative PKB induced eosinophil development in vivo. Inhibitory phosphorylation of C/EBP alpha on Thr222/226 was abrogated upon PKB activation in hematopoietic progenitors. Ectopic expression of a nonphosphorylatable C/EBP alpha mutant inhibited eosinophil differentiation ex vivo, whereas neurtrophil development was induced, demonstrating the importance of PKB-mediated C/EBPa. phosphorylation in regulation of granulopoiesis. These results identify an important novel role for PKB in regulation of cell fate choices during hematopoietic lineage commitment.

Activation of granulocyte effector functions, such as induction of the respiratory burst and migration, are regulated by a variety of relatively ill-defined signaling pathways. Recently, we identified a novel Ca2+/calmodulin-dependent kinase I-like kinase, CKLiK, which exhibits restricted mRNA expression to human granulocytes. Using a novel antibody generated against the C-terminus of CKLiK, CKLiK was detected in CD34+-derived neutrophils and eosinophils, as well as in mature peripheral blood granulocytes. Activation of human granulocytes by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF), but not the phorbol ester PMA (phorbol 12-myristate-13-acetate), resulted in induction of CKLiK activity, in parallel with a rise of intracellular Ca2+ [Ca2+]i. To study the functionality of CKLiK in human granulocytes, a cell-permeable CKLiK peptide inhibitor (CKLiK297-321) was generated which was able to inhibit kinase activity in a dose-dependent manner. The effect of this peptide was studied on specific granulocyte effector functions such as phagocytosis, respiratory burst, migration, and adhesion. Phagocytosis of Aspergillus fumigatus particles was reduced in the presence of CKLiK297-321 and fMLP-induced reactive oxygen species (ROS) production was potently inhibited by CKLiK297-321 in a dose-dependent manner. Furthermore, fMLP-induced neutrophil migration on albumin-coated surfaces was perturbed, as well as β2-integrin-mediated adhesion. These findings suggest a critical role for CKLiK in modulating chemoattractant-induced functional responses in human granulocytes.