Bayes Lines Tool (BLT): a SQL-script for analyzing diagnostic test results with an application to SARS-CoV-2-testing.

The performance of diagnostic tests crucially depends on the disease prevalence, test sensitivity, and test specificity. However, these quantities are often not well known when tests are performed outside defined routine lab procedures which make the rating of the test results somewhat problematic. A current example is the mass testing taking place within the context of the world-wide SARS-CoV-2 crisis. Here, for the first time in history, laboratory test results have a dramatic impact on political decisions. Therefore, transparent, comprehensible, and reliable data is mandatory. It is in the nature of wet lab tests that their quality and outcome are influenced by multiple factors reducing their performance by handling procedures, underlying test protocols, and analytical reagents. These limitations in sensitivity and specificity have to be taken into account when calculating the real test results. As a resolution method, we have developed a Bayesian calculator, the Bayes Lines Tool (BLT), for analyzing disease prevalence, test sensitivity, test specificity, and, therefore, true positive, false positive, true negative, and false negative numbers from official test outcome reports. The calculator performs a simple SQL (Structured Query Language) query and can easily be implemented on any system supporting SQL. We provide an example of influenza test results from California, USA, as well as two examples of SARS-CoV-2 test results from official government reports from The Netherlands and Germany-Bavaria, to illustrate the possible parameter space of prevalence, sensitivity, and specificity consistent with the observed data. Finally, we discuss this tool's multiple applications, including its putative importance for informing policy decisions.

Natural Knockouts: Natural Selection Knocked Out.

In functional genomics studies, research is dedicated to unveiling the function of genes using gene-knockouts, model organisms in which a gene is artificially inactivated. The idea is that, by knocking out the gene, the provoked phenotype would inform us about the function of the gene. Still, the function of many genes cannot be elucidated, because disruption of conserved sequences, including protein-coding genes, often does not directly affect the phenotype. Since the phenomenon was first observed in the early nineties of the last century, these so-called 'no-phenotype knockouts' have met with great skepticism and resistance by died-in-the-wool selectionists. Still, functional genomics of the late 20th and early 21st centuries has taught us two important lessons. First, two or more unrelated genes can often substitute for each other; and second, some genes are only present in the genome in a silent state. In the laboratory, the disruption of such genes does not negatively influence reproductive success, and does not show measurable fitness effects of the species. The genes are redundant. Genetic redundancy, one of the big surprises of modern biology, can thus be defined as the condition in which the inactivation of a gene is selectively neutral. The no-phenotype knockout is not just a freak of the laboratory. Genetic variants known as homozygous loss-of-function (HLOF) variants are of considerable scientific and clinical interest, as they represent experiments of nature qualifying as "natural knockouts". Such natural knockouts challenge the conventional NeoDarwinian appraisal that genetic information is the result of natural selection acting on random genetic variation.

Hypoxia exerts dualistic effects on inflammatory and proliferative responses of healthy and asthmatic primary human bronchial smooth muscle cells.

BACKGROUND: For oxygen supply, airway wall cells depend on diffusion though the basement membrane, as well as on delivery by micro-vessels. In the asthmatic lung, local hypoxic conditions may occur due to increased thickness and altered composition of the basement membrane, as well as due to edema of the inflamed airway wall. OBJECTIVE: In our study we investigated the effect of hypoxia on proliferation and pro-inflammatory and pro-angiogenic parameter production by human bronchial smooth muscle cells (BSMC). Furthermore, conditioned media of hypoxia-exposed BSMC was tested for its ability to induce sprout outgrowth from endothelial cells spheroids. METHODS: BSMC were cultured in RPMI1640 (5% FCS) under normoxic (21% O2) and hypoxic (1% and 5% O2) conditions. Proliferation was determined by cell count and Western blot analysis for cyclin E and Proliferating Cell Nuclear Antigen (PCNA). Secretion of IL-6, IL-8, ENA-78 and VEGF-A was analyzed by ELISA. BSMC conditioned medium was tested for its angiogenic capacity by endothelial cell (EC)-spheroid in vitro angiogenesis assay. RESULTS: Proliferation of BSMC obtained from asthmatic and non-asthmatic patients was significantly reduced in the presence of 1% O2, whereas 5% O2 reduced proliferation of asthmatic BSMC only. Hypoxia induced HIF-1α expression in asthmatic and non-asthmatic BSMC, which coincided with significantly increased release of IL-6, IL-8 and VEGF-A, but not ENA-78. Finally, endothelial sprout outgrowth from EC spheroids was increased when exposed to hypoxia conditioned BSMC medium. CONCLUSION: Hypoxia had dualistic effects on proliferative and inflammatory responses of asthmatic and non-asthmatic BSMC. First, hypoxia reduced BSMC proliferation. Second, hypoxia induced a pro-inflammatory, pro-angiogenic response. BSMC and EC may thus be promising new targets to counteract and/or alleviate airway wall remodeling.

Bronchial smooth muscle cells of asthmatics promote angiogenesis through elevated secretion of CXC-chemokines (ENA-78, GRO-α, and IL-8).

BACKGROUND: Airway wall remodelling is a key pathology of asthma. It includes thickening of the airway wall, hypertrophy and hyperplasia of bronchial smooth muscle cells (BSMC), as well as an increased vascularity of the sub-epithelial cell layer. BSMC are known to be the effector cells of bronchoconstriction, but they are increasingly recognized as an important source of inflammatory mediators and angiogenic factors. OBJECTIVE: To compare the angiogenic potential of BSMC of asthmatic and non-asthmatic patients and to identify asthma-specific angiogenic factors. METHODS: Primary BSMC were isolated from human airway tissue of asthmatic and non-asthmatic patients. Conditioned medium (CM) collected from BSMC isolates was tested for angiogenic capacity using the endothelial cell (EC)-spheroid in vitro angiogenesis assay. Angiogenic factors in CM were quantified using a human angiogenesis antibody array and enzyme linked immunosorbent assay. RESULTS: Induction of sprout outgrowth from EC-spheroids by CM of BSMC obtained from asthma patients was increased compared with CM of control BSMC (twofold, p < 0.001). Levels of ENA-78, GRO-α and IL-8 were significantly elevated in CM of BSMC from asthma patients (p < 0.05 vs. non-asthmatic patients). SB 265610, a competitive antagonist of chemokine (CXC-motif) receptor 2 (CXCR2), attenuated the increased sprout outgrowth induced by CM of asthma patient-derived BSMC. CONCLUSIONS: BSMC isolated from asthma patients exhibit increased angiogenic potential. This effect is mediated through the CXCR2 ligands (ENA78, GRO-α and IL-8) produced by BSMC. IMPLICATIONS: CXCR2 ligands may play a decisive role in directing the neovascularization in the sub-epithelial cell layers of the lungs of asthma patients. Counteracting the CXCR2-mediated neovascularization by pharmaceutical compounds may represent a novel strategy to reduce airway remodelling in asthma.

Exploring the transcription factor activity in high-throughput gene expression data using RLQ analysis.

BACKGROUND: Interpretation of gene expression microarray data in the light of external information on both columns and rows (experimental variables and gene annotations) facilitates the extraction of pertinent information hidden in these complex data. Biologists classically interpret genes of interest after retrieving functional information from a subset of genes of interest. Transcription factors play an important role in orchestrating the regulation of gene expression. Their activity can be deduced by examining the presence of putative transcription factors binding sites in the gene promoter regions. RESULTS: In this paper we present the multivariate statistical method RLQ which aims to analyze microarray data where additional information is available on both genes and samples. As an illustrative example, we applied RLQ methodology to analyze transcription factor activity associated with the time-course effect of steroids on the growth of primary human lung fibroblasts. RLQ could successfully predict transcription factor activity, and could integrate various other sources of external information in the main frame of the analysis. The approach was validated by means of alternative statistical methods and biological validation. CONCLUSIONS: RLQ provides an efficient way of extracting and visualizing structures present in a gene expression dataset by directly modeling the link between experimental variables and gene annotations.

Asthma and COPD - The C/EBP Connection.

Asthma and chronic obstructive pulmonary disease (COPD) are the two most prominent chronic inflammatory lung diseases with increasing prevalence. Both diseases are associated with mild or severe remodeling of the airways. In this review, we postulate that the pathologies of asthma and COPD may result from inadequate responses and/or a deregulated balance of a group of cell differentiation regulating factors, the CCAAT/Enhancer Binding Proteins (C/EBPs). In addition, we will argue that the exposure to environmental factors, such as house dust mite and cigarette smoke, changes the response of C/EBPs and are different in diseased cells. These novel insights may lead to a better understanding of the etiology of the diseases and may provide new aspects for therapies.

Calreticulin is a negative regulator of bronchial smooth muscle cell proliferation.

Background. Calreticulin controls the C/EBPαp42/p30 at the translational level trough a cis-regulatory CNG rich loop in the CEBPA mRNA. We determined the effects of steroids and long-acting beta-agonists on the p42/p30 ratio and on calreticulin expression in primary human bronchial smooth muscle (BSM) cells. Methods. The effects of budesonide (10(-8) M) and formoterol (10(-8) M) were studied in BSM cells pre-treated with siRNA targeting calreticulin. The expression of C/EBPα and calreticulin was determined by immuno-blotting. Automated cell counts were performed to measure proliferation. Results. All tested BSM cell lines (n = 5) expressed C/EBPα and calreticulin. In the presence of 5% FBS, the p42/p30 ratio significantly decreased (n = 3, P < 0.05) and coincided with BSM cell proliferation. High levels of calreticulin were associated with a decreased p42/p30 isoform ratio. FBS induced the expression of calreticulin (n = 3, P < 0.05), which was further increased by formoterol. siRNA targeting calreticulin increased the p42/p30 ratio in non-stimulated BSM cells and significantly inhibited the proliferation of PDGF-BB-stimulated BSM cells (n = 5, P < 0.05). Neither budesonide nor formoterol restored the p42 isoform expression. Conclusions. Our data show calreticulin is a negative regulator of C/EBPα protein expression in BSM cells. Modulation of calreticulin levels may provide a novel target to reduce BSM remodeling.

Impaired translation of CCAAT/enhancer binding protein alpha mRNA in bronchial smooth muscle cells of asthmatic patients.

BACKGROUND: Bronchial smooth muscle (BSM) cells of asthmatic patients have an impaired expression of CCAAT/enhancer binding protein (C/EBP) alpha, which is associated with increased proliferation. OBJECTIVE: We sought to assess the translational regulation of CEBPA mRNA in cultured BSM cells of healthy control subjects (n = 11) and asthmatic patients (n = 12). METHODS: Translation efficiency was studied by using a translation control reporter system driven by the control elements present in the CEBPA mRNA. Translation efficiency was determined by the ratio of 2 artificial hemagglutinin (HA.11) proteins: p23 and p12. We also analyzed levels of proteins that control translation of CEBPA mRNA, namely heterogeneous nuclear ribonucleoprotein E2, calreticulin, eukaryotic translation initiation factor (eIF4E), and 4E binding protein. RESULTS: Compared with healthy control subjects, BSM cells of asthmatic patients proliferate faster (2.1-fold) and are primed for IL-6 secretion. Real-time RT-PCR showed that BSM cells of asthmatic patients express normal levels of CEBPA mRNA, whereas they express lower levels of C/EBPalpha (p42). Transient transfections with the translation control reporter system construct showed a disturbed p12/p23 ratio in BSM cells of asthmatic patients relative to healthy control subjects, which coincided with lower levels of eIF4E. CONCLUSION: BSM cells of asthmatic patients have normal levels of CEBPA mRNA but inadequately reinitiate the translation into C/EBPalpha. Impaired translation control upstream of eIF4E might underlie the observed increased proliferation and priming of BSM cells of asthmatic patients.

Disease-specific expression and regulation of CCAAT/enhancer-binding proteins in asthma and chronic obstructive pulmonary disease.

BACKGROUND: CCAAT/enhancer-binding proteins (C/EBPs) control cell proliferation; lack of C/EBPalpha correlates with increased proliferation of bronchial smooth muscle cells (BSMCs) of asthmatic patients. OBJECTIVE: We sought to assess disease-specific expression of C/EBPalpha, beta, delta, and epsilon and the effects of budesonide (10(-8) mol/L) and formoterol (10(-8) mol/L). METHODS: Expression and function of C/EBPalpha, beta, delta, and epsilon BSMCs of control subjects (n = 9), asthmatic patients (n = 12), and patients with chronic obstructive pulmonary disease (COPD; n = 10) were determined. RESULTS: The control group expressed C/EBPalpha, beta, delta, and epsilon, which were upregulated by serum (5%). Budesonide completely inhibited C/EBPalpha and beta expression; formoterol increased C/EBPalpha expression (2-fold). C/EBPdelta and epsilon expression were not affected by the drugs. The asthmatic group did not appropriately express C/EBPalpha. Basal levels of C/EBPbeta, delta, and epsilon were upregulated by serum (5%). Budesonide and formoterol increased C/EBPbeta levels (3.4-fold and 2.5-fold, respectively), leaving C/EBPalpha, delta, and epsilon levels unaffected. The COPD group normally expressed C/EBPalpha, beta, and epsilon, which were upregulated by serum treatment (5%). Basal levels of C/EBPdelta were downregulated by serum in 7 of 10 BSMC lines. Budesonide inhibited C/EBPalpha and beta expression, upregulated C/EBPdelta (3.2-fold), and had no effect on C/EBPepsilon. Formoterol upregulated C/EBPalpha expression (3-fold) but not the other C/EBPs. Protein analysis and electrophoretic mobility shift assay confirmed the disease-specific expression pattern of C/EBPalpha in asthmatic patients and C/EBPdelta in patients with COPD. CONCLUSIONS: The expression and regulation of C/EBPs in BSMCs of asthmatic patients and patients with COPD seems disease specific. Budesonide and formoterol modulate C/EBP expression in a drug- and disease-specific pattern. CLINICAL IMPLICATIONS: The data could provide a method to discriminate between asthma and COPD at an early disease stage.

Asthma: is it due to an abnormal airway smooth muscle cell?

Asthma is an airway disease highly prevalent in westernized countries and of unknown etiology. Often, asthma is associated with atopy, but not all atopic individuals have asthma. Some patients with asthma outgrow symptoms, whereas many others acquire asthma later in life. Still other patients suffer from asthma their entire life. How can we explain these different patterns? It may be that asthma should be regarded as the clinical manifestation of a group of diseases with similar pathology due to a common factor. In this Pulmonary Perspective, we propose that an aberrant phenotype of airway smooth muscle (ASM) cells could be sufficient to explain the pathology of asthma. We will argue an abnormal ASM cell is a prerequisite to the development of asthma. Our postulate is that inadequate levels of C/EBPalpha, a protein that is pivotal for the suppression of both inflammation and proliferation responses, confer on ASM cells an activated phenotype that is more susceptible to mitogenic and contractile stimuli.

House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms.

House dust mite allergens (HDM) cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of Dermatophagoides pteronissinus with protease activity (Der p 1) and unknown enzymatic activity (Der p 2, Der p 5) induce biological responses in a human airway-derived epithelial cell line (A549), and if so, to elucidate the underlying mechanism(s) of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca2+ levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR)1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca2+ mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca2+ mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells.

Dysfunctional interaction of C/EBPalpha and the glucocorticoid receptor in asthmatic bronchial smooth-muscle cells.

BACKGROUND: Increased proliferation of bronchial smooth-muscle cells may lead to increased muscle mass in the airways of patients with asthma. The antiproliferative effect of glucocorticoids in bronchial smooth-muscle cells in subjects without asthma is mediated by a complex of the glucocorticoid receptor and the CCAAT/enhancer binding protein alpha (C/EBPalpha). We examined the signaling pathway controlling the inhibitory effect of glucocorticoids on cell proliferation and interleukin-6 synthesis in bronchial smooth-muscle cells of subjects with asthma and those without asthma. METHODS: Lines of bronchial smooth-muscle cells were established from cells from 20 subjects with asthma, 8 subjects with emphysema, and 26 control subjects. Cell proliferation was determined by means of cell counts and [3H]thymidine incorporation. Signal transduction was studied by means of an electrophoretic DNA mobility-shift assay, a supershift electrophoretic-mobility assay, immunoblotting, use of C/EBPalpha antisense oligonucleotides, and use of a human C/EBPalpha expression vector. Interleukin-6 release was determined by means of an enzyme-linked immunosorbent assay. RESULTS: Glucocorticoids activated the glucocorticoid receptor and inhibited serum-induced secretion of interleukin-6 in bronchial smooth-muscle cells from both subjects with asthma and those without asthma; however, glucocorticoids inhibited proliferation only in bronchial smooth-muscle cells from subjects without asthma. C/EBPalpha protein was detected by immunoblotting in all bronchial smooth-muscle cells from subjects without asthma but not in those with asthma, whereas the protein was expressed in lymphocytes from both groups of subjects. C/EBPalpha antisense oligonucleotides or the glucocorticoid-receptor inhibitor mifepristone reversed the antiproliferative effect of glucocorticoids in bronchial smooth-muscle cells from subjects without asthma. When bronchial smooth-muscle cells from subjects with asthma were transiently transfected with an expression vector for human C/EBPalpha, two forms of the protein were expressed, and subsequent administration of glucocorticoids inhibited cell proliferation. CONCLUSIONS: We hypothesize that a cell-type-specific absence of C/EBPalpha is responsible for the enhanced proliferation of bronchial smooth-muscle cells derived from subjects with asthma and that it explains the failure of glucocorticoids to inhibit proliferation in vitro.

Cyclosporine A mediates fibroproliferation through epithelial cells.

BACKGROUND: Obliterative bronchiolitis (OB) is the major cause of morbidity and mortality after lung transplantation. The potential role of immunosuppressive drugs in the development of OB is uncertain, but there are limited data indicating that cyclosporine A (CsA) may have a direct fibrogenic effect on various human cell types. Epithelium-fibroblast interactions have been suggested to play a crucial role in the course of fibroproliferation, which is a major feature of OB. METHODS: We studied the effect of CsA and FK506 on primary human lung fibroblast proliferation in a human epithelial-fibroblast interactive model. RESULTS: Clinically relevant concentrations of CsA (0.1-1 microg/mL) and FK506 (0.001-0.01 microg/mL) did not affect fibroblast proliferation in monocultures. Conditioned medium (CM) from untreated epithelial cells (Calu-3) stimulated fibroblast proliferation. CM from FK506-treated (0.001-0.1 microg/mL) epithelial cells had no significant additive effect on fibroblast proliferation compared with CM of untreated epithelial cells. In contrast, CM obtained from epithelial cells treated with 0.1 microg/mL CsA significantly enhanced fibroblast proliferation compared with CM of untreated epithelial cells. This proliferative effect of 0.1 microg/mL CsA was mediated by epithelial-derived factors greater than 100 kDa. CONCLUSIONS: These data demonstrate that a clinically relevant concentration of CsA stimulates fibroblast proliferation through mediators produced by airway epithelial cells, raising the possibility that CsA may contribute to the development of OB after lung transplantation.

Polarized Th1 and Th2 cells are less responsive to negative feedback by receptors coupled to the AC/cAMP system compared to freshly isolated T cells.

1. The adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP) system is known to negatively regulate transcriptional activity of T cells, thereby possibly modulating T-cell-mediated responses at the sites of inflammation. Effects of cAMP have been widely studied in freshly isolated T cells and T-cell clones; yet, effects in differentiated Th1 and Th2 cells are largely unknown. 2. To obtain differentiated T helper cells, we activated naive T cells for 1 week in the presence of IL-12 plus alpha-IL-4 to generate Th1-type cells and in the presence of IL-4 plus alpha-IL-12 to generate Th2-type cells. 3. We demonstrate that, in contrast to freshly isolated T cells, the production of Th1 (IFN-gamma) and Th2 (IL-4, IL-5) cytokines in polarized T helper cells is not strictly controlled by the activation of AC/cAMP-linked beta(2)-adrenergic and prostaglandin (PG)E(2) receptors. 4. In Th2 cells, PGE(2) could still activate the G(s) protein-coupled AC/cAMP system and subsequently induce CREB phosphorylation, whereas PGE(2) was unable to activate the cAMP-dependent pathway in Th1 cells. In both Th1 and Th2 cells, the induction of CREB phosphorylation by beta(2)-agonist fenoterol was impaired. 5. The loss of control over cytokine production by cAMP elevating agents in differentiated Th1 and Th2 subsets may have important implications for the regulation of Th1- and Th2-mediated diseases, in particular those associated with the ongoing immune responses.

Asthma and the CCAAT-enhancer binding proteins: a holistic view on airway inflammation and remodeling.

Asthma is an airway disease with increasing prevalence characterized by intermittent reversible airway obstruction, airway inflammation, and airway wall remodeling. The disease is generally triggered by inhalation of allergens, but nonallergic asthma triggers are quite common. The pathogenesis of asthma is well documented, and a great deal of research has been carried out to elucidate the underlying mechanisms. A multitude of articles have focused on cells alleged to be involved in the pathogenesis, including circulating cells from the immunologic compartment (ie, eosinophils and T lymphocytes) and resident cells, such as fibroblasts, airway smooth muscle cells, and, more recently, the airway epithelium. Despite the enormous amount of research, it is still unclear what exactly causes asthma. A general feature of most studies is an enhanced activation status of asthmatic cells, suggesting a general defect with respect to regulation of cellular responses. Here we discuss the ubiquitous transcription factor family of CCAAT-enhancer binding proteins (C/EBPs) and its involvement in inflammation and proliferation. We propose that an imbalance of C/EBP isoform expression might lead to an enhanced activity of asthmatic cells and provide an overall hypothesis that both airway inflammation and remodeling can be conceived as the result of an imbalance of C/EBP isoform expression.

Interleukin-6 promotes the production of interleukin-4 and interleukin-5 by interleukin-2-dependent and -independent mechanisms in freshly isolated human T cells.

T helper 2 (Th2) cytokines [interleukin (IL)-4 and IL-5] play a central role in the development of allergic immune responses. After allergen provocation, the expression of Th2 cytokines is rapidly up-regulated in atopy and asthma. IL-6 is a multifunctional cytokine that is able to direct Th2 immune responses and is secreted by multiple tissue cell types. This study shows that IL-6 induces up-regulation of IL-4 and IL-5 after short (5 min) preincubation periods in freshly isolated, alpha-CD3/alpha-CD28-stimulated T cells. After longer preincubation periods with IL-6 (12 and 24 hr), the priming effect on IL-4 production gradually disappears, whereas the effect on IL-5 becomes more pronounced. In contrast, a small but significant inhibitory effect is found on the production of the Th1 cytokine interferon-gamma. Additional experiments indicate that the long-term priming effect of IL-6 on IL-5 production is dependent on IL-2 signalling. This is not the case for the short-term IL-6 effect on IL-5 secretion, where the p38 mitogen-activated protein kinase-dependent induction of activator protein-1 DNA-binding activity is involved, independent of signal transducer and activator of transcription 3 phosphorylation. In summary, these data demonstrate that the short-term and long-term priming effects of IL-6 on Th2 cytokine production are regulated by different mechanisms.