CD74 is a functional MIF receptor on activated CD4+ T cells.

Next to its classical role in MHC II-mediated antigen presentation, CD74 was identified as a high-affinity receptor for macrophage migration inhibitory factor (MIF), a pleiotropic cytokine and major determinant of various acute and chronic inflammatory conditions, cardiovascular diseases and cancer. Recent evidence suggests that CD74 is expressed in T cells, but the functional relevance of this observation is poorly understood. Here, we characterized the regulation of CD74 expression and that of the MIF chemokine receptors during activation of human CD4+ T cells and studied links to MIF-induced T-cell migration, function, and COVID-19 disease stage. MIF receptor profiling of resting primary human CD4+ T cells via flow cytometry revealed high surface expression of CXCR4, while CD74, CXCR2 and ACKR3/CXCR7 were not measurably expressed. However, CD4+ T cells constitutively expressed CD74 intracellularly, which upon T-cell activation was significantly upregulated, post-translationally modified by chondroitin sulfate and could be detected on the cell surface, as determined by flow cytometry, Western blot, immunohistochemistry, and re-analysis of available RNA-sequencing and proteomic data sets. Applying 3D-matrix-based live cell-imaging and receptor pathway-specific inhibitors, we determined a causal involvement of CD74 and CXCR4 in MIF-induced CD4+ T-cell migration. Mechanistically, proximity ligation assay visualized CD74/CXCR4 heterocomplexes on activated CD4+ T cells, which were significantly diminished after MIF treatment, pointing towards a MIF-mediated internalization process. Lastly, in a cohort of 30 COVID-19 patients, CD74 surface expression was found to be significantly upregulated on CD4+ and CD8+ T cells in patients with severe compared to patients with only mild disease course. Together, our study characterizes the MIF receptor network in the course of T-cell activation and reveals CD74 as a novel functional MIF receptor and MHC II-independent activation marker of primary human CD4+ T cells.

Engineering an inducible leukemia-associated fusion protein enables large-scale ex vivo production of functional human phagocytes.

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches.

Linking brain activity across scales with simultaneous opto- and electrophysiology.

The brain enables adaptive behavior via the dynamic coordination of diverse neuronal signals across spatial and temporal scales: from fast action potential patterns in microcircuits to slower patterns of distributed activity in brain-wide networks. Understanding principles of multiscale dynamics requires simultaneous monitoring of signals in multiple, distributed network nodes. Combining optical and electrical recordings of brain activity is promising for collecting data across multiple scales and can reveal aspects of coordinated dynamics invisible to standard, single-modality approaches. We review recent progress in combining opto- and electrophysiology, focusing on mouse studies that shed new light on the function of single neurons by embedding their activity in the context of brain-wide activity patterns. Optical and electrical readouts can be tailored to desired scales to tackle specific questions. For example, fast dynamics in single cells or local populations recorded with multi-electrode arrays can be related to simultaneously acquired optical signals that report activity in specified subpopulations of neurons, in non-neuronal cells, or in neuromodulatory pathways. Conversely, two-photon imaging can be used to densely monitor activity in local circuits while sampling electrical activity in distant brain areas at the same time. The refinement of combined approaches will continue to reveal previously inaccessible and under-appreciated aspects of coordinated brain activity.

Plant MDL proteins synergize with the cytokine MIF at CXCR2 and CXCR4 receptors in human cells.

Mammalian macrophage migration inhibitory factor (MIF) and its paralog, D-dopachrome tautomerase, are multifunctional inflammatory cytokines. Plants have orthologous MIF and D-dopachrome tautomerase-like (MDL) proteins that mimic some of the effects of MIF on immune cells in vitro. We explored the structural and functional similarities between the three Arabidopsis thaliana MDLs and MIF. X-ray crystallography of the MDLs revealed high structural similarity between MDL and MIF homotrimers and suggested a potential explanation for the lack of tautomerase activity in the MDLs. MDL1 and MDL2 interacted with each other and with MIF in vitro, in yeast, and in plant leaves and formed hetero-oligomeric complexes with MIF in vitro. The MDLs stimulated signaling through the MIF receptors CXCR2 or CXCR4 and enhanced the responses to MIF in a yeast reporter system, in human neutrophils, and in human lung epithelial cells. Pharmacological inhibitors that disrupted MIF activity or prevented the formation of MIF-MDL hetero-oligomers blocked the observed synergism. These findings demonstrate that MDLs can enhance cellular responses to MIF, which may have functional implications in tissues exposed to MDLs from the diet or environment.

More than random responding: Empirical evidence for the validity of the (Extended) Crosswise Model.

The Randomized Response Technique (Warner, Journal of the American Statistical Association, 60, 63-69, 1965) has been developed to control for socially desirable responses in surveys on sensitive attributes. The Crosswise Model (CWM; Yu et al., Metrika, 67, 251-263, 2008) and its extension, the Extended Crosswise Model (ECWM; Heck et al., Behavior Research Methods, 50, 1895-1905, 2018), are advancements of the Randomized Response Technique that have provided promising results in terms of improved validity of the obtained prevalence estimates compared to estimates based on conventional direct questions. However, recent studies have raised the question as to whether these promising results might have been primarily driven by a methodological artifact in terms of random responses rather than a successful control of socially desirable responding. The current study was designed to disentangle the influence of successful control of socially desirable responding and random answer behavior on the validity of (E)CWM estimates. To this end, we orthogonally manipulated the direction of social desirability (undesirable vs. desirable) and the prevalence (high vs. low) of sensitive attributes. Our results generally support the notion that the ECWM successfully controls social desirability bias and is inconsistent with the alternative account that ECWM estimates are distorted by a substantial influence of random responding. The results do not rule out a small proportion of random answers, especially when socially undesirable attributes with high prevalence are studied, or when high randomization probabilities are applied. Our results however do rule out that random responding is a major factor that can account for the findings attesting to the improved validity of (E)CWM as compared with DQ estimates.

Heterocomplexes between the atypical chemokine MIF and the CXC-motif chemokine CXCL4L1 regulate inflammation and thrombus formation.

To fulfil its orchestration of immune cell trafficking, a network of chemokines and receptors developed that capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within this network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine-chemokine interactions extend to ACKs and that MIF forms heterocomplexes with classical chemokines. We tested this hypothesis by using an unbiased chemokine protein array. Platelet chemokine CXCL4L1 (but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12) was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation was observed to inhibit MIF-elicited T-cell chemotaxis as assessed by transwell migration assay and in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ, as well as MIF-mediated monocyte adhesion on aortic endothelial cell monolayers under flow stress conditions. Of note, CXCL4L1 blocked binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. Because MIF and CXCL4L1 are platelet-derived products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and in clinical human thrombus sections obtained from peripheral artery disease (PAD) in patients undergoing thrombectomy. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction that adds to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures that can be exploited to modulate inflammation and thrombosis.

Nothing but the truth? Effects of faking on the validity of the crosswise model.

In self-reports, socially desirable responding threatens the validity of prevalence estimates for sensitive personal attitudes and behaviors. Indirect questioning techniques such as the crosswise model attempt to control for the influence of social desirability bias. The crosswise model has repeatedly been found to provide more valid prevalence estimates than direct questions. We investigated whether crosswise model estimates are also less susceptible to deliberate faking than direct questions. To this end, we investigated the effect of "fake good" instructions on responses to direct and crosswise model questions. In a sample of 1,946 university students, 12-month prevalence estimates for a sensitive road traffic behavior were higher and thus presumably more valid in the crosswise model than in a direct question. Moreover, "fake good" instructions severely impaired the validity of the direct questioning estimates, whereas the crosswise model estimates were unaffected by deliberate faking. Participants also reported higher levels of perceived confidentiality and a lower perceived ease of faking in the crosswise model compared to direct questions. Our results corroborate previous studies finding the crosswise model to be an effective tool for counteracting the detrimental effects of positive self-presentation in surveys on sensitive issues.

A database and deep learning toolbox for noise-optimized, generalized spike inference from calcium imaging.

Inference of action potentials ('spikes') from neuronal calcium signals is complicated by the scarcity of simultaneous measurements of action potentials and calcium signals ('ground truth'). In this study, we compiled a large, diverse ground truth database from publicly available and newly performed recordings in zebrafish and mice covering a broad range of calcium indicators, cell types and signal-to-noise ratios, comprising a total of more than 35 recording hours from 298 neurons. We developed an algorithm for spike inference (termed CASCADE) that is based on supervised deep networks, takes advantage of the ground truth database, infers absolute spike rates and outperforms existing model-based algorithms. To optimize performance for unseen imaging data, CASCADE retrains itself by resampling ground truth data to match the respective sampling rate and noise level; therefore, no parameters need to be adjusted by the user. In addition, we developed systematic performance assessments for unseen data, openly released a resource toolbox and provide a user-friendly cloud-based implementation.

Controlled growth of ordered monolayers of N-heterocyclic carbenes on silicon.

N-Heterocyclic carbenes (NHCs) are promising modifiers and anchors for surface functionalization and offer some advantages over thiol-based systems. Because of their strong binding affinity and high electron donation, NHCs can dramatically change the properties of the surfaces to which they are bonded. Highly ordered NHC monolayers have so far been limited to metal surfaces. Silicon, however, remains the element of choice in semiconductor devices and its modification is therefore of utmost importance for electronic industries. Here, a comprehensive study on the adsorption of NHCs on silicon is presented. We find covalently bound NHC molecules in an upright adsorption geometry and demonstrate the formation of highly ordered monolayers exhibiting good thermal stability and strong work function reductions. The structure and ordering of the monolayers is controlled by the substrate geometry and reactivity and in particular by the NHC side groups. These findings pave the way towards a tailor-made organic functionalization of silicon surfaces and, thanks to the high modularity of NHCs, new electronic and optoelectronic applications.

Functional analysis of information rates conveyed by rat whisker-related trigeminal nuclei neurons.

The rat whisker system connects the tactile environment with the somatosensory thalamocortical system using only two synaptic stages. Encoding properties of the first stage, the primary afferents with somas in the trigeminal ganglion (TG), has been well studied, whereas much less is known from the second stage, the brainstem trigeminal nuclei (TN). The TN are a computational hub giving rise to parallel ascending tactile pathways and receiving feedback from many brain sites. We asked the question, whether encoding properties of TG neurons are kept by two trigeminal nuclei, the principalis (Pr5) and the spinalis interpolaris (Sp5i), respectively giving rise to two "lemniscal" and two "nonlemniscal" pathways. Single units were recorded in anesthetized rats while a single whisker was deflected on a band-limited white noise trajectory. Using information theoretic methods and spike-triggered mixture models (STM), we found that both nuclei encode the stimulus locally in time, i.e., stimulus features more than 10 ms in the past do not significantly influence spike generation. They further encode stimulus kinematics in multiple, distinct response fields, indicating encoding characteristics beyond previously described directional responses. Compared with TG, Pr5 and Sp5i gave rise to lower spike and information rates, but information rate per spike was on par with TG. Importantly, both brainstem nuclei were found to largely keep encoding properties of primary afferents, i.e. local encoding and kinematic response fields. The preservation of encoding properties in channels assumed to serve different functions seems surprising. We discuss the possibility that it might reflect specific constraints of frictional whisker contact with object surfaces.NEW & NOTEWORTHY We studied two trigeminal nuclei containing the second neuron on the tactile pathway of whisker-related tactile information in rats. We found that the subnuclei, traditionally assumed to give rise to functional tactile channels, nevertheless transfer primary afferent information with quite similar properties in terms of integration time and kinematic profile. We discuss whether such commonality may be due the requirement to adapt to physical constraints of frictional whisker contact.

Macrophage Migration Inhibitory Factor (MIF) Plasma Concentration in Critically Ill COVID-19 Patients: A Prospective Observational Study.

Mortality in critically ill coronavirus disease 2019 (COVID-19) patients is high and pharmacological treatment strategies remain limited. Early-stage predictive biomarkers are needed to identify patients with a high risk of severe clinical courses and to stratify treatment strategies. Macrophage migration inhibitory factor (MIF) was previously described as a potential predictor for the outcome of critically ill patients and for acute respiratory distress syndrome (ARDS), a hallmark of severe COVID-19 disease. This prospective observational study evaluates the predictive potential of MIF for the clinical outcome after severe COVID-19 infection. Plasma MIF concentrations were measured in 36 mechanically ventilated COVID-19 patients over three days after intensive care unit (ICU) admission. Increased compared to decreased MIF was significantly associated with aggravated organ function and a significantly lower 28-day survival (sequential organ failure assessment (SOFA) score; 8.2 ± 4.5 to 14.3 ± 3, p = 0.009 vs. 8.9 ± 1.9 to 12 ± 2, p = 0.296; survival: 56% vs. 93%; p = 0.003). Arterial hypertension was the predominant comorbidity in 85% of patients with increasing MIF concentrations (vs. decreasing MIF: 39%; p = 0.015). Without reaching significance, more patients with decreasing MIF were able to improve their ARDS status (p = 0.142). The identified association between an early MIF response, aggravation of organ function and 28-day survival may open future perspectives for biomarker-based diagnostic approaches for ICU management of COVID-19 patients.

Do they really wash their hands? Prevalence estimates for personal hygiene behaviour during the COVID-19 pandemic based on indirect questions.

BACKGROUND: During the COVID-19 pandemic, billions of people have to change their behaviours to slow down the spreading of the virus. Protective measures include self-isolation, social (physical) distancing and compliance with personal hygiene rules, particularly regular and thorough hand washing. Prevalence estimates for the compliance with the COVID-19 measures are often based on direct self-reports. However, during a health crisis there is strong public pressure to comply with health and safety regulations so that people's responding in direct self-reports may be seriously compromised by social desirability. METHODS: In an online survey, an indirect questioning technique was used to test whether the prevalence of hygiene practices may be lower than in conventional surveys when confidentiality of responding is guaranteed. The Extended Crosswise Model is an indirect questioning technique that guarantees the confidentiality of responding. To the degree that direct self-reports are biased by social desirability, prevalence estimates of hygiene practices such as thorough hand washing based on the Extended Crosswise Model should be lower than those based on direct self-reports. RESULTS: We analysed data of 1434 participants. In the direct questioning group 94.5% of the participants claimed to practice proper hand hygiene; in the indirect questioning group a significantly lower estimate of only 78.1% was observed. CONCLUSIONS: These results indicate that estimates of the degree of commitment to measures designed to counter the spread of the disease may be significantly inflated by social desirability in direct self-reports. Indirect questioning techniques with higher levels of confidentiality seem helpful in obtaining more realistic estimates of the degree to which people follow the recommended personal hygiene measures. More realistic estimates of compliance can help to inform and to adjust public information campaigns on COVID-19 hygiene recommendations.

Controlling social desirability bias: An experimental investigation of the extended crosswise model.

Indirect questioning techniques such as the crosswise model aim to control for socially desirable responding in surveys on sensitive personal attributes. Recently, the extended crosswise model has been proposed as an improvement over the original crosswise model. It offers all of the advantages of the original crosswise model while also enabling the detection of systematic response biases. We applied the extended crosswise model to a new sensitive attribute, campus islamophobia, and present the first experimental investigation including an extended crosswise model, and a direct questioning control condition, respectively. In a paper-pencil questionnaire, we surveyed 1,361 German university students using either a direct question or the extended crosswise model. We found that the extended crosswise model provided a good model fit, indicating no systematic response bias and allowing for a pooling of the data of both groups of the extended crosswise model. Moreover, the extended crosswise model yielded significantly higher estimates of campus Islamophobia than a direct question. This result could either indicate that the extended crosswise model was successful in controlling for social desirability, or that response biases such as false positives or careless responding have inflated the estimate, which cannot be decided on the basis of the available data. Our findings highlight the importance of detecting response biases in surveys implementing indirect questioning techniques.

Designed CXCR4 mimic acts as a soluble chemokine receptor that blocks atherogenic inflammation by agonist-specific targeting.

Targeting a specific chemokine/receptor axis in atherosclerosis remains challenging. Soluble receptor-based strategies are not established for chemokine receptors due to their discontinuous architecture. Macrophage migration-inhibitory factor (MIF) is an atypical chemokine that promotes atherosclerosis through CXC-motif chemokine receptor-4 (CXCR4). However, CXCR4/CXCL12 interactions also mediate atheroprotection. Here, we show that constrained 31-residue-peptides ('msR4Ms') designed to mimic the CXCR4-binding site to MIF, selectively bind MIF with nanomolar affinity and block MIF/CXCR4 without affecting CXCL12/CXCR4. We identify msR4M-L1, which blocks MIF- but not CXCL12-elicited CXCR4 vascular cell activities. Its potency compares well with established MIF inhibitors, whereas msR4M-L1 does not interfere with cardioprotective MIF/CD74 signaling. In vivo-administered msR4M-L1 enriches in atherosclerotic plaques, blocks arterial leukocyte adhesion, and inhibits atherosclerosis and inflammation in hyperlipidemic Apoe-/- mice in vivo. Finally, msR4M-L1 binds to MIF in plaques from human carotid-endarterectomy specimens. Together, we establish an engineered GPCR-ectodomain-based mimicry principle that differentiates between disease-exacerbating and -protective pathways and chemokine-selectively interferes with atherosclerosis.

Can detailed instructions and comprehension checks increase the validity of crosswise model estimates?

The crosswise model is an indirect questioning technique designed to control for socially desirable responding. Although the technique has delivered promising results in terms of improved validity in survey studies of sensitive issues, recent studies have indicated that the crosswise model may sometimes produce false positives. Hence, we investigated whether an insufficient understanding of the crosswise model instructions might be responsible for these false positives and whether ensuring a deeper understanding of the model and surveying more highly educated respondents reduces the problem of false positives. To this end, we experimentally manipulated the amount of information respondents received in the crosswise model instructions. We compared a crosswise model condition with only brief instructions and a crosswise model condition with detailed instructions and additional comprehension checks. Additionally, we compared the validity of crosswise model estimates between a higher- and a lower-educated subgroup of respondents. Our results indicate that false positives among highly educated respondents can be reduced when detailed instructions and comprehension checks are employed. Since false positives can also occur in direct questioning, they do not appear to be a specific flaw of the crosswise model, but rather a more general problem of self-reports on sensitive topics. False negatives were found to occur for all questioning techniques, but were less prevalent in the crosswise model than in the direct questioning condition. We highlight the importance of comprehension checks when applying indirect questioning and emphasize the necessity of developing instructions suitable for lower-educated respondents.

On the validity of non-randomized response techniques: an experimental comparison of the crosswise model and the triangular model.

Non-randomized response techniques (NRRTs) such as the crosswise model and the triangular model (CWM and TRM; Yu et al. Metrika, 67, 251-263, 2008) have been developed to control for socially desirable responding in surveys on sensitive personal attributes. We present the first study to directly compare the validity of the CWM and TRM and contrast their performance with a conventional direct questioning (DQ) approach. In a paper-pencil survey of 1382 students, we obtained prevalence estimates for two sensitive attributes (xenophobia and rejection of further refugee admissions) and one nonsensitive control attribute with a known prevalence (the first letter of respondents' surnames). Both NRRTs yielded descriptively higher prevalence estimates for the sensitive attributes than DQ; however, only the CWM estimates were significantly higher. We attribute the higher prevalence estimates for the CWM to its response symmetry, which is lacking in the TRM. Only the CWM provides symmetric answer options, meaning that there is no "safe" alternative respondents can choose to distance themselves from being carriers of the sensitive attribute. Prevalence estimates for the nonsensitive control attribute with known prevalence confirmed that neither method suffered from method-specific bias towards over- or underestimation. Exploratory moderator analyses further suggested that the sensitive attributes were perceived as more sensitive among politically left-oriented than among politically right-oriented respondents. Based on our results, we recommend using the CWM over the TRM in future studies on sensitive personal attributes.

Studying the Pro-Migratory Effects of MIF.

Macrophage migration inhibitory factor (MIF) is an upstream regulator of innate immunity and dysregulated MIF is a key mediator of acute and chronic inflammatory processes, autoimmune and cardiovascular diseases, as well as cancer. MIF is a pleiotropic cytokine with chemokine-like functions that has been designated as an atypical chemokine (ACK). It orchestrates leukocyte recruitment and migration into inflamed tissues through non-cognate interactions with the classical chemokine receptors CXCR2 and CXCR4, pathways that are further facilitated by MIF's cognate receptor CD74. Here, we describe two complementary methods that can be used to characterize immune cell migration and motility responses controlled by MIF and its receptors. These are the Transwell filter migration assay, also known as modified Boyden chamber assay, a two-dimensional (2D) device, and a matrix-based three-dimensional (3D) chemotaxis assay. The Transwell system is primarily suitable to study chemotactic cell transmigration responses toward a chemoattractant such as MIF through a porous filter membrane. The 3D chemotaxis setup enables for the cellular tracking of migration, invasion, and motility of single cells using live cell imaging.

Detecting nonadherence without loss in efficiency: A simple extension of the crosswise model.

In surveys concerning sensitive behavior or attitudes, respondents often do not answer truthfully, because of social desirability bias. To elicit more honest responding, the randomized-response (RR) technique aims at increasing perceived and actual anonymity by prompting respondents to answer with a randomly modified and thus uninformative response. In the crosswise model, as a particularly promising variant of the RR, this is achieved by adding a second, nonsensitive question and by prompting respondents to answer both questions jointly. Despite increased privacy protection and empirically higher prevalence estimates of socially undesirable behaviors, evidence also suggests that some respondents might still not adhere to the instructions, in turn leading to questionable results. Herein we propose an extension of the crosswise model (ECWM) that makes it possible to detect several types of response biases with adequate power in realistic sample sizes. Importantly, the ECWM allows for testing the validity of the model's assumptions without any loss in statistical efficiency. Finally, we provide an empirical example supporting the usefulness of the ECWM.

On the comprehensibility and perceived privacy protection of indirect questioning techniques.

On surveys that assess sensitive personal attributes, indirect questioning aims at increasing respondents' willingness to answer truthfully by protecting confidentiality. However, the assumption that subjects understand questioning procedures fully and trust them to protect their privacy is rarely tested. In a scenario-based design, we compared four indirect questioning procedures in terms of their comprehensibility and perceived privacy protection. All indirect questioning techniques were found to be less comprehensible by respondents than a conventional direct question used for comparison. Less-educated respondents experienced more difficulties when confronted with any indirect questioning technique. Regardless of education, the crosswise model was found to be the most comprehensible among the four indirect methods. Indirect questioning in general was perceived to increase privacy protection in comparison to a direct question. Unexpectedly, comprehension and perceived privacy protection did not correlate. We recommend assessing these factors separately in future evaluations of indirect questioning.