Phospholipase D1 mediates lymphocyte adhesion and migration in experimental autoimmune encephalomyelitis

Lymphocyte adhesion and subsequent trafficking across endothelial barriers are essential steps in various immune‐mediated disorders of the CNS, including MS. The molecular mechanisms underlying these processes, however, are still unknown. Phospholipase D1 (PLD1), an enzyme that generates phosphatidic acid through hydrolysis of phosphatidylcholine and additionally yields choline as a product, has been described as regulator of the cell mobility. By using PLD1‐deficient mice, we investigated the functional significance of PLD1 for lymphocyte adhesion and migration in vitro and after myelin oligodendrocyte glycoprotein (MOG)_35–55‐induced EAE, a model of human MS. The lack of PLD1 reduced chemokine‐mediated static adhesion of lymphocytes to the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM‐1) and intercellular adhesion molecule 1 (ICAM‐1) in vitro, and was accompanied by a decreased migratory capacity in both blood brain barrier and cell migration models. Importantly, PLD1 is also relevant for the recruitment of immune cells into the CNS in vivo since disease severity after EAE was significantly attenuated in PLD1‐deficient mice. Furthermore, PLD1 expression could be detected on lymphocytes in MS patients. Our findings suggest a critical function of PLD1‐dependent intracellular signaling cascades in regulating lymphocyte trafficking during autoimmune CNS inflammation.     

Gain‐of‐Function Mutation in STIM1 (P.R304W) Is Associated with Stormorken Syndrome

Stormorken syndrome is a rare autosomal dominant disorder characterized by a phenotype that includes miosis, thrombocytopenia/thrombocytopathy with bleeding time diathesis, intellectual disability, mild hypocalcemia, muscle fatigue, asplenia, and ichthyosis. Using targeted sequencing and whole‐exome sequencing, we identified the c.910C > T transition in a STIM1 allele (p.R304W) only in patients and not in their unaffected family members. STIM1 encodes stromal interaction molecule 1 protein (STIM1), which is a finely tuned endoplasmic reticulum Ca²⁺ sensor. The effect of the mutation on the structure of STIM1 was investigated by molecular modeling, and its effect on function was explored by calcium imaging experiments. Results obtained from calcium imaging experiments using transfected cells together with fibroblasts from one patient are in agreement with impairment of calcium homeostasis. We show that the STIM1 p.R304W variant may affect the conformation of the inhibitory helix and unlock the inhibitory state of STIM1. The p.R304W mutation causes a gain of function effect associated with an increase in both resting Ca²⁺ levels and store‐operated calcium entry. Our study provides evidence that Stormorken syndrome may result from a single‐gene defect, which is consistent with Mendelian‐dominant inheritance.     

Partner choice and fidelity stabilize coevolution in a Cretaceous-age defensive symbiosis

Many insects rely on symbiotic microbes for survival, growth, or reproduction. Over evolutionary timescales, the association with intracellular symbionts is stabilized by partner fidelity through strictly vertical symbiont transmission, resulting in congruent host and symbiont phylogenies. However, little is known about how symbioses with extracellular symbionts, representing the majority of insect-associated microorganisms, evolve and remain stable despite opportunities for horizontal exchange and de novo acquisition of symbionts from the environment. Here we demonstrate that host control over symbiont transmission (partner choice) reinforces partner fidelity between solitary wasps and antibiotic-producing bacteria and thereby stabilizes this Cretaceous-age defensive mutualism. Phylogenetic analyses show that three genera of beewolf wasps (Philanthus, Trachypus, and Philanthinus) cultivate a distinct clade of Streptomyces bacteria for protection against pathogenic fungi. The symbionts were acquired from a soil-dwelling ancestor at least 68 million years ago, and vertical transmission via the brood cell and the cocoon surface resulted in host–symbiont codiversification. However, the external mode of transmission also provides opportunities for horizontal transfer, and beewolf species have indeed exchanged symbiont strains, possibly through predation or nest reuse. Experimental infection with nonnative bacteria reveals that—despite successful colonization of the antennal gland reservoirs—transmission to the cocoon is selectively blocked. Thus, partner choice can play an important role even in predominantly vertically transmitted symbioses by stabilizing the cooperative association over evolutionary timescales.Cooperation is ubiquitous in nature, yet it presents a conundrum to evolutionary biology because acts that are beneficial to the receiver but costly to the actor should not be favored by natural selection (1). In interspecific associations (i.e., symbioses), the two most important models to explain the maintenance of cooperation are partner fidelity and partner choice (2, 3). In partner-fidelity associations, host and symbiont interact repeatedly and reward cooperating individuals while punishing cheaters, thereby reinforcing mutually beneficial interactions (2, 4). In partner-choice associations, individuals may interact only once, but one member can select its partner in advance of any possible exploitation (2, 4). Partner choice appears to select for cooperative strains among environmentally acquired microbial symbionts, e.g., the bioluminescent Vibrio fischeri bacteria of squids (5), the nitrogen-fixing rhizobia of legumes (6), and mycorrhizal fungi of plants (7). By contrast, partner fidelity is generally assumed to be the major stabilizing force in the widespread and ecologically important vertically transmitted symbioses of insects (4).However, localization and transmission routes of mutualistic bacteria in insects are diverse, and the differences across symbiotic systems have important implications for the evolutionary trajectory of the associations. Symbionts with an obligate intracellular lifestyle are usually tightly integrated into the host’s metabolism (e.g., ref. 8) and development (9), and the mutual interdependence of both partners coincides with perfect vertical symbiont transmission. Over evolutionary timescales, the high degree of partner fidelity results in host–symbiont cocladogenesis, and, concordantly, phylogenies of hosts and their intracellular symbionts are often found to be congruent (10–13). Although such a pattern is also observed for some extracellular symbioses with especially tight host–symbiont integration (14, 15), the ability of many extracellularly transmitted symbionts to spend part of their life cycle outside of the host’s body is often reflected in more or less extensive horizontal transmission or de novo acquisition of symbionts from the environment (16, 17). In these cases, partner choice mechanisms are expected to ensure specificity in the establishment and maintenance of the association (18). The nature of such control mechanisms, however, remains poorly understood.Although many of the well-studied mutualistic associations in insects have a nutritional basis (19, 20), an increasing number of symbioses for the defense of the host against predators (21), parasitoids (22), or pathogens (23–25) have recently been discovered. Among defensive symbionts, Actinobacteria are particularly prevalent, probably due to their ubiquity in the soil and their ability to produce secondary metabolites with antibiotic properties (23). Antibiotic-producing actinobacterial symbionts have been discovered on the cuticle of leaf-cutting ants (26), in the fungal galleries of a bark beetle (27), and in the antennae and on cocoons of beewolf wasps (28). While in the former two cases the symbionts have been implicated in the defense of the hosts’ nutritional resources against competing fungi (26, 27), the beewolves’ bacteria protect the offspring in the cocoon against pathogenic microorganisms (28, 29).Beewolves are solitary wasps in the genera Philanthus, Trachypus, and Philanthinus (Hymenoptera, Crabronidae, Philanthini). They engage in a defensive alliance with the Actinobacterium ‘Candidatus Streptomyces philanthi’ (CaSP) (28, 30, 31), which is cultivated by female beewolves in specialized antennal gland reservoirs (32). The uniqueness and complexity of the glands suggest a long history of host adaptation towards cultivating its actinobacterial symbionts (32). From the antennae, the streptomycetes are secreted into the brood cell, taken up by the larva, and incorporated into its cocoon (33), where they provide protection against pathogenic fungi and bacteria (28) by producing at least nine different antimicrobial compounds (29). Weeks or months later, eclosing adult females acquire the bacteria from the cocoon surface (33), thus completing the vertical transmission of CaSP. However, this mode of transmission provides opportunities for the horizontal transfer of symbionts among beewolf species or the de novo uptake of bacteria from the environment. Despite these opportunities, a monophyletic clade of CaSP strains has previously been found in 31 species of beewolves, suggesting an ancient and highly coevolved relationship (30, 31, 34).Here we combine cophylogenetic analyses of beewolves and their vertically transmitted defensive symbionts with experimental manipulation of symbiont infection status and subsequent observations of transmission from female antennal gland reservoirs into the brood cell to (i) reconstruct the coevolutionary history of the symbiosis, (ii) estimate the age of the symbiosis, (iii) elucidate the ancestral lifestyle of the symbionts, and (iv) assess the importance of partner fidelity and partner choice for the long-term stability of the association.     

Single whole‐leg compression ultrasound for exclusion of deep vein thrombosis in symptomatic ambulatory patients: a prospective observational cohort study

International guidance has recently recommended serial proximal compression ultrasound (CUS) as first line imaging for suspected deep vein thrombosis (DVT). Single whole‐leg CUS is a routine alternative diagnostic strategy that can reduce repeated attendances and identify alternative pathology. We conducted a prospective observational cohort study. Consecutive ambulatory, adult patients with suspected DVT and negative or inconclusive whole‐leg CUS had anticoagulation withheld and were followed for 3 months. The primary outcome was a predefined clinically relevant adverse event rate. Secondary outcomes included technical failure, alternative diagnoses and all cause mortality. 212 patients agreed to participate and completed follow up. One patient was subsequently diagnosed with an isolated distal DVT. The adverse event rate was thus 1/212, 0·47% (95% confidence interval [CI] 0·08–2·62). Technical imaging failure occurred in 11·3% of cases (95% CI 7·7–16·3). Several potential predictors of an inconclusive result were identified on multivariate analysis. 150 (70·8%) patients were provided with a documented alternative diagnosis. Patients who have anticoagulation withheld following a negative or inconclusive whole‐leg CUS for suspected DVT have a low rate of adverse events. Technical failure remains an issue: several factors were significantly associated with inconclusive results and may warrant an alternative diagnostic approach.     

Local proliferation of macrophages in adipose tissue during obesity-induced inflammation

Aims/hypothesis

Obesity is frequently associated with low-grade inflammation of adipose tissue (AT), and the increase in adipose tissue macrophages (ATMs) is linked to an increased risk of type 2 diabetes. Macrophages have been regarded as post-mitotic, but recent observations have challenged this view. In this study, we tested the hypothesis that macrophages proliferate within AT in diet-induced obesity in mice and humans.

Methods

We studied the expression of proliferation markers by immunofluorescence, PCR and flow cytometry in three different models of mouse obesity as well as in humans (n?=?239). The cell fate of dividing macrophages was assessed by live imaging of AT explants.

Results

We show that ATMs undergo mitosis within AT, predominantly within crown-like structures (CLS). We found a time-dependent increase in ATM proliferation when mice were fed a high-fat diet. Upregulation of CD206 and CD301 in proliferating ATMs indicated preferential M2 polarisation. Live imaging within AT explants from mice revealed that macrophages emigrate out of the CLS to become resident in the interstitium. In humans, we confirmed the increased expression of proliferation markers of CD68⁺ macrophages in CLS and demonstrated a higher mRNA expression of the proliferation marker Ki67 in AT from obese patients.

Conclusions/interpretation

Local proliferation contributes to the increase in M2 macrophages in AT. Our data confirm CLS as the primary site of proliferation and a new source of ATMs and support a model of different recruitment mechanisms for classically activated (M1) and alternatively activated (M2) macrophages in obesity.     

Illness perceptions predict exercise capacity and psychological well-being after pulmonary rehabilitation in COPD patients

Objective

Patients' beliefs about their disease have been associated with emotional adjustment and outcomes in several severe illnesses. The aim of the present study was to investigate whether illness perceptions before pulmonary rehabilitation influence exercise capacity and quality of life after rehabilitation in patients suffering from chronic obstructive pulmonary disease (COPD).

Methods

Ninety-six patients with COPD (GOLD III/IV) were approached on admission to rehabilitation and reassessed before discharge. Assessment included medical measures of FEV?% predicted, and exercise capacity (6 min walk test). Additionally, depressive symptoms (Hospital Anxiety and Depression scale), anxiety (COPD specific anxiety questionnaire) and quality of life (Short Form 36 health survey (SF-36)) were assessed. Illness beliefs were measured by the Revised Illness Perception Questionnaire (IPQ-R).

Results

Exercise capacity and psychological well-being (SF-36) of patients improved after rehabilitation programme, while physical functioning (SF-36) did not change. Additionally, patients showed significantly lower levels of depressive symptoms, COPD specific anxiety and negative perceptions of their illness after pulmonary rehabilitation compared to baseline. In the hierarchical multiple regression analyses, after controlling for socio-demographic data, psychological variables, illness severity and baseline scores of the corresponding variables, it was shown that illness perceptions before rehabilitation predicted exercise capacity and psychological well-being, both assessed at the end of treatment.

Conclusion

COPD patients' perceptions about their illness before rehabilitation influence exercise capacity and quality of life (psychological well-being) after treatment. Therefore it might be relevant to identify and change maladaptive illness perceptions in order to improve medical and psychological outcome in COPD.     

Validity of standard gamble estimated quality of life in acute venous thrombosis

Introduction

The standard gamble is considered the ‘gold standard’ technique for measuring quality of life. We recently used the standard gamble to estimate quality of life in acute venous thrombosis, and found unexpected variability in the responses. The current study aimed to explore the reasons for variability by comparing the standard gamble technique in patients with acute venous thrombosis to other quality of life measurement tools.

Materials and Methods

Thrombosis clinic patients treated for venous thrombosis were eligible to participate. Patients evaluated their current health state by performing a standard gamble interview, reporting on a visual analogue scale, completing the SF-36 and disease specific questionnaires (PEmb-Qol and VEINES-QOL/Sym). Validity was assessed by correlating the standard gamble utilities with the other methods. Test-retest reliability, responsiveness and acceptability were also assessed.

Results

Forty-four patients were interviewed, with 16 attending for a repeat interview. The median standard gamble utility was 0.97 (0.84-1.0), SF-6D 0.64 (0.59 - 0.80) and visual analogue score 70 (60 - 80). Participants with pulmonary embolism had lower standard gamble estimates than those with deep vein thrombosis. There was good discriminant validity in that the standard gamble estimates were not associated with risk taking behavior, negative outlook, sex or education. Test-retest reliability with the standard gamble was moderate and there was evidence of a ceiling effect.

Conclusions

Standard gamble utilities are higher than other methods of measuring quality of life in venous thrombosis. The choice of utility values adopted in studies will impact on future economic studies.