Circulating immune profile in granulomatosis with polyangiitis reveals distinct patterns related to disease activity.

Granulomatosis with polyangiitis (GPA) is an autoimmune disorder characterized by recurrent relapses that can cause severe tissue damage and life-threatening organ dysfunction. Multiple immune cells and cytokines/chemokines are involved in the different stages of the disease. Immune profiling of patients may be useful for tracking disease activity, however, reliable immune signatures for GPA activity are lacking. In this study, we examined circulating immune profiles in GPA patients during active and remission disease states to identify potential immune patterns associated with disease activity. The distribution and phenotypic characteristics of major circulating immune cells, and the profiles of circulating cytokines/chemokines, were studied on cryopreserved peripheral blood mononuclear cells from GPA patients (active, n = 20; remission, n = 20) and healthy controls (n = 20) leveraging a 40-color optimized multicolor immunofluorescence panel (OMIP-69) and in serum using a 46-plex Luminex multiplex assay, respectively. Deep phenotyping uncovered a distinct composition of major circulating immune cells in active GPA and GPA in remission, with the most significant findings emerging within the monocyte compartment. Our detailed analysis revealed circulating monocyte diversity beyond the conventional monocyte subsets. We identified eight classical monocyte populations, two intermediate monocyte populations, and one non-classical monocyte population. Notably, active GPA had a higher frequency of CD45RA+CCR5+CCR6-CCR7+/lowCD127-HLA-DR+CD2- classical monocytes and a lower frequency of CD45RA-CCR5-/lowCCR6-CCR7-CD127-HLA-DR+CD2+/- classical monocytes, which both strongly correlated with disease activity. Furthermore, serum levels of CXCL1, CXCL2, and CCL20, all linked to monocyte biology, were elevated in active GPA and correlated strongly with disease activity. These findings shed light on the circulating immune profile of GPA and may lead to immune signature profiles for assessing disease activity. Monocytes in particular may be studied further as potential markers for monitoring GPA.

The relationship between medical student engagement in the provision of the school's education programme and learning outcomes.

INTRODUCTION: Student engagement in the provision of the school's education programme (educational student engagement) plays an important role in quality assurance in medical education. However, little is known whether this specific type of student engagement has effects on the learning outcomes for the involved medical students. METHODS: This study was based on a national-wide survey in China among medical students with 123,055 responses. The questionnaire was designed using international and Chinese national standards. T-test, analysis of variance, multivariate regression, and regression with interaction terms were used. RESULTS: Educational student engagement was positively associated with medical students' learning outcomes in Clinical Practice, Science and Scholarship, Health and Society, and Professionalism. Besides, the influence was heterogeneous among participants at different learning phases. Learning outcomes in Clinical Practice were strongly associated with educational student engagement efficiently at the Clinical Medical Education and the Clerkship Rotation phases, and learning outcomes in Science and Scholarship were best correlated with the Clerkship Rotation phase. CONCLUSION: Educational student engagement is positively associated with the learning outcomes, with the greatest effect on learning outcomes in Clinical Practice and the least effect in Professionalism. Besides, it has a greater impact on medical students at senior learning phases.

Social environment affects sensory gene expression in ant larvae.

Social insects depend on communication to regulate social behaviour. This also applies to their larvae, which are commonly exposed to social interactions and can react to social stimulation. However, how social insect larvae sense their environment is not known. Using RNAseq, we characterized expression of sensory-related genes in larvae of the ant Formica fusca, upon exposure to two social environments: isolation without contact to other individuals, and stimulation via the presence of other developing individuals. Expression of key sensory-related genes was higher following social stimulation, and larvae expressed many of the same sensory-related genes as adult ants and larvae of other insects, including genes belonging to the major insect chemosensory gene families. Our study provides first insights into the molecular changes associated with social information perception in social insect larvae.

Implications of estimating road traffic serious injuries from hospital data.

To determine accurately the number of serious injuries at EU level and to compare serious injury rates between different countries it is essential to use a common definition. In January 2013, the High Level Group on Road Safety established the definition of serious injuries as patients with an injury level of MAIS3+(Maximum Abbreviated Injury Scale). Whatever the method used for estimating the number or serious injuries, at some point it is always necessary to use hospital records. The aim of this paper is to understand the implications for (1) in/exclusion criteria applied to case selection and (2) a methodological approach for converting ICD (International Classification of Diseases/Injuries) to MAIS codes, when estimating the number of road traffic serious injuries from hospital data. A descriptive analysis with hospital data from Spain and the Netherlands was carried out to examine the effect of certain choices concerning in- and exclusion criteria based on codes of the ICD9-CM and ICD10. The main parameters explored were: deaths before and after 30 days, readmissions, and external injury causes. Additionally, an analysis was done to explore the impact of using different conversion tools to derive MAIS3 + using data from Austria, Belgium, France, Germany, Netherlands, and Spain. Recommendations are given regarding the in/exclusion criteria and when there is incomplete data to ascertain a road injury, weighting factors could be used to correct data deviations and make more real estimations.

Longitudinal analysis of varicella-zoster virus-specific antibodies in systemic lupus erythematosus: No association with subclinical viral reactivations or lupus disease activity.

Systemic lupus erythematosus (SLE) patients are at high risk of herpes zoster. Previously, we found increased immunoglobulin (Ig)G levels against varicella-zoster virus (VZV) in SLE patients compared to controls, while antibody levels against diphtheria and cellular immunity to VZV were decreased. We aimed to test our hypothesis that increased VZV-IgG levels in SLE result from subclinical VZV reactivations, caused by stress because of lupus disease activity or immunosuppressive drug use. Methods Antibody levels to VZV (IgG, IgA, IgM), total IgG and VZV-DNA were longitudinally determined in the serum of 34 SLE patients, using enzyme-linked immunosorbent assay and polymerase chain reaction. Clinical data were retrieved from medical records. Reactivation of VZV was defined as an at least fivefold rise in VZV-IgG or presence of VZV-IgM or VZV-DNA. Generalized estimating equations (GEE) were used to longitudinally analyse associations between antibody levels, lupus disease activity and medication use. Systemic Lupus Erythematosus Disease Activity Index, anti-double-stranded DNA and complement levels were used as indicators of lupus disease activity. Results A VZV reactivation was determined in 11 patients (33%). In at least five of them, herpes zoster was clinically overt. No association between SLE disease activity or medication use and VZV-specific antibody levels was found. There was a weak association between total IgG and VZV-IgG. Conclusions Our results indicate that increased VZV-IgG levels in SLE do not result from frequent subclinical VZV reactivations, and are not associated with lupus disease activity. Increased VZV-IgG can only partially be explained by hypergammaglobulinaemia.

Health burden of serious road injuries in the Netherlands.

BACKGROUND: The consequences of injuries in terms of disabilities and health burden are relevant for policy making. This article provides an overview of the current knowledge on this topic and discusses the health burden of serious road injuries in The Netherlands. METHODS: The overview of current knowledge on disabilities following a road crash is based on a literature review. The health burden of serious road injuries is quantified in terms of years lived with disability (YLD), by combining incidence data from the Dutch hospital discharge register with information about temporary and lifelong disability. RESULTS: Literature shows that road traffic injuries can have a major impact on victims' physical and psychological well-being and functioning. Reported proportions of people with disability vary between 11 and 80% depending on the type of casualties, time elapsed since the crash, and the health impacts considered. Together, all casualties involving serious injuries in The Netherlands in 2009 account for about 38,000 YLD, compared to 25,000 years of life lost (YLL) of fatalities. Ninety percent of the burden of injury is due to lifelong consequences that are experienced by 20% of all those seriously injured in road accidents. Lower leg injuries and head injuries represent a high share in the total burden of injury as have cyclists that are injured in a crash without a motorized vehicle. Pedestrians and powered 2-wheeler users show the highest burden of injury per casualty. CONCLUSION: Given their major impacts and contribution to health burden, road policy making should also be aimed at reducing the number of serious road injuries and limiting the resulting health impacts.

Effectiveness of low speed autonomous emergency braking in real-world rear-end crashes.

This study set out to evaluate the effectiveness of low speed autonomous emergency braking (AEB) technology in current model passenger vehicles, based on real-world crash experience. The validating vehicle safety through meta-analysis (VVSMA) group comprising a collaboration of government, industry consumer organisations and researchers, pooled data from a number of countries using a standard analysis format and the established MUND approach. Induced exposure methods were adopted to control for any extraneous effects. The findings showed a 38 percent overall reduction in rear-end crashes for vehicles fitted with AEB compared to a comparison sample of similar vehicles. There was no statistical evidence of any difference in effect between urban (≤60 km/h) and rural (>60 km/h) speed zones. Areas requiring further research were identified and widespread fitment through the vehicle fleet is recommended.

Inbreeding-related trade-offs in stress resistance in the ant Formica exsecta.

Inbred individuals and populations are predicted to suffer from inbreeding depression, especially in times of stress. Under natural conditions, organisms are exposed to more than one stressor at any one time, highlighting the importance of stress resistance traits. We studied how inbreeding- and immunity-related traits are correlated under different dietary conditions in the ant Formica exsecta. Its natural diet varies in the amount and nature of plant secondary compounds and the level of free radicals, all of which require detoxification to maintain organismal homeostasis. We found that inbreeding decreased general antibacterial activity under dietary stress, suggesting inbreeding-related physiological trade-offs.

Potential mechanisms explaining why hydrolyzed casein-based diets outclass single amino acid-based diets in the prevention of autoimmune diabetes in diabetes-prone BB rats.

BACKGROUND: It remains controversial whether avoidance of dietary diabetogenic triggers, such as cow's milk proteins, can prevent type 1 diabetes in genetically susceptible individuals. Here, different extensive casein hydrolysates (HC) and single amino acid (AA) formulations were tested for their effect on mechanisms underlying autoimmune diabetes pathogenesis in diabetes-prone BioBreeding rats. Intestinal integrity, gut microbiota composition and mucosal immune reactivity were studies to assess whether these formulations have differential effects in autoimmune diabetes prevention. METHODS: Diabetes-prone BioBreeding rats received diets in which the protein fraction was exchanged for the different hydrolysates or AA compositions, starting from weaning until the end of the experiment (d150). Diabetes development was monitored, and faecal and ileal samples were collected. Gut microbiota composition and cytokine/tight junction mRNA expression were measured by quantitative polymerase chain reaction. Cytokine levels of ileum explant cultures were measured by ELISA, and intestinal permeability was measured in vivo by lactulose-mannitol assay. RESULTS: Both HC-diet fed groups revealed remarkable reduction of diabetes incidence with the most pronounced effect in Nutramigen®-fed animals. Interestingly, AA-fed rats only showed delayed autoimmune diabetes development. Furthermore, both HC-fed groups had improved intestinal barrier function when compared with control chow or AA-fed animals. Interestingly, higher IL-10 levels were measured in ileum tissue explants from Nutramigen®-fed rats. Beneficial gut microbiota changes (increased Lactobacilli and reduced Bacteroides spp. levels) were found associated especially with HC-diet interventions. CONCLUSIONS: Casein hydrolysates were found superior to AA-mix in autoimmune diabetes prevention. This suggests the presence of specific peptides that beneficially affect mechanisms that may play a critical role in autoimmune diabetes pathogenesis.

Sick ants become unsociable.

Parasites represent a severe threat to social insects, which form high-density colonies of related individuals, and selection should favour host traits that reduce infection risk. Here, using a carpenter ant (Camponotus aethiops) and a generalist insect pathogenic fungus (Metarhizium brunneum), we show that infected ants radically change their behaviour over time to reduce the risk of colony infection. Infected individuals (i) performed less social interactions than their uninfected counterparts, (ii) did not interact with brood anymore and (iii) spent most of their time outside the nest from day 3 post-infection until death. Furthermore, infected ants displayed an increased aggressiveness towards non-nestmates. Finally, infected ants did not alter their cuticular chemical profile, suggesting that infected individuals do not signal their physiological status to nestmates. Our results provide evidence for the evolution of unsociability following pathogen infection in a social animal and suggest an important role of inclusive fitness in driving such evolution.

Case-based e-learning to improve the attitude of medical students towards occupational health, a randomised controlled trial.

OBJECTIVES: Undergraduate medical teaching in occupational health (OH) is a challenge in universities around the world. Case-based e-learning with an attractive clinical context could improve the attitude of medical students towards OH. The study question is whether case-based e-learning for medical students is more effective in improving knowledge, satisfaction and a positive attitude towards OH than non-case-based textbook learning. METHODS: Participants, 141 second year medical students, were randomised to either case-based e-learning or text-based learning. Outcome measures were knowledge, satisfaction and attitude towards OH, measured at baseline, directly after the intervention, after 1 week and at 3-month follow-up. RESULTS: Of the 141 participants, 130 (92%) completed the questionnaires at short-term follow-up and 41 (29%) at 3-month follow-up. At short-term follow-up, intervention and control groups did not show a significant difference in knowledge nor satisfaction but attitude towards OH was significantly more negative in the intervention group (F=4.041, p=0.047). At 3-month follow-up, there were no significant differences between intervention and control groups for knowledge, satisfaction and attitude. CONCLUSIONS: We found a significant decrease in favourable attitude during the internship in the experimental group compared with the control group. There were no significant differences in knowledge or satisfaction between case-based e-learning and text-based learning. The attitude towards OH should be further investigated as an outcome of educational programmes.

Restoration of impaired intestinal barrier function by the hydrolysed casein diet contributes to the prevention of type 1 diabetes in the diabetes-prone BioBreeding rat.

AIMS/HYPOTHESIS: Impaired intestinal barrier function is observed in type 1 diabetes patients and animal models of the disease. Exposure to diabetogenic antigens from the intestinal milieu due to a compromised intestinal barrier is considered essential for induction of the autoimmune process leading to type 1 diabetes. Since a hydrolysed casein (HC) diet prevents autoimmune diabetes onset in diabetes-prone (DP)-BioBreeding (BB) rats, we studied the role of the HC diet on intestinal barrier function and, therefore, prevention of autoimmune diabetes onset in this animal model. METHODS: DP-BB rats were fed the HC diet from weaning onwards and monitored for autoimmune diabetes development. Intestinal permeability was assessed in vivo by lactulose-mannitol test and ex vivo by measuring transepithelial electrical resistance (TEER). Levels of serum zonulin, a physiological tight junction modulator, were measured by ELISA. Ileal mRNA expression of Myo9b, Cldn1, Cldn2 and Ocln (which encode the tight junction-related proteins myosin IXb, claudin-1, claudin-2 and occludin) and Il-10, Tgf-ß (also known as Il10 and Tgfb, respectively, which encode regulatory cytokines) was analysed by quantitative PCR. RESULTS: The HC diet reduced autoimmune diabetes by 50% in DP-BB rats. In DP-BB rats, prediabetic gut permeability negatively correlated with the moment of autoimmune diabetes onset. The improved intestinal barrier function that was induced by HC diet in DP-BB rats was visualised by decreasing lactulose:mannitol ratio, decreasing serum zonulin levels and increasing ileal TEER. The HC diet modified ileal mRNA expression of Myo9b, and Cldn1 and Cldn2, but left Ocln expression unaltered. CONCLUSIONS/INTERPRETATION: Improved intestinal barrier function might be an important intermediate in the prevention of autoimmune diabetes by the HC diet in DP-BB rats. Effects on tight junctions, ileal cytokines and zonulin production might be important mechanisms for this effect.

Prolonged exclusive breastfeeding reduces autoimmune diabetes incidence and increases regulatory T-cell frequency in bio-breeding diabetes-prone rats.

BACKGROUND: Previously, we reported that exclusive breastfeeding delayed and partially protected bio-breeding diabetes-prone (BBDP) rats from spontaneous autoimmune diabetes development. To investigate whether this protection results from modulation of the (mucosal) immune system, the present study was designed to analyse the effect of nutrition early in life on the immune status of BBDP rats. METHODS: The breastfeeding period of BBDP pups was extended or not, while allowing half of the pups to eat during that period whereas the other half received only breast milk. Cytokine profiles as well as naturally occurring regulatory T-cell frequencies were measured over time in the mesenteric lymph nodes (MLNs) and spleen. RESULTS: Prolonged exclusive breastfeeding partially protects against autoimmune diabetes development and resulted in elevated levels of natural regulatory T cells (CD4(+) CD25(+) FoxP3(+)) in MLNs and spleen directly after weaning and throughout life. Stimulation of MLN cells from rats that ingested solid food during the nursing period showed massive secretion of interferon gamma (IFN-gamma), interleukin (IL)-4 and IL-10, whereas MLN cells from exclusive breastfed rats did not. In contrast, transforming growth factor beta (TGF-ss) was secreted equally by all groups. CONCLUSIONS: Prolonged exclusive breastfeeding partially protects BBDP rats from autoimmune diabetes development. Interestingly, ingestion of solid food during the weaning period completely abolishes this protective effect. The protective effect of exclusive breastfeeding correlates with higher levels of naturally occurring regulatory T cells throughout life and low cytokine secretion at weaning.

Staphylococcal toxic-shock-syndrome-toxin-1 as a risk factor for disease relapse in Wegener's granulomatosis.

OBJECTIVES: Nasal carriage of Staphylococcus aureus constitutes a risk factor for disease exacerbation in Wegener's granulomatosis (WG). We hypothesized that staphylococcal superantigens (SAg) are a determinant of S. aureus-related risk for disease relapse in WG. METHODS: In a retrospective longitudinal cohort study in 62 WG patients, we investigated the presence of the staphylococcal SAg genes sea, seb, sec, sed, see, tsst-1 and eta in S. aureus strains isolated from WG patients during an observation period of seven years. Subsequently, we assessed whether relapses of WG were associated with the presence of SAg-positive staphylococci. RESULTS: Of 1718 swab cultures analysed, 709 (41.2%) were S. aureus-positive. Fifty-one patients carried S. aureus, of whom 37 (72.5%) patients carried at least one SAg-positive S. aureus strain. Of the 709 S. aureus-positive cultures, 326 (46%) contained at least one SAg gene. Except for see, all assessed SAg genes were detected. sea was found most frequently, followed by sec, tsst-1 and eta and finally, by sed and seb. Using a multivariate, time-dependent Cox regression analysis we found that the presence of S. aureus was associated with relapses of WG (RR 3.2; 95% CI 1.2-8.4). The risk for relapse was modulated by the presence and type of SAg, with tsst-1 being associated with an increased risk for relapse (RR 13.3, 95% CI 4.2-42.6). CONCLUSION: The risk for relapse of WG increases with the presence of tsst-1-positive S. aureus. Eradication of tsst-1-positive S. aureus in WG may show whether disease relapses can be prevented.

Antibiotic treatment partially protects against type 1 diabetes in the Bio-Breeding diabetes-prone rat. Is the gut flora involved in the development of type 1 diabetes?

AIMS/HYPOTHESIS: Accumulating data suggest that the gut immune system plays a role in the development of type 1 diabetes. The intestinal flora is essential for the development of the (gut) immune system and the establishment of tolerance. It has been reported that oral administration of food and bacterial antigens early in life suppresses later development of diabetes in the Bio-Breeding diabetes-prone (BB-DP) rat. This study was designed to investigate the possible relationship between the development of diabetes and the composition of intestinal flora. MATERIALS AND METHODS: The intestinal flora of BB-DP rats, a rat model for type 1 diabetes, was characterised long before the clinical onset of diabetes by fluorescent in situ hybridisation. In a separate experiment, BB-DP rats were treated with antibiotics and the effect on diabetes incidence and level of insulitis was analysed. RESULTS: We observed a difference in bacterial composition between rats that eventually did and those that did not develop diabetes. This difference was detectable long before clinical onset of the disease. Rats that did not develop diabetes at a later age displayed a lower amount of Bacteroides sp. Modulation of the intestinal flora through antibiotic treatment decreased the incidence and delayed the onset of diabetes. A combination of antibiotic treatment and a protective hydrolysed casein diet completely prevented diabetes in the BB-DP rat. CONCLUSIONS/INTERPRETATION: Our data suggest that the intestinal flora is involved in the development of type 1 diabetes. Factors influencing composition of the intestinal flora could be a target for therapeutic intervention.

Neonatal oral administration of DiaPep277, combined with hydrolysed casein diet, protects against Type 1 diabetes in BB-DP rats. An experimental study.

AIMS/HYPOTHESIS: Environmental factors such as diet and bacterial antigens play an important role in the onset of Type 1 diabetes. Different self-antigens are suggested to play a role in the development of diabetes. Antibodies against the 60-kDa heat shock protein 60, which have a high homology to bacterial heat shock protein 65, have been found in the circulation at the onset of diabetes in humans and in pre-diabetic NOD-mice. One of the immunodominant epitopes in autoimmune diabetes is p277, a specific peptide of human heat shock protein 60 corresponding to positions 437-460. In this study we investigated whether neonatal oral administration of DiaPep277 (a synthetic peptide analogue of p277) affected the development of diabetes in the BioBreeding-Diabetes Prone (BB-DP) rat, and whether this could potentiate the effect of a protective hydrolysed casein-diet. METHODS: BB-DP rats were orally inoculated once per day with placebo or DiaPep277 at days 4, 5, 6 and 7 of life. At the age of 21 days rats were weaned on to a conventional, cereal-based diet or on to the hydrolysed casein-diet. RESULTS: The development of diabetes in animals receiving DiaPep277 in combination with the hydrolysed casein-diet was delayed by 17 days, and a relative reduction of the incidence by 64% was seen. Non-diabetic animals did not show any sign of insulitis. CONCLUSIONS/INTERPRETATION: Short-term neonatal feeding with p277 in early life, combined with diet adaptation, appears to provide a procedure to significantly reduce the development of Type 1 diabetes in later life.

Interactions of commensal gut microbes with subsets of B- and T-cells in the murine host.

Although mechanisms operative in the induction and maintenance of specific, adaptive immunity, including 'cognate' B/T interactions, have been extensively studied and defined, we still know little about the mechanisms operative in developing and maintaining B- and T-cell dependent 'natural' immunity. Particularly, we are still rather ignorant concerning gut microbial/gut or systemic APC, T cell and B cell interactions that lead to lymphoid cell mediated 'natural' immunity: specific or broadly reactive, activation via TCR and BCR and/or via other receptors such as the TLR series, and whether T/B interactions are operative at this level? Here we will address: (1) the general role of gut microbes in the development and maintenance of the intestinal, humoral immune system; (2) the general role of gut microbes in the development of B1 cell mediated, 'natural' gut IgA and the dependence of these B1 cells on bystander T cell help; (3) the relative contributions of B1 versus B2 cells to gut 'natural' and specific IgA responses; (4) the role for particular 'normal' gut microbes in the initiation of inflammatory bowel diseases (IBD) in mice with a dysregulated immune system; and (5) the possible roles of gut microbes in facilitating oral tolerance, a mechanism likely operative in forestalling or ameliorating IBD. A central theme of this paper is to attempt to define the specificities of activated, functional CD4+ T cells in the gut for Ags of particular, usually benign gut microbes. We will also consider the still-unresolved issue of whether the contributions of B1-derived IgA in the gut to the 'natural' Ab pool are Ag-selected and driven to proliferation/differentiation or whether the main stimuli are not via BCRs but rather other receptors (TLRs, etc.). The main experimental approach has been to use antigen-free, germ-free, or gnotobiotic (mono- or oligo-associated with precisely known bacterial species) mice.

Staphylococcal superantigens and T cell expansions in Wegener's granulomatosis.

In Wegener's granulomatosis (WG), a form of autoimmune systemic vasculitis, chronic carriage of Staphylococcus aureus constitutes a risk factor for the development of exacerbations. Circulating T cells in this disease are persistently activated, suggesting the presence of a chronic stimulus. A causal link between chronic carriage of S. aureus and chronic T cell activation in WG is conceivable, because S. aureus produces superantigens (SAg), which are potent T cell stimulators. Superantigenic stimulation of T cells results in expansion of T cell subsets expressing SAg-binding T cell receptor V-beta (Vbeta) chains. In the present study we hypothesized that in WG the presence of staphylococcal SAg is accompanied by expansion of SAg-reacting T cell subsets. We tested our hypothesis in a cross-sectional and a longitudinal study in which the association between seven staphylococcal SAg genes [typed by poplymerase chain reaction (PCR)], eight SAg-binding Vbeta chains and four SAg-non-binding Vbeta chains (assessed by flow-cytometry) was assessed. Both studies showed that T cell expansions were present at a significantly higher rate in WG patients than in healthy individuals, but were not associated with the presence of either S. aureus or its SAg. Moreover, T cell expansions were generally of small extent, and did not appear simultaneously in both CD4 and CD8 subsets. We conclude that in WG S. aureus effects its supposed pathogenic function by a mechanism other than superantigenic T cell activation.