Cytometry profiling of ex vivo recall responses to Coxiella burnetii in previously naturally exposed individuals reveals long-term changes in both adaptive and innate immune cellular compartments.

Introduction: Q fever, caused by the intracellular bacterium Coxiella burnetii, is considered an occupational and biodefense hazard and can result in debilitating long-term complications. While natural infection and vaccination induce humoral and cellular immune responses, the exact nature of cellular immune responses to C. burnetii is incompletely understood. The current study seeks to investigate more deeply the nature of long-term cellular recall responses in naturally exposed individuals by both cytokine release assessment and cytometry profiling. Methods: Individuals exposed during the 2007-2010 Dutch Q fever outbreak were grouped in 2015, based on a C. burnetii-specific IFNγ release assay (IGRA), serological status, and self-reported clinical symptoms during initial infection, into asymptomatic IGRA-negative/seronegative controls, and three IGRA-positive groups (seronegative/asymptomatic; seropositive/asymptomatic and seropositive/symptomatic). Recall responses following in vitro re-stimulation with heat-inactivated C. burnetii in whole blood, were assessed in 2016/2017 by cytokine release assays (n=55) and flow cytometry (n=36), and in blood mononuclear cells by mass cytometry (n=36). Results: Cytokine release analysis showed significantly elevated IL-2 responses in all seropositive individuals and elevated IL-1ß responses in those recovered from symptomatic infection. Comparative flow cytometry analysis revealed significantly increased IFNγ, TNFα and IL-2 recall responses by CD4 T cells and higher IL-6 production by monocytes from symptomatic, IGRA-positive/seropositive individuals compared to controls. Mass cytometry profiling and unsupervised clustering analysis confirmed recall responses in seropositive individuals by two activated CD4 T cell subsets, one characterized by a strong Th1 cytokine profile (IFNγ+IL-2+TNFα+), and identified C. burnetii-specific activation of CD8 T cells in all IGRA-positive groups. Remarkably, increased C. burnetii-specific responses in IGRA-positive individuals were also observed in three innate cell subpopulations: one characterized by an IFNγ+IL-2+TNFα+ Th1 cytokine profile and lack of canonical marker expression, and two IL-1ß-, IL-6- and IL-8-producing CD14+ monocyte subsets that could be the drivers of elevated secretion of innate cytokines in pre-exposed individuals. Discussion: These data highlight that there are long-term increased responses to C. burnetii in both adaptive and innate cellular compartments, the latter being indicative of trained immunity. These findings warrant future studies into the protective role of these innate responses and may inform future Q fever vaccine design.

Natural Exposure- and Vaccination-Induced Profiles of Ex Vivo Whole Blood Cytokine Responses to Coxiella burnetii.

Q fever is a zoonotic disease caused by the highly infectious Gram-negative coccobacillus, Coxiella burnetii (C. burnetii). The Q fever vaccine Q-VAX® is characterised by high reactogenicity, requiring individuals to be pre-screened for prior exposure before vaccination. To date it remains unclear whether vaccine side effects in pre-exposed individuals are associated with pre-existing adaptive immune responses to C. burnetii or are also a function of innate responses to Q-VAX®. In the current study, we measured innate and adaptive cytokine responses to C. burnetii and compared these among individuals with different pre-exposure status. Three groups were included: n=98 Dutch blood bank donors with unknown exposure status, n=95 Dutch village inhabitants with known natural exposure status to C. burnetii during the Dutch Q fever outbreak of 2007-2010, and n=96 Australian students receiving Q-VAX® vaccination in 2021. Whole blood cytokine responses following ex vivo stimulation with heat-killed C. burnetii were assessed for IFNγ, IL-2, IL-6, IL-10, TNFα, IL-1ß, IP-10, MIP-1α and IL-8. Serological data were collected for all three cohorts, as well as data on skin test and self-reported vaccine side effects and clinical symptoms during past infection. IFNγ, IP-10 and IL-2 responses were strongly elevated in individuals with prior C. burnetii antigen exposure, whether through infection or vaccination, while IL-1ß, IL-6 and TNFα responses were slightly increased in naturally exposed individuals only. High dimensional analysis of the cytokine data identified four clusters of individuals with distinct cytokine response signatures. The cluster with the highest levels of adaptive cytokines and antibodies comprised solely individuals with prior exposure to C. burnetii, while another cluster was characterized by high innate cytokine production and an absence of C. burnetii-induced IP-10 production paired with high baseline IP-10 levels. Prior exposure status was partially associated with these signatures, but could not be clearly assigned to a single cytokine response signature. Overall, Q-VAX® vaccination and natural C. burnetii infection were associated with comparable cytokine response signatures, largely driven by adaptive cytokine responses. Neither individual innate and adaptive cytokine responses nor response signatures were associated retrospectively with clinical symptoms during infection or prospectively with side effects post-vaccination.

Asymmetric Mach-Zehnder Interferometric Biosensing for Quantitative and Sensitive Multiplex Detection of Anti-SARS-CoV-2 Antibodies in Human Plasma.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has once more emphasized the urgent need for accurate and fast point-of-care (POC) diagnostics for outbreak control and prevention. The main challenge in the development of POC in vitro diagnostics (IVD) is to combine a short time to result with a high sensitivity, and to keep the testing cost-effective. In this respect, sensors based on photonic integrated circuits (PICs) may offer advantages as they have features such as a high analytical sensitivity, capability for multiplexing, ease of miniaturization, and the potential for high-volume manufacturing. One special type of PIC sensor is the asymmetric Mach-Zehnder Interferometer (aMZI), which is characterized by a high and tunable analytical sensitivity. The current work describes the application of an aMZI-based biosensor platform for sensitive and multiplex detection of anti-SARS-CoV-2 antibodies in human plasma samples using the spike protein (SP), the receptor-binding domain (RBD), and the nucleocapsid protein (NP) as target antigens. The results are in good agreement with several CE-IVD marked reference methods and demonstrate the potential of the aMZI biosensor technology for further development into a photonic IVD platform.

Evaluation of a Human T Cell-Targeted Multi-Epitope Vaccine for Q Fever in Animal Models of Coxiella burnetii Immunity.

T cell-mediated immunity plays a central role in the control and clearance of intracellular Coxiella burnetii infection, which can cause Q fever. Therefore, we aimed to develop a novel T cell-targeted vaccine that induces pathogen-specific cell-mediated immunity to protect against Q fever in humans while avoiding the reactogenicity of the current inactivated whole cell vaccine. Human HLA class II T cell epitopes from C. burnetii were previously identified and selected by immunoinformatic predictions of HLA binding, conservation in multiple C. burnetii isolates, and low potential for cross-reactivity with the human proteome or microbiome. Epitopes were selected for vaccine inclusion based on long-lived human T cell recall responses to corresponding peptides in individuals that had been naturally exposed to the bacterium during a 2007-2010 Q fever outbreak in the Netherlands. Multiple viral vector-based candidate vaccines were generated that express concatemers of selected epitope sequences arranged to minimize potential junctional neo-epitopes. The vaccine candidates caused no antigen-specific reactogenicity in a sensitized guinea pig model. A subset of the vaccine epitope peptides elicited antigenic recall responses in splenocytes from C57BL/6 mice previously infected with C. burnetii. However, immunogenicity of the vaccine candidates in C57BL/6 mice was dominated by a single epitope and this was insufficient to confer protection against an infection challenge, highlighting the limitations of assessing human-targeted vaccine candidates in murine models. The viral vector-based vaccine candidates induced antigen-specific T cell responses to a broader array of epitopes in cynomolgus macaques, establishing a foundation for future vaccine efficacy studies in this large animal model of C. burnetii infection.

Whole Blood Interferon γ Release Is a More Sensitive Marker of Prior Exposure to Coxiella burnetii Than Are Antibody Responses.

For the zoonotic disease Q fever, serological analysis plays a dominant role in the diagnosis of Coxiella burnetii infection and in pre-screening for past exposure prior to vaccination. A number of studies suggest that assessment of C. burnetii-specific T-cell IFNγ responses may be a more sensitive tool to assess past exposure. In this study, we assessed the performance of a whole blood C. burnetii IFNγ release assay in comparison to serological detection in an area of high Q fever incidence in 2014, up to seven years after initial exposure during the Dutch Q fever outbreak 2007-2010. In a cohort of >1500 individuals from the Dutch outbreak village of Herpen, approximately 60% had mounted IFNγ responses to C. burnetii. This proportion was independent of the Coxiella strain used for stimulation and much higher than the proportion of individuals scored sero-positive using the serological gold standard immunofluorescence assay. Moreover, C. burnetii-specific IFNγ responses were found to be more durable than antibody responses in two sub-groups of individuals known to have sero-converted as of 2007 or previously reported to the municipality as notified Q fever cases. A novel ready-to-use version of the IFNγ release assay assessed in a subgroup of pre-exposed individuals in 2021 (10-14 years post exposure) proved again to be more sensitive than serology in detecting past exposure. These data demonstrate that C. burnetii-induced IFNγ release is indeed a more sensitive and durable marker of exposure to C. burnetii than are serological responses. In combination with a simplified assay version suitable for implementation in routine diagnostic settings, this makes the assessment of IFNγ responses a valuable tool for exposure screening to obtain epidemiological data, and to identify previously exposed individuals in pre-vaccination screens.

Two-tiered SARS-CoV-2 seroconversion screening in the Netherlands and stability of nucleocapsid, spike protein domain 1 and neutralizing antibodies.

BACKGROUND: Serological testing in the COVID-19 pandemic is mainly implemented to gain sero-epidemiological data, but can also retrospectively inform about suspected SARS-CoV-2 infection. METHOD: We verified and applied a two-tiered testing strategy combining a SARS-CoV-2 receptor-binding domain (RBD)-specific lateral flow assay (LFA) with a nucleocapsid protein (NCP) IgG ELISA to assess seroconversion in n = 7241 individuals. The majority had experienced symptoms consistent with COVID-19, but had no access to RT-PCR testing. Longitudinal follow-up in n = 97 LFA + individuals was performed up to 20 weeks after initial infection using NCP and spike protein S1 domain (S1) IgG ELISAs and a surrogate virus neutralization test (sVNT). RESULTS: Individuals reporting symptoms from January 2020 onwards showed seroconversion, as did a considerable proportion of asymptomatic individuals. Seroconversion for symptomatic and asymptomatic individuals was higher in an area with a known infection cluster compared to a low incidence area. Overall, 94% of individuals with a positive IgG result by LFA were confirmed by NCP ELISA. The proportion of ELISA-confirmed LFA results declined over time, in line with contracting NCP IgG titres during longitudinal follow-up. Neutralizing antibody activity was considerably more stable than S1 and NCP IgG titres, and both reach a plateau after approximately 100 d. The sVNT proved to be not only highly specific, but also more sensitive than the specificity-focussed two-tiered serology approach. CONCLUSIONS: Our results demonstrate the high specificity of two-tiered serology testing and highlight the sVNT used as a valuable tool to support modelling of SARS-CoV-2 transmission dynamics, complement molecular testing and provide relevant information to individuals.

Evaluation of pathogen specific urinary peptides in tick-borne illnesses.

Mass spectrometry enhanced by nanotechnology can achieve previously unattainable sensitivity for characterizing urinary pathogen-derived peptides. We utilized mass spectrometry enhanced by affinity hydrogel particles (analytical sensitivity = 2.5 pg/mL) to study tick pathogen-specific proteins shed in the urine of patients with (1) erythema migrans rash and acute symptoms, (2) post treatment Lyme disease syndrome (PTLDS), and (3) clinical suspicion of tick-borne illnesses (TBI). Targeted pathogens were Borrelia, Babesia, Anaplasma, Rickettsia, Ehrlichia, Bartonella, Francisella, Powassan virus, tick-borne encephalitis virus, and Colorado tick fever virus. Specificity was defined by 100% amino acid sequence identity with tick-borne pathogen proteins, evolutionary taxonomic verification for related pathogens, and no identity with human or other organisms. Using a cut off of two pathogen peptides, 9/10 acute Lyme Borreliosis patients resulted positive, while we identified zero false positive in 250 controls. Two or more pathogen peptides were identified in 40% of samples from PTLDS and TBI patients (categories 2 and 3 above, n = 59/148). Collectively, 279 distinct unique tick-borne pathogen derived peptides were identified. The number of pathogen specific peptides was directly correlated with presence or absence of symptoms reported by patients (ordinal regression pseudo-R2 = 0.392, p = 0.010). Enhanced mass spectrometry is a new tool for studying tick-borne pathogen infections.

Coxiella burnetii Epitope-Specific T-Cell Responses in Patients with Chronic Q Fever.

Infection with Coxiella burnetii, the causative agent of Q fever, can result in life-threatening persistent infection. Reactogenicity hinders worldwide implementation of the only licensed human Q fever vaccine. We previously demonstrated long-lived immunoreactivity in individuals with past symptomatic and asymptomatic Coxiella infection (convalescents) to promiscuous HLA class II C. burnetii epitopes, providing the basis for a novel T-cell targeted subunit vaccine. In this study, we investigated in a cohort of 22 individuals treated for persistent infection (chronic Q fever) whether they recognize the same set of epitopes or distinct epitopes that could be candidates for a therapeutic vaccine or aid in the diagnosis of persistent infection. In cultured enzyme-linked immunosorbent spot (ELISpot) assays, individuals with chronic Q fever showed strong class II epitope-specific responses that were largely overlapping with the peptide repertoire identified previously for convalescents. Five additional peptides were recognized more frequently by chronic subjects, but there was no combination of epitopes uniquely recognized by or nonreactive in subjects with chronic Q fever. Consistent with more recent/prolonged exposure, we found, however, stronger ex vivo responses by direct ELISpot to both whole-cell C. burnetii and individual peptides in chronic patients than in convalescents. In conclusion, we have validated and expanded a previously published set of candidate epitopes for a novel T-cell targeted subunit Q fever vaccine in treated patients with chronic Q fever and demonstrated that they successfully mounted a T-cell response comparable to that of convalescents. Finally, we demonstrated that individuals treated for chronic Q fever mount a broader ex vivo response to class II epitopes than convalescents, which could be explored for diagnostic purposes.

Promiscuous Coxiella burnetii CD4 Epitope Clusters Associated With Human Recall Responses Are Candidates for a Novel T-Cell Targeted Multi-Epitope Q Fever Vaccine.

Coxiella burnetii, the causative agent of Q fever, is a Gram-negative intracellular bacterium transmitted via aerosol. Regulatory approval of the Australian whole-cell vaccine Q-VAX® in the US and Europe is hindered by reactogenicity in previously exposed individuals. The aim of this study was to identify and rationally select C. burnetii epitopes for design of a safe, effective, and less reactogenic T-cell targeted human Q fever vaccine. Immunoinformatic methods were used to predict 65 HLA class I epitopes and 50 promiscuous HLA class II C. burnetii epitope clusters, which are conserved across strains of C. burnetii. HLA binding assays confirmed 89% of class I and 75% of class II predictions, and 11 HLA class II epitopes elicited IFNγ responses following heterologous DNA/DNA/peptide/peptide prime-boost immunizations of HLA-DR3 transgenic mice. Human immune responses to the predicted epitopes were characterized in individuals naturally exposed to C. burnetii during the 2007-2010 Dutch Q fever outbreak. Subjects were divided into three groups: controls with no immunological evidence of previous infection and individuals with responses to heat-killed C. burnetii in a whole blood IFNγ release assay (IGRA) who remained asymptomatic or who experienced clinical Q fever during the outbreak. Recall responses to C. burnetii epitopes were assessed by cultured IFNγ ELISpot. While HLA class I epitope responses were sparse in this cohort, we identified 21 HLA class II epitopes that recalled T-cell IFNγ responses in 10-28% of IGRA+ subjects. IGRA+ individuals with past asymptomatic and symptomatic C. burnetii infection showed a comparable response pattern and cumulative peptide response which correlated with IGRA responses. None of the peptides elicited reactogenicity in a C. burnetii exposure-primed guinea pig model. These data demonstrate that a substantial proportion of immunoinformatically identified HLA class II epitopes show long-lived immunoreactivity in naturally infected individuals, making them desirable candidates for a novel human multi-epitope Q fever vaccine.

Standardized guinea pig model for Q fever vaccine reactogenicity.

Historically, vaccination with Coxiella burnetii whole cell vaccines has induced hypersensitivity reactions in humans and animals that have had prior exposure to the pathogen as a result of infection or vaccination. Intradermal skin testing is routinely used to evaluate exposure in humans, and guinea pig hypersensitivity models have been developed to characterize the potential for reactogenicity in vaccine candidates. Here we describe a refinement of the guinea pig model using an alternate vaccine for positive controls. An initial comparative study used viable C. burnetii to compare the routes of sensitizing exposure of guinea pigs (intranasal vs intraperitoneal), evaluation of two time points for antigen challenge (21 and 42 days) and an assessment of two routes (intradermal and subcutaneous) of challenge using the ruminant vaccine Coxevac as the antigenic control. Animals sensitized by intraperitoneal exposure exhibited slightly larger gross reactions than did those sensitized by intranasal exposure, and reactions were more pronounced when skin challenge was performed at 42 days compared to 21 days post-sensitization. The intradermal route proved to be the optimal route of reactogenicity challenge. Histopathological changes at injection sites were similar to those previously reported and a scoring system was developed to compare reactions between groups receiving vaccine by intradermal versus subcutaneous routes. Based on the comparative study, a standardized protocol for assessment of vaccine reactogenicity in intranasally-sensitized animals was tested in a larger confirmatory study. Results suggest that screens utilizing a group size of n = 3 would achieve 90% power for detecting exposure-related reactogenic responses of the magnitude induced by Coxevac using either of two outcome measures.

Human TLR10 is an anti-inflammatory pattern-recognition receptor.

Toll-like receptor (TLR)10 is the only pattern-recognition receptor without known ligand specificity and biological function. We demonstrate that TLR10 is a modulatory receptor with mainly inhibitory effects. Blocking TLR10 by antagonistic antibodies enhanced proinflammatory cytokine production, including IL-1ß, specifically after exposure to TLR2 ligands. Blocking TLR10 after stimulation of peripheral blood mononuclear cells with pam3CSK4 (Pam3Cys) led to production of 2,065 ± 106 pg/mL IL-1ß (mean ± SEM) in comparison with 1,043 ± 51 pg/mL IL-1ß after addition of nonspecific IgG antibodies. Several mechanisms mediate the modulatory effects of TLR10: on the one hand, cotransfection in human cell lines showed that TLR10 acts as an inhibitory receptor when forming heterodimers with TLR2; on the other hand, cross-linking experiments showed specific induction of the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra, 16 ± 1.7 ng/mL, mean ± SEM). After cross-linking anti-TLR10 antibody, no production of IL-1ß and other proinflammatory cytokines could be found. Furthermore, individuals bearing TLR10 polymorphisms displayed an increased capacity to produce IL-1ß, TNF-α, and IL-6 upon ligation of TLR2, in a gene-dose-dependent manner. The modulatory effects of TLR10 are complex, involving at least several mechanisms: there is competition for ligands or for the formation of heterodimer receptors with TLR2, as well as PI3K/Akt-mediated induction of the anti-inflammatory cytokine IL-1Ra. Finally, transgenic mice expressing human TLR10 produced fewer cytokines when challenged with a TLR2 agonist. In conclusion, to our knowledge we demonstrate for the first time that TLR10 is a modulatory pattern-recognition receptor with mainly inhibitory properties.

In vivo knockdown of TAK1 accelerates bone marrow proliferation/differentiation and induces systemic inflammation.

TAK1 (TGF-ß Activated Kinase 1) is a MAPK kinase kinase, which activates the p38- and JNK-MAPK and NF-κB pathways downstream of receptors such as Toll-Like-, cytokine- and T-cell and B-cell receptors. Representing such an important node in the pro-inflammatory signal-transduction network, the function of TAK1 has been studied extensively. TAK1 knock-out mice are embryonic lethal, while conditional knock-out mice demonstrated either a pro- or anti-inflammatory function. To study the function of TAK1 protein in the adult immune system, we generated and characterized a transgenic mouse expressing TAK1 shRNA under the control of a doxycycline-inducible promoter. Following treatment of TAK-1 shRNA transgenic mice with doxycycline an effective knockdown of TAK1 protein levels was observed in lymphoid organs and cells in the peritoneal cavity (>50% down regulation). TAK1 knockdown resulted in significant changes in leukocyte populations in blood, bone marrow, spleen and peritoneal cavity. Upon TAK1 knockdown mice demonstrated splenomegaly, signs of systemic inflammation (increased levels of circulating cytokines and increase in cellularity of the B-cell areas and in germinal center development in the follicles) and degenerative changes in heart, kidneys and liver. Not surprisingly, TAK1-Tg mice treated with LPS or anti-CD3 antibodies showed enhanced cytokine/chemokine secretion. Finally, analysis of progenitor cells in the bone marrow upon doxycycline treatment showed increased proliferation and differentiation of myeloid progenitor cells. Given the similarity of the phenotype with TGF-ß genetic models, our data suggest that in our model the function of TAK1 in TGF-ß signal-transduction is overruling its function in pro-inflammatory signaling.

Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling.

BACKGROUND: T lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli and pathway inhibitors. Results from these experiments were validated in a human experimental setting using whole blood and purified CD4+ Tcells. RESULTS: Calcium-dependent activation of T cells using CD3/CD28 and PMA/CD3 stimulation induced a Th1 expression profile reflected by increased expression of T-bet, RUNX3, IL-2, and IFNγ, whereas calcium-independent activation via PMA/CD28 induced a Th2 expression profile which included GATA3, RXRA, CCL1 and Itk. Knock down with siRNA and gene expression profiling in the presence of selective kinase inhibitors showed that proximal kinases Lck and PKCθ are crucial signaling hubs during T helper cell activation, revealing a clear role for Lck in Th1 development and for PKCθ in both Th1 and Th2 development. Medial signaling via MAPkinases appeared to be less important in these pathways, since specific inhibitors of these kinases displayed a minor effect on gene expression. Translation towards a primary, whole blood setting and purified human CD4+ T cells revealed that PMA/CD3 stimulation induced a more pronounced Th1 specific, Lck and PKCθ dependent IFNγ production, whereas PMA/CD28 induced Th2 specific IL-5 and IL-13 production, independent of Lck activation. PMA/CD3-mediated skewing towards a Th1 phenotype was also reflected in mRNA expression of the master transcription factor Tbet, whereas PMA/CD28-mediated stimulation enhanced GATA3 mRNA expression in primary human CD4+ Tcells. CONCLUSIONS: This study identifies stimulatory pathways and gene expression profiles for in vitro skewing of T helper cell activation. PMA/CD3 stimulation enhances a Th1-like response in an Lck and PKCθ dependent fashion, whereas PMA/CD28 stimulation results in a Th2-like phenotype independent of the proximal TCR-tyrosine kinase Lck. This approach offers a robust and fast translational in vitro system for skewed T helper cell responses in Jurkat T cells, primary human CD4+ Tcells and in a more complex matrix such as human whole blood.

Cannabinoid Receptor 2 Deficiency in Haematopoietic cells Aggravates Early Atherosclerosis in LDL Receptor Deficient Mice.

OBJECTIVE: The cannabinoid receptor 2 (CB2) has been implicated to play a role in various inflammatory processes. Since atherosclerosis is currently considered a chronic inflammatory disease, we studied the effect of haematopoietic CB2 deficiency on atherosclerosis development. METHODS AND RESULTS: To investigate the effect of CB2 deficiency in immune cells on atherogenesis in vivo, a bone marrow transplantation was performed in irradiated LDL receptor deficient mice (LDLr(-/-)), using CB2 deficient (CB2(-/-)) or wildtype (WT) donor mice. After 12 weeks on a high fat-high cholesterol diet, en face analysis showed that atherosclerosis in the aortic arch was significantly increased in CB2(-/-) transplanted animals (6.40 ± 3.21%) as compared to WT transplanted mice (3.85 ± 1.61%). Although the total lesion area in the aortic root was not significantly different between WT and CB2(-/-) transplanted mice (0.45 ± 0.13 mm(2) and 0.51 ± 0.17 mm(2), respectively), CB2(-/-) transplanted mice showed a significantly larger plaque area (0.13 ± 0.07 mm(2)) than WT transplanted mice (0.08 ± 0.05 mm(2)) in the aortic valve in which atherogenesis is in an earlier stage than in the other aortic valves. CONCLUSIONS: Lack of endocannabinoid signaling via the CB2 receptor aggravates early atherosclerosis development in LDLr(-/-) mice, suggesting that CB2 specific activation may prevent the development of atherosclerosis.

Identification of CXCL13 as a marker for rheumatoid arthritis outcome using an in silico model of the rheumatic joint.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by inflammation and joint destruction, with the degree of damage varying greatly among patients. Prediction of disease severity using known clinical and serologic risk factors is inaccurate. This study was undertaken to identify new serologic markers for RA severity using an in silico model of the rheumatic joint. METHODS: An in silico model of a prototypical rheumatic joint was used to predict candidate markers associated with erosiveness. The following 4 markers were chosen for validation: tartrate-resistant acid phosphatase 5b (TRAP-5b), N-telopeptide of type I collagen (NTX), angiopoietin 2 (Ang-2), and CXCL13. Serum from 74 RA patients was used to study whether radiologic joint destruction (total erosion score and total Sharp/van der Heijde score [SHS]) after 4 years of disease was associated with serum levels at the time of diagnosis. Serum marker levels were determined using enzyme-linked immunosorbent assays. For confirmation, baseline serum levels were analyzed for an association with progression of joint damage over 7 years of followup in a cohort of 155 patients with early RA. RESULTS: Comparison of high and low quartiles of erosion score and SHS at 4 years showed a difference in baseline serum CXCL13 level (P = 0.011 and P = 0.018, respectively). In the confirmation cohort, elevated baseline CXCL13 levels were associated with increased rates of joint destruction during 7 years of followup (P < 0.001 unadjusted and P ≤ 0.004 with adjustment for C-reactive protein level). Analyzing anti-CCP-2-positive and anti-CCP-2­negative RA separately yielded a significant result only in the anti-CCP-2-negative group (P ≤ 0.001). CONCLUSION: Our findings indicate that CXCL13 is a novel serologic marker predictive of RA severity.This marker was identified with the help of an in silicomodel of the RA joint.

Regulatory T cells and toll-like receptors: regulating the regulators.

Regulatory T cells (Treg) play a crucial role in maintaining control of leucocytes. Several studies have shown that in vivo Treg depletion results in autoimmune syndromes like thyroiditis, gastritis, diabetes mellitus and colitis, but at the same time, may also result in improved anti-tumour vaccination. Although Treg are recognised to maintain peripheral tolerance in healthy individuals, recent research has shown that Treg also suppress immune responses during infections to prevent tissue damage. How the Treg themselves are regulated is still under investigation. Their suppressive activity must be regulated in order to allow for the effective elimination of pathogens. Until recently, this control of Treg function was found to be through modulation via cytokines or by stimulation via co-stimulatory molecules on antigen-presenting cells. It is now demonstrated, however, that the presence of pathogens can be communicated to Treg directly through toll-like receptors (TLRs). Up until now, Treg have been reported to respond to ligands for TLR2, 4, 5 and 8, and different TLRs can have alternative effects on Treg resulting in more suppression or, in contrast, abrogation of suppression. As TLRs can also recognise endogenous proteins, such as heat shock proteins, it is tempting to speculate on the role of these proteins in modulating Treg function during chronic inflammation. In this review, we will discuss the implications of TLR engagement on Treg and any consequences this may have for chronic autoinflammatory diseases like rheumatoid arthritis (RA).