Aberrant cell signalling in PBMCs upon IFN-α stimulation in primary Sjögren's syndrome patients associates with type I interferon signature.

Primary Sjögren's syndrome (pSS) is a complex systemic autoimmune disease with heterogeneous disease manifestations. Genetic predisposition, hormonal and environmental factors are all thought to contribute to disease etiology and pathogenesis. A better understanding of the disease pathogenesis is required in order to establish new targeted therapies. We analysed MAPK/ERK and JAK/STAT signalling networks in peripheral blood mononuclear cells (PBMCs) upon stimulation with interferon alpha 2b (IFN-α2b) by flow cytometry to define potentially dysfunctional intracellular signalling pathways involved in disease pathogenesis. Cells derived from pSS patients displayed small but significant increases in basal phosphorylation levels of numerous signalling proteins compared to cells from healthy donors. The phosphorylation profiles following stimulation with IFNα2b differed significantly between pSS patients and healthy donors, especially regarding STAT1 Y701. PCA further grouped patients according to clinical characteristics. Type I IFN induced gene expression was found to negatively correlate with the IFN-α2b induced phosphorylation of STAT3 S727 in T cells and positively with pSTAT1 Y701 in B cells. Increases in pSTAT1 Y701 were associated with the presence of autoantibodies. Our results indicate involvement of both STAT3 S727 and STAT1 Y701 pathways in pSS patients. Therapies targeting these pathways might therefore be beneficial for certain subgroups of patients.

Contrasting expression pattern of RNA-sensing receptors TLR7, RIG-I and MDA5 in interferon-positive and interferon-negative patients with primary Sjögren's syndrome.

OBJECTIVE: The interferon (IFN) type I signature is present in over half of patients with primary Sjögren's syndrome (pSS) and associated with higher disease-activity and autoantibody presence. Plasmacytoid dendritic cells (pDCs) are considered as the main source of enhanced IFN type I expression. The objective of this study was to unravel the molecular pathways underlying IFN type I bioactivity in pDCs of patients with pSS. METHODS: Blood samples from 42 healthy controls (HC) and 115 patients with pSS were stratified according to their IFN type I signature. CD123+BDCA4+ pDCs and CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs). Genome-wide microarray analysis was conducted on sorted pDCs in a small sample set, followed by validation of differentially expressed genes of interest in pDCs and monocytes. RESULTS: We found an upregulation of endosomal toll-like receptor (TLR) 7, but not TLR9, in IFN-positive (IFNpos) pDCs (p<0.05) and monocytes (p=0.024). Additionally, the downstream signalling molecules MyD88, RSAD2 and IRF7 were upregulated, as were the cytoplasmic RNA-sensing receptors DDX58/retinoic acid inducible gene-I (RIG-I) and IFIH1/melanoma differentiation associated gene-5 (MDA5). In vitro triggering of the TLR7-pathway in HC PBMCs induced upregulation of DDX58/RIG-I and IFIH1/MDA5, and downregulated TLR9. The upregulation of TLR7, its downstream signalling pathway, DDX58/RIG-I and IFIH1/MDA5 were confined to patients with IFN-positive pSS. IFN-negative patients had a contrasting expression pattern-TLR7 normal, and decreased TLR9, RIG-I and MDA5. CONCLUSIONS: Here we conclude a contrasting expression pattern of the RNA-sensing receptors TLR7, RIG-I and MDA5 in pDCs and monocytes of patients with IFNpos pSS. This profile could explain the pathogenic IFN production and might reveal novel therapeutic targets in these patients.

The 2011 Nobel Prize in physiology or medicine.

The 2011 Noble Prize in Physiology or Medicine was awarded to Bruce A. Beutler, Jules A. Hoffmann and Ralph M. Steinman for their groundbreaking research within immunology. Bruce A. Beutler and Jules A. Hoffmann were recognized for their discoveries on Toll and Toll-like receptor activation of innate immunity in fruit fly and mammals, respectively. Ralph M. Steinman received the award for the discovery of dendritic cells, a cell type bridging innate and adaptive immunity, and how these cells orchestrate immune responses.

Pituitary Gonadotropin Gene Expression During Induced Onset of Postsmolt Maturation in Male Atlantic Salmon: In Vivo and Tissue Culture Studies.

Precocious male maturation causes reduced welfare and increased production costs in Atlantic salmon (Salmo salar) aquaculture. The pituitary produces and releases follicle-stimulating hormone (Fsh), the gonadotropin triggering puberty in male salmonids. However, little is known about how Fsh production is regulated in Atlantic salmon. We examined, in vivo and ex vivo, transcriptional changes of gonadotropin-related genes accompanying the initial steps of testis maturation, in pituitaries of males exposed to photoperiod and temperature conditions promoting maturation (constant light and 16°C). Pituitary fshb, lhb and gnrhr2bba transcripts increased in vivo in maturing males (gonado-somatic index > 0.1%). RNA sequencing (RNAseq) analysis using pituitaries from genetically similar males carrying the same genetic predisposition to mature, but differing by responding or not responding to stimulatory environmental conditions, revealed 144 differentially expressed genes, ~2/3rds being up-regulated in responders, including fshb and other pituitary hormones, steroid-related and other puberty-associated transcripts. Functional enrichment analyses confirmed gene involvement in hormone/steroid production and gonad development. In ex vivo studies, whole pituitaries were exposed to a selection of hormones and growth factors. Gonadotropin-releasing hormone (Gnrh), 17ß-estradiol (E2) and 11-ketotestosterone (11-KT) up-regulated gnrhr2bba and lhb, while fshb was up-regulated by Gnrh but down-regulated by 11-KT in pituitaries from immature males. Also pituitaries from maturing males responded to Gnrh and sex steroids by increased gnrhr2bba and lhb transcript levels, but fshb expression remained unchanged. Growth factors (inhibin A, activin A and insulin-like growth factor 1) did not change gnrhr2bba, lhb or fshb transcript levels in pituitaries either from immature or maturing males. Additional pituitary ex vivo studies on candidates identified by RNAseq showed that these transcripts were preferentially regulated by Gnrh and sex steroids, but not by growth factors, and that Gnrh/sex steroids were less effective when incubating pituitaries from maturing males. Our results suggest that a yet to be characterized mechanism up-regulating fshb expression in the salmon pituitary is activated in response to stimulatory environmental conditions prior to morphological signs of testis maturation, and that the transcriptional program associated with this mechanism becomes unresponsive or less responsive to most stimulators ex vivo once males had entered pubertal developmental in vivo.

A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis.

The dsRNA-binding protein Staufen was the first RNA-binding protein proven to play a role in RNA localization in Drosophila. A mammalian homolog, Staufen1 (Stau1), has been implicated in dendritic RNA localization in neurons, translational control, and mRNA decay. However, the precise mechanisms by which it fulfills these specific roles are only partially understood. To determine its physiological functions, the murine Stau1 gene was disrupted by homologous recombination. Homozygous stau1(tm1Apa) mutant mice express a truncated Stau1 protein lacking the functional RNA-binding domain 3. The level of the truncated protein is significantly reduced. Cultured hippocampal neurons derived from stau1(tm1Apa) homozygous mice display deficits in dendritic delivery of Stau1-EYFP and beta-actin mRNA-containing ribonucleoprotein particles (RNPs). Furthermore, these neurons have a significantly reduced dendritic tree and develop fewer synapses. Homozygous stau1(tm1Apa) mutant mice are viable and show no obvious deficits in development, fertility, health, overall brain morphology, and a variety of behavioral assays, e.g., hippocampus-dependent learning. However, we did detect deficits in locomotor activity. Our data suggest that Stau1 is crucial for synapse development in vitro but not critical for normal behavioral function.

Comparison of anadromous and landlocked Atlantic salmon genomes reveals signatures of parallel and relaxed selection across the Northern Hemisphere.

Most Atlantic salmon (Salmo salar L.) populations follow an anadromous life cycle, spending early life in freshwater, migrating to the sea for feeding, and returning to rivers to spawn. At the end of the last ice age ~10,000 years ago, several populations of Atlantic salmon became landlocked. Comparing their genomes to their anadromous counterparts can help identify genetic variation related to either freshwater residency or anadromy. The objective of this study was to identify consistently divergent loci between anadromous and landlocked Atlantic salmon strains throughout their geographical distribution, with the long-term aim of identifying traits relevant for salmon aquaculture, including fresh and seawater growth, omega-3 metabolism, smoltification, and disease resistance. We used a Pool-seq approach (n = 10-40 individuals per population) to sequence the genomes of twelve anadromous and six landlocked Atlantic salmon populations covering a large part of the Northern Hemisphere and conducted a genomewide association study to identify genomic regions having been under different selection pressure in landlocked and anadromous strains. A total of 28 genomic regions were identified and included cadm1 on Chr 13 and ppargc1a on Chr 18. Seven of the regions additionally displayed consistently reduced heterozygosity in fish obtained from landlocked populations, including the genes gpr132, cdca4, and sertad2 on Chr 15. We also found 16 regions, including igf1 on Chr 17, which consistently display reduced heterozygosity in the anadromous populations compared to the freshwater populations, indicating relaxed selection on traits associated with anadromy in landlocked salmon. In conclusion, we have identified 37 regions which may harbor genetic variation relevant for improving fish welfare and quality in the salmon farming industry and for understanding life-history traits in fish.

Levels of plasmacytoid dendritic cells and type-2 myeloid dendritic cells are reduced in peripheral blood of patients with primary Sjogren's syndrome.

OBJECTIVE: Sjögren's syndrome (SS) is a lymphoproliferative autoimmune disease, characterised by dryness of the mouth and eyes. Dendritic cells (DC) are potent antigen-presenting cells crucial for initiating and maintaining primary immune responses. This study quantified interferon-producing plasmacytoid DC (pDC) and two myeloid DC subsets (mDC1 and mDC2) in peripheral blood (PB) from primary SS (pSS) patients and healthy controls. METHODS: Blood samples from 31 pSS patients and 28 gender and age-matched healthy controls were analysed by flow cytometry using the Miltenyi Blood DC enumeration kit. The presence of pDC in salivary glands (SG) from pSS patients was analysed by immunohistochemistry. RESULTS: Patients with pSS had significantly less pDC and mDC2 in PB compared with healthy controls. Moreover, pDC are present in SG from patients with pSS. CONCLUSION: Patients with pSS have alterations among DC populations in PB, and pDC are present in the SG, suggesting a potential role of these cells in SS.

Levels of dendritic cell populations and regulatory T cells vary significantly between two commonly used mouse strains.

Dendritic cells (DC) are a heterogeneous group of professional antigen-presenting cells (APC) involved in both initiating immune responses and maintaining tolerance. Roughly, DC can be divided into plasmacytoid DC (pDC) and conventional DC (cDC). By controlling regulatory T cells (Treg), DC can influence the outcome of both immunity and autoimmunity. Since the use of mice as in vivo models became a practical tool for researchers studying pathological events in all kind of human diseases, we decided to compare levels of cDC, pDC and Treg in both spleen and blood between two inbred mouse strains. Here we show that two commonly used mouse strains, BALB/c and C57BL/10J mice, have significantly different levels of distinct CD11c(+)/CD4(-)/CD8a(+), CD11c(+)/CD4(+)/CD8a(-) and CD11c(+)/CD4(-)/CD8a(-) cDC populations, pDC and Treg. Therefore, we emphasize the importance of considering the proper model when comparing data sets from different mouse strains.

Single Cell Based Phosphorylation Profiling Identifies Alterations in Toll-Like Receptor 7 and 9 Signaling in Patients With Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is associated with polymorphisms and mRNA expression profiles that are indicative of an exaggerated innate and type I IFN immune response. Excessive activation potential of signaling pathways may play a role in this profile, but the intracellular signaling profile of the disease is not well characterized. To gain insights into potentially dysfunctional intracellular signaling profiles of pSS patients we conducted an exploratory analysis of MAPK/ERK and JAK/STAT signaling networks in peripheral blood mononuclear cells (PBMC) from 25 female pSS patients and 25 female age-matched healthy donors using phospho-specific flow cytometry. We analyzed unstimulated samples, as well as samples during a 4 h time period following activation of Toll-like receptor (TLR) 7 and 9. Expression levels of MxA, IFI44, OAS1, GBP1, and GBP2 in PBMC were analyzed by real-time PCR. Cytokine levels in plasma were determined using a 25-plex Luminex-assay. Principal component analysis (PCA) showed that basal phosphorylation profiles could be used to differentiate pSS patients from healthy donor samples by stronger intracellular signaling pathway activation in NK and T cells relative to B cells. Stimulation of PBMC with TLR7 and -9 ligands showed significant differences in the phosphorylation profiles between samples from pSS patients and healthy donors. Including clinical parameters such as extraglandular manifestations (EGM), we observed stronger responses of NF-κB and STAT3 S727 in B cells from EGM-negative patients compared to EGM-positive patients and healthy controls. Plasma cytokine levels were correlated to the basal phosphorylation levels in these patients. In addition, 70% of the patients had a positive IFN score. These patients differed from the IFN score negative patients regarding their phosphorylation profiles and their plasma cytokine levels. In conclusion, we here report increased signaling potentials in peripheral B cells of pSS patients in response to TLR7 and -9 stimulation through STAT3 S727 and NF-κB that correlate with a type I IFN signature. Induction of these pathways could contribute to the generation of a type I IFN signature in pSS. Patients displaying elevated potentiation of STAT3 S727 and NF-κB signaling could therefore benefit from therapies targeting these pathways.

Reduced glucose transporter-1 in brain derived circulating endothelial cells in mild Alzheimer's disease patients.

Patients with Alzheimer's disease (AD) have blood-brain barrier (BBB) dysfunction. Methods to study cells of the BBB in vivo would facilitate analyses of neurovascular damage in early AD. Thus, we conducted a pilot study to investigate if brain-derived endothelial cells (BDCECs) could be identified from a cell population of circulating endothelial cells (CECs). Peripheral blood was sampled from early AD patients (n = 9), patients with vascular diseases (myocardial infarction (n = 8) and ischemic stroke (n = 8)), and healthy controls (n = 8). We enumerated CD34+/CD146+/CD45- cells (CECs) and Glucose transporter-1 (Glut1+ CECs (BDCECs)) by flow cytometry. We found that BDCECs formed a separate, aggregate cell population. Glut1 expression on BDCECs, measured by the median fluorescence intensity, was significantly decreased in patients with AD compared to both the healthy controls and patients with myocardial infarction ((p < .05, Kruskal-Wallis, Dunn's post hoc test). We found no significant differences in cell numbers. Our study shows that isolation of BDCECs offers a promising non-invasive tool to investigate cells derived from the BBB. Downregulation of Glut1 at the mild stages of AD suggests that agents that increase Glut1 levels may be therapeutic candidates to improve energy availability to the brain.

Antibodies to Signaling Molecules and Receptors in Alzheimer's Disease are Associated with Psychomotor Slowing, Depression, and Poor Visuospatial Function.

BACKGROUND: Alzheimer's disease (AD) is associated with several antibodies as well as signaling molecules and receptors. These may be detrimental in the presence of a disrupted blood-brain barrier (BBB). OBJECTIVE: To investigate whether the levels of antibodies toward 33 signaling molecules involved in neurotransmitter, vascular, and immune functions were associated with AD and, within the AD group; cognitive function and mood. METHODS: Antibodies in sera from patients with mild AD [(n = 91) defined as a Mini-Mental State Examination ≥ 20 or a Clinical Dementia Rating Scale≤1] and healthy controls (n = 102) were measured with enzyme-linked immunosorbent assays. Levels in AD and controls were compared by Mann-Whitney test. In the AD group, associations between antibodies and psychometric test scores were analyzed by robust regression. The false discovery threshold was set to 0.05. RESULTS: Antibodies to serotonin receptors [5-HT2AR (effect size (r) = 0.21, p = 0.004), 5-HT2CR (r = 0.25, p = 0.0005) and 5-HT7R (r = 0.21, p = 0.003)], vascular endothelial growth factor receptor 1 [VEGFR1 (r = 0.29, p < 0.001)] and immune-receptors (Stabilin-1 (r = 0.23, p = 0.001) and C5aR1 (r = 0.21, p = 0.004) were higher in AD. Psychomotor speed was associated with D1R-abs (ß 0.49, p < 0.001), depression with ETAR-abs (ß 0.31, p < 0.001), and visuospatial function with 5-HT1AR-abs (ß 0.27, p = 0.004) despite similar antibody levels compared to controls. CONCLUSIONS: Antibody levels to VEGFR1, serotonergic receptors, and receptors in the immune system were increased in AD. Antibodies at similar levels as in controls were associated cognitive dysfunction and depression in AD.

An optimized multiplex flow cytometry protocol for the analysis of intracellular signaling in peripheral blood mononuclear cells.

Phosphoflow cytometry is increasingly being used as a tool for the discovery of biomarkers used in the treatment and monitoring of disease and therapy. The ability to measure numerous phospho-protein targets simultaneously at a single cell level accurately and rapidly provides significant advantages over other methods. We here discuss important considerations required to successfully implement these methods. Three different blood collection tubes (lithium-heparin tubes, CPT with sodium citrate and CPT with sodium heparin) were evaluated, with PBMC isolated through lithium-heparin tubes/lymphoprep displaying reduced basal and increased stimulation induced phosphorylation compared to the other two methods. Further, we provide a protocol outlining an 8 color assay developed for the study of intracellular signaling in peripheral blood mononuclear cells. The assay allows for the quantitative measurement of the phospho-proteins ERK1/2, NF-κB p65, Stat1 (Y701), Stat1 (S727), Stat3 (Y705), Stat3 (S727), Stat4 (Y693), p38 and Stat5 (Y694), as well as the identification of T cells, B cells, natural killer cells and monocytes. The assay additionally incorporates fluorescent cell barcoding, reducing assay costs and increasing throughput while increasing data robustness. Inter-assay precision was assessed over a month long period for all experimental variables (phospho-protein measured, cell type and stimulant). Coefficient of variations (CVs) calculated from process triplicates of normalized median fluorescence intensity (MFI) of the phospho-proteins displayed median CVs under 10% when grouped according to cell type, stimulation agent and phospho-protein measured, while the CV for each triplicate did not exceed 20%.

The clinical relevance of animal models in Sjögren's syndrome: the interferon signature from mouse to man.

Mouse models have been widely used to elucidate the pathogenic mechanisms of human diseases. The advantages of using these models include the ability to study different stages of the disease with particular respect to specific target organs, to focus on the role of specific pathogenic factors and to investigate the effect of possible therapeutic interventions. Sjögren's syndrome (SS) is a systemic autoimmune disease, characterised by lymphocytic infiltrates in the salivary and lacrimal glands. To date, effective therapy is not available and treatment has been mainly symptomatic. Ongoing studies in murine models are aimed at developing more effective and targeted therapies in SS. The heterogeneity of SS will most probably benefit from optimising therapies, tailored to specific subgroups of the disease. In this review, we provide our perspective on the importance of subdividing SS patients according to their interferon signature, and recommend choosing appropriate mouse models for interferon-positive and interferon-negative SS subtypes. Murine models better resembling human-disease phenotypes will be essential in this endeavour.

Altered phenotype and Stat1 expression in Toll-like receptor 7/8 stimulated monocyte-derived dendritic cells from patients with primary Sjögren's syndrome.

INTRODUCTION: Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system, involved in both initiating immune responses and maintaining tolerance. Dysfunctional and via toll-like receptor (TLR) ligands activated DC have been implicated in the development of autoimmune diseases, but their role in the etiology of Sjögren's syndrome, a chronic inflammatory autoimmune disease characterized by progressive mononuclear cell infiltration in the exocrine glands, has not been revealed yet. Therefore, the aim of this study was to investigate phenotype and functional properties of immature and TLR7/8 stimulated monocyte-derived DC (moDC) of patients with primary Sjögren's syndrome (pSS) and compare them to healthy controls. METHODS: The phenotype, apoptosis susceptibility and endocytic capacity of moDC were analyzed by flow cytometry. Secretion of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and multiplex Luminex analyses in moDC cell culture supernatants. The expression of TLR7 was analyzed by flow cytometry and real-time quantitative polymerase chain reaction (qPCR). Expression of Ro/Sjögren's syndrome-associated autoantigen A (Ro52/SSA), interferon regulatory factor 8 (IRF-8), Bim, signal transduction and activators of transcription (Stat) 1, p-Stat1 (Tyrosin 701), p-Stat1 (Serin 727), Stat3, pStat3 (Tyrosin 705) and glyceraldehyde 3-phosphatase dehydrogenase (GAPDH) was measured by Western blotting. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) family members were quantified using the ELISA-based TransAM NF-κB family kit. RESULTS: We could not detect differences in expression of co-stimulatory molecules and maturation markers such as cluster of differentiation (CD) 86, CD80, CD40 or CD83 on moDC from patients compared to healthy controls. Moreover, we could not observe variations in apoptosis susceptibility, Bim and Ro52/SSA expression and the endocytic capacity of the moDC. However, we found that moDC from pSS patients expressed increased levels of the major histocompatibility complex (MHC) class II molecule human leukocyte antigen (HLA)-DR. We also found significant differences in cytokine production by moDC, where increased interleukin (IL)-12p40 secretion in mature pSS moDC correlated with increased RelB expression. Strikingly, moDC from pSS patients matured for 48 hours with TLR7/8 ligand CL097 expressed significantly less Stat1. CONCLUSION: Our results suggest a role for moDC in the pathogenesis of Sjögren's syndrome.

Dendritic cell populations in patients with self-reported food hypersensitivity.

Self-reported hypersensitivity to food is a common condition and many of these patients have indications of intestinal immune activation. Dendritic cells (DCs) are recognized as the most potent antigen-presenting cells involved in both initiating immune responses and maintaining tolerance. The aims of this study were to evaluate the DC populations with their phenotype and T cell stimulatory capacity in patients with food hypersensitivity and to study its relationship with atopic disease. Blood samples from 10 patients with self-reported food hypersensitivity, divided into atopic and nonatopic subgroups, and 10 gender- and age-matched healthy controls were analyzed by flow cytometry using the Miltenyi Blood Dendritic cells kit. Monocyte-derived DCs (moDCs) were evaluated concerning their phenotype and T cell stimulatory capacity. DC populations and cell surface markers were not significantly different between patients and healthy controls, but moDCs from atopic patients expressed significantly more CD38 compared to moDCs from nonatopic patients. Moreover, lipopolysaccharide stimulated moDCs from atopic patients produced significantly more interleukin-10 compared to nonatopic patients. CD38 expression was correlated to total serum immunoglobulin E levels. These findings support the notion of immune activation in some patients with self-reported food hypersensitivity. They need to be confirmed in a larger cohort.

The complexity of Sjögren's syndrome: novel aspects on pathogenesis.

In Sjögren's syndrome, like in most other autoimmune diseases, the enigma leading to a pathogenic attack against self has not yet been solved. By definition, the disease must be mediated by specific immune reactions against endogenous tissues to qualify as an autoimmune disease. In Sjögren's syndrome the autoimmune response is directed against the exocrine glands, which, as histopathological hallmark of the disease, display persistent and progressive focal mononuclear cell infiltrates. Clinically, the disease in most patients is manifested by two severe symptoms: dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). A number of systemic features have also been described and the presence of autoantibodies against the ubiquitously expressed ribonucleoprotein particles Ro (Sjögren's-syndrome-related antigen A - SSA) and La (SSB) further underline the systemic nature of Sjögren's syndrome. The original explanatory concept for the pathogenesis of Sjögren's syndrome proposed a specific, self-perpetuating, immune mediated loss of acinar and ductal cells as the principal cause of salivary gland hypofunction. Although straightforward and plausible, the hypothesis, however, falls short of accommodating several Sjögren's syndrome-related phenomena and experimental findings. Consequently, researchers considered immune-mediated salivary gland dysfunction prior to glandular destruction and atrophy as potential molecular mechanisms underlying the symptoms of dryness in Sjögren's syndrome. Accordingly, apoptosis, fibrosis and atrophy of the salivary glands would represent consequences of salivary gland hypofunction. The emergence of advanced bio-analytical platforms further enabled the identification of potential biomarkers with the intent to improve Sjögren's syndrome diagnosis, promote the development of prognostic tools for Sjögren's syndrome and the long-term goal to identify possible processes for therapeutic treatment interventions. In addition, such approaches allowed us to glimpse at the apparent complexity of Sjögren's syndrome.