CD40 activation induces NREM sleep and modulates genes associated with sleep homeostasis

The T-cell derived cytokine CD40 ligand is overexpressed in patients with autoimmune diseases. Through activation of its receptor, CD40 ligand leads to a tumor necrosis factor (TNF) receptor 1 (TNFR1) dependent impairment of locomotor activity in mice. Here we report that this effect is explained through a promotion of sleep, which was specific to non-rapid eye movement (NREM) sleep while REM sleep was suppressed. The increase in NREM sleep was accompanied by a decrease in EEG delta power during NREM sleep and by a decrease in the expression of transcripts in the cerebral cortex known to be associated with homeostatic sleep drive, such as Homer1a, Early growth response 2, Neuronal pentraxin 2, and Fos-like antigen 2. The effect of CD40 activation was mimicked by peripheral TNF injection and prevented by the TNF blocker etanercept. Our study indicates that sleep-wake dysregulation in autoimmune diseases may result from CD40 induced TNF:TNFR1 mediated alterations of molecular pathways, which regulate sleep-wake behavior.

The metabolomic window into hepatobiliary disease

The emergent discipline of metabolomics has attracted considerable research effort in hepatology. Here we review the metabolomic data for non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), alcoholic liver disease (ALD), hepatitis B and C, cholecystitis, cholestasis, liver transplantation, and acute hepatotoxicity in animal models. A metabolomic window has permitted a view into the changing biochemistry occurring in the transitional phases between a healthy liver and hepatocellular carcinoma or cholangiocarcinoma. Whether provoked by obesity and diabetes, alcohol use or oncogenic viruses, the liver develops a core metabolomic phenotype (CMP) that involves dysregulation of bile acid and phospholipid homeostasis. The CMP commences at the transition between the healthy liver (Phase 0) and NAFLD/NASH, ALD or viral hepatitis (Phase 1). This CMP is maintained in the presence or absence of cirrhosis (Phase 2) and whether or not either HCC or CCA (Phase 3) develops. Inflammatory signalling in the liver triggers the appearance of the CMP. Many other metabolomic markers distinguish between Phases 0, 1, 2 and 3. A metabolic remodelling in HCC has been described but metabolomic data from all four Phases demonstrate that the Warburg shift from mitochondrial respiration to cytosolic glycolysis foreshadows HCC and may occur as early as Phase 1. The metabolic remodelling also involves an upregulation of fatty acid β-oxidation, also beginning in Phase 1. The storage of triglycerides in fatty liver provides high energy-yielding substrates for Phases 2 and 3 of liver pathology. The metabolomic window into hepatobiliary disease sheds new light on the systems pathology of the liver.

Phenotypic and molecular characterization of hyperpigmented group B Streptococci.

Group B Streptococcus (GBS) causes invasive infections in neonates, older adults and patients with comorbidities. β-hemolysin/cytolysin is an important GBS virulence factor. It is encoded by the cyl operon and confers GBS hemolytic activity. Isolates displaying hyperpigmentation are typically hyperhemolytic. Comparison of clonally identical isolates displaying different levels of pigmentation has shown transcriptional dysregulation due to mutations in components of the control of the virulence S/R (CovS/R) regulatory system. In addition, hyperpigmented isolates show decreased CAMP factor and decreased capsule thickness. In analogy to findings in group A Streptococcus, a pivotal role of CovS/R has been proposed in the host-pathogen interaction of invasive GBS infection. However, corresponding investigations on multiple clinical GBS isolates have not been performed. We prospectively collected hyperpigmented isolates found in a diagnostic laboratory and performed phenotypic, molecular and transcriptional analyses. In the period from 2008 to 2012, we found 10 isolates obtained from 10 patients. The isolates reflected both invasive pathogens and colonizers. In three cases, clonally identical but phenotypically different variants were also found. Hence, the analyses included 13 isolates. No capsular serotype was found to be significantly more frequent. Bacterial pigments were analyzed via spectrophotometry and for their hemolytic activity. Data obtained for typical absorbance spectra peaks correlated significantly with hemolytic activity. Molecular analysis of the cyl operon showed that it was conserved in all isolates. The covR sequence displayed mutations in five isolates; in one isolate, the CovR binding site to cylX was abrogated. Our results on clinical isolates support previous findings on CovR-deficient isogenic mutants, but suggest that - at least in some clinical isolates - for β-hemolysin/cytolysin and CAMP factor production, other molecular pathways may be involved.

Double seronegative myasthenia gravis with antiphospholipid syndrome: a case report

INTRODUCTION Myasthenia gravis is an autoimmune disease characterized by fluctuating muscle weakness. It is often associated with other autoimmune disorders, such as thyroid disease, rheumatoid arthritis, systemic lupus erythematosus, and antiphospholipid syndrome. Many aspects of autoimmune diseases are not completely understood, particularly when they occur in association, which suggests a common pathogenetic mechanism. CASE PRESENTATION We report a case of a 42-year-old Caucasian woman with antiphospholipid syndrome, in whom myasthenia gravis developed years later. She tested negative for both antibodies against the acetylcholine receptor and against muscle-specific receptor tyrosine-kinase, but had typical decremental responses at the repetitive nerve stimulation testing, so that a generalized myasthenia gravis was diagnosed. Her thromboplastin time and activated partial thromboplastin time were high, anticardiolipin and anti-β2 glycoprotein-I antibodies were slightly elevated, as a manifestation of the antiphospholipid syndrome. She had a good clinical response when treated with a combination of pyridostigmine, prednisone and azathioprine. CONCLUSIONS Many patients with myasthenia gravis test positive for a large variety of auto-antibodies, testifying of an immune dysregulation, and some display mild T-cell lymphopenia associated with hypergammaglobulinemia and B-cell hyper-reactivity. Both of these mechanisms could explain the occurrence of another autoimmune condition, such as antiphospholipid syndrome, but further studies are necessary to shed light on this matter.Clinicians should be aware that patients with an autoimmune diagnosis such as antiphospholipid syndrome who develop signs and neurological symptoms suggestive of myasthenia gravis are at risk and should prompt an emergent evaluation by a specialist.

The interplay between host genetic variation, viral replication and microbial translocation in untreated HIV-infected individuals.

Systemic immune activation, a major determinant of HIV disease progression, is the result of a complex interplay between viral replication, dysregulation of the immune system, and microbial translocation due to gut mucosal damage. While human genetic variants influencing HIV viral load have been identified, it is unknown to what extent the host genetic background contributes to inter-individual differences in other determinants of HIV pathogenesis like gut damage and microbial translocation. Using samples and data from 717 untreated participants in the Swiss HIV Cohort Study and a genome-wide association study design, we searched for human genetic determinants of plasma levels of intestinal fatty-acid binding protein (I-FABP/FABP2), a marker of gut damage, and of soluble sCD14 (sCD14), a marker of LPS bioactivity and microbial translocation. We also assessed the correlations between HIV viral load, sCD14 and I-FABP. While we found no genome-wide significant determinant of the tested plasma markers, we observed strong associations between sCD14 and both HIV viral load and I-FABP, shedding new light on the relationships between processes that drive progression of untreated HIV infection.

Ribosomal and Immune Transcripts Associate with Relapse in Acquired ADAMTS13-Deficient Thrombotic Thrombocytopenic Purpura.

Approximately 40% of patients who survive acute episodes of thrombotic thrombocytopenic purpura (TTP) associated with severe acquired ADAMTS13 deficiency experience one or more relapses. Risk factors for relapse other than severe ADAMTS13 deficiency and ADAMTS13 autoantibodies are unknown. ADAMTS13 autoantibodies, TTP episodes following infection or type I interferon treatment and reported ensuing systemic lupus erythematosus in some patients suggest immune dysregulation. This cross-sectional study asked whether autoantibodies against RNA-binding proteins or peripheral blood gene expression profiles measured during remission are associated with history of prior relapse in acquired ADAMTS13-deficient TTP. Peripheral blood from 38 well-characterized patients with autoimmune ADAMTS13-deficient TTP in remission was examined for autoantibodies and global gene expression. A subset of TTP patients (9 patients, 24%) exhibited a peripheral blood gene signature composed of elevated ribosomal transcripts that associated with prior relapse. A non-overlapping subset of TTP patients (9 patients, 24%) displayed a peripheral blood type I interferon gene signature that associated with autoantibodies to RNA-binding proteins but not with history of relapse. Patients who had relapsed bimodally expressed higher HLA transcript levels independently of ribosomal transcripts. Presence of any one potential risk factor (ribosomal gene signature, elevated HLA-DRB1, elevated HLA-DRB5) associated with relapse (OR = 38.4; p = 0.0002) more closely than any factor alone or all factors together. Levels of immune transcripts typical of natural killer (NK) and T lymphocytes positively correlated with ribosomal gene expression and number of prior episodes but not with time since the most recent episode. Flow cytometry confirmed elevated expression of cell surface markers encoded by these transcripts on T and/or NK cell subsets of patients who had relapsed. These data associate elevated ribosomal and immune transcripts with relapse history in acquired, ADAMTS13-deficient TTP.

Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions

BACKGROUND Dysregulation of the coagulation system due to inflammatory responses and cross-species molecular incompatibilities represents a major obstacle to successful xenotransplantation. We hypothesized that complement inhibition mediated by transgenic expression of human CD46 in pigs might also regulate the coagulation and fibrinolysis cascades and tested this in ex vivo human-to-pig xenoperfusions. METHODS Forelimbs of wild-type and hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused for 12 hours with whole heparinized human blood. Muscle biopsies were stained for galactose-α1,3-galactose, immunoglobulin M, immunoglobulin G, complement, fibrin, tissue factor, fibrinogen-like protein 2, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI)-1. The PAI-1/tPA complexes, D-dimers, and prothrombin fragment F1 + 2 were measured in plasma samples after ex vivo xenoperfusion. RESULTS No differences of galactose expression or deposition of immunoglobulin M and immunoglobulin G were found in xenoperfused tissues of wild type and transgenic limbs. In contrast, significantly lower deposition of C5b-9 (P < 0.0001), fibrin (P = 0.009), and diminished expression of tissue factor (P = 0.005) and fibrinogen-like protein 2 (P = 0.028) were found in xenoperfused tissues of transgenic limbs. Levels of prothrombin fragment F1 + 2 (P = 0.031) and D-dimers (P = 0.044) were significantly lower in plasma samples obtained from transgenic as compared to wild-type pig limb perfusions. The expression of the fibrinolytic marker tPA was significantly higher (P = 0.009), whereas PAI-1 expression (P = 0.022) and PAI-1/tPA complexes in plasma (P = 0.015) were lower after transgenic xenoperfusion as compared to wild-type xenoperfusions. CONCLUSIONS In this human-to-pig xenoperfusion model, complement inhibition by transgenic hCD46 expression led to a significant inhibition of procoagulant and antifibrinolytic pathways.

Methylation of NR3C1 is related to maternal PTSD, parenting stress and maternal medial prefrontal cortical activity in response to child separation among mothers with histories of violence exposure.

Prior research has shown that mothers with Interpersonal violence-related posttraumatic stress disorder (IPV-PTSD) report greater difficulty in parenting their toddlers. Relative to their frequent early exposure to violence and maltreatment, these mothers display dysregulation of their hypothalamic pituitary adrenal axis (HPA-axis), characterized by hypocortisolism. Considering methylation of the promoter region of the glucocorticoid receptor gene NR3C1 as a marker for HPA-axis functioning, with less methylation likely being associated with less circulating cortisol, the present study tested the hypothesis that the degree of methylation of this gene would be negatively correlated with maternal IPV-PTSD severity and parenting stress, and positively correlated with medial prefrontal cortical (mPFC) activity in response to video-stimuli of stressful versus non-stressful mother-child interactions. Following a mental health assessment, 45 mothers and their children (ages 12-42 months) participated in a behavioral protocol involving free-play and laboratory stressors such as mother-child separation. Maternal DNA was extracted from saliva. Interactive behavior was rated on the CARE-Index. During subsequent fMRI scanning, mothers were shown films of free-play and separation drawn from this protocol. Maternal PTSD severity and parenting stress were negatively correlated with the mean percentage of methylation of NR3C1. Maternal mPFC activity in response to video-stimuli of mother-child separation versus play correlated positively to NR3C1 methylation, and negatively to maternal IPV-PTSD and parenting stress. Among interactive behavior variables, child cooperativeness in play was positively correlated with NR3C1 methylation. Thus, the present study is the first published report to our knowledge, suggesting convergence of behavioral, epigenetic, and neuroimaging data that form a psychobiological signature of parenting-risk in the context of early life stress and PTSD.

Spatial genomic heterogeneity within localized, multifocal prostate cancer

Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.

T-cell reprogramming through targeted CD4-coreceptor and T-cell receptor expression on maturing thymocytes by latent Circoviridae family member porcine circovirus type 2 cell infections in the thymus

Although porcine circovirus type 2 (PCV2)-associated diseases have been evaluated for known immune evasion strategies, the pathogenicity of these viruses remained concealed for decades. Surprisingly, the same viruses that cause panzootics in livestock are widespread in young, unaffected animals. Recently, evidence has emerged that circovirus-like viruses are also linked to complex diseases in humans, including children. We detected PCV2 genome-carrying cells in fetal pig thymi. To elucidate virus pathogenicity, we developed a new pig infection model by in vivo transfection of recombinant PCV2 and the immunosuppressant cofactor cyclosporine A. Using flow cytometry, immunofluorescence and fluorescence in situ hybridization, we found evidence that PCV2 dictates positive and negative selection of maturing T cells in the thymus. We show for the first time that PCV2-infected cells reside at the corticomedullary junction of the thymus. In diseased animals, we found polyclonal deletion of single positive cells (SPs) that may result from a loss of major histocompatibility complex class-II expression at the corticomedullary junction. The percentage of PCV2 antigen-presenting cells correlated with the degree of viremia and, in turn, the severity of the defect in thymocyte maturation. Moreover, the reversed T-cell receptor/CD4-coreceptor expression dichotomy on thymocytes at the CD4(+)CD8(interm) and CD4SP cell stage is viremia-dependent, resulting in a specific hypo-responsiveness of T-helper cells. We compare our results with the only other better-studied member of Circoviridae, chicken anemia virus. Our data show that PCV2 infection leads to thymocyte selection dysregulation, adding a valuable dimension to our understanding of virus pathogenicity.

Inactivation of the p53-KLF4-CEBPA Axis in Acute Myeloid Leukemia.

PURPOSE In acute myeloid leukemia (AML), the transcription factors CEBPA and KLF4 as well as the universal tumor suppressor p53 are frequently deregulated. Here, we investigated the extent of dysregulation, the molecular interactions, and the mechanisms involved. EXPERIMENTAL DESIGN One hundred ten AML patient samples were analyzed for protein levels of CEBPA, KLF4, p53, and p53 modulators. Regulation of CEBPA gene expression by KLF4 and p53 or by chemical p53 activators was characterized in AML cell lines. RESULTS We found that CEBPA gene transcription can be directly activated by p53 and KLF4, suggesting a p53-KLF4-CEBPA axis. In AML patient cells, we observed a prominent loss of p53 function and concomitant reduction of KLF4 and CEBPA protein levels. Assessment of cellular p53 modulator proteins indicated that p53 inactivation in leukemic cells correlated with elevated levels of the nuclear export protein XPO1/CRM1 and increase of the p53 inhibitors MDM2 and CUL9/PARC in the cytoplasm. Finally, restoring p53 function following treatment with cytotoxic chemotherapy compounds and p53 restoring non-genotoxic agents induced CEBPA gene expression, myeloid differentiation, and cell-cycle arrest in AML cells. CONCLUSIONS The p53-KLF4-CEBPA axis is deregulated in AML but can be functionally restored by conventional chemotherapy and novel p53 activating treatments. Clin Cancer Res; 22(3); 746-56. ©2015 AACR.

Neuropeptide Y restores non-receptor-mediated vasoconstrictive action in superior mesenteric arteries in portal hypertension

BACKGROUND & AIMS Vascular hyporeactivity to vasoconstrictors contributes to splanchnic arterial vasodilatation and hemodynamic dysregulation in portal hypertension. Neuropeptide Y (NPY), a sympathetic cotransmitter, has been shown to improve adrenergic vascular contractility in portal hypertensive rats and markedly attenuate hyperdynamic circulation. To further characterize the NPY-effects in portal hypertension, we investigated its role for non-receptor-mediated vasoconstriction in the superior mesenteric artery (SMA) of portal vein ligated (PVL) and sham-operated rats. METHODS Ex vivo SMA perfusion of PVL and sham rats was used to analyse the effects of NPY on pressure response to non-receptor-mediated vasoconstriction. Dose-response curves to KCl (30-300 mM) were used to bypass G protein-coupled receptor mechanisms. Potential involvement of the cyclooxygenase-pathway was tested by non-selective cyclooxygenase-inhibition using indomethacin. RESULTS KCl-induced vascular contractility but not vascular sensitivity was significantly attenuated in PVL rats as compared with sham rats. Administration of NPY resulted in an augmentation of KCl-evoked vascular sensitivity being not different between study groups. However, KCl-induced vascular contractility was markedly more enhanced in PVL rats, thus, vascular response was no more significantly different between PVL and sham rats after addition of NPY. Administration of indomethacin abolished the NPY-induced enhancement of vasoconstriction. CONCLUSIONS Receptor-independent vascular contractility is impaired in mesenteric arteries in portal hypertension. NPY improves non-receptor mediated mesenteric vasoconstriction more effective in portal hypertension than in healthy conditions correcting splanchnic vascular hyporesponsiveness. This beneficial vasoactive action of NPY adds to its well known more pronounced effects on adrenergic vasoconstriction in portal hypertension making it a promising therapeutic agent in portal hypertension.

Alcoholic Cirrhosis Increases Risk for Autoimmune Diseases: A Nationwide Registry-Based Cohort Study

BACKGROUND & AIMS Alcoholic cirrhosis is associated with hyperactivation and dysregulation of the immune system. In addition to its ability to increase risk for infections, it also may increase the risk for autoimmune diseases. We studied the incidence of autoimmune diseases among patients with alcoholic cirrhosis vs controls in Denmark. METHODS We collected data from nationwide health care registries to identify and follow up all citizens of Denmark diagnosed with alcoholic cirrhosis from 1977 through 2010. Each patient was matched with 5 random individuals from the population (controls) of the same sex and age. The incidence rates of various autoimmune diseases were compared between patients with cirrhosis and controls and adjusted for the number of hospitalizations in the previous year (a marker for the frequency of clinical examination). RESULTS Of the 24,679 patients diagnosed with alcoholic cirrhosis, 532 developed an autoimmune disease, yielding an overall increased adjusted incidence rate ratio (aIRR) of 1.36 (95% confidence interval [CI], 1.24-1.50). The strongest associations were with Addison's disease (aIRR, 2.47; 95% CI, 1.04-5.85), inflammatory bowel disease (aIRR, 1.56; 95% CI, 1.26-1.92), celiac disease (aIRR, 5.12; 95% CI, 2.58-10.16), pernicious anemia (aIRR, 2.35; 95% CI, 1.50-3.68), and psoriasis (aIRR, 4.06; 95% CI, 3.32-4.97). There was no increase in the incidence rate for rheumatoid arthritis (aIRR, 0.89; 95% CI, 0.69-1.15); the incidence rate for polymyalgia rheumatica decreased in patients with alcoholic cirrhosis compared with controls (aIRR, 0.47; 95% CI, 0.33-0.67). CONCLUSIONS Based on a nationwide cohort study of patients in Denmark, alcoholic cirrhosis is a risk factor for several autoimmune diseases.

Sociodemographic, behavioral and genetic determinants of allostatic load in a Swiss population-based study.

Allostatic load (AL) is a marker of physiological dysregulation which reflects exposure to chronic stress. High AL has been related to poorer health outcomes including mortality. We examine here the association of socioeconomic and lifestyle factors with AL. Additionally, we investigate the extent to which AL is genetically determined. We included 803 participants (52% women, mean age 48±16years) from a population and family-based Swiss study. We computed an AL index aggregating 14 markers from cardiovascular, metabolic, lipidic, oxidative, hypothalamus-pituitary-adrenal and inflammatory homeostatic axes. Education and occupational position were used as indicators of socioeconomic status. Marital status, stress, alcohol intake, smoking, dietary patterns and physical activity were considered as lifestyle factors. Heritability of AL was estimated by maximum likelihood. Women with a low occupational position had higher AL (low vs. high OR=3.99, 95%CI [1.22;13.05]), while the opposite was observed for men (middle vs. high OR=0.48, 95%CI [0.23;0.99]). Education tended to be inversely associated with AL in both sexes(low vs. high OR=3.54, 95%CI [1.69;7.4]/OR=1.59, 95%CI [0.88;2.90] in women/men). Heavy drinking men as well as women abstaining from alcohol had higher AL than moderate drinkers. Physical activity was protective against AL while high salt intake was related to increased AL risk. The heritability of AL was estimated to be 29.5% ±7.9%. Our results suggest that generalized physiological dysregulation, as measured by AL, is determined by both environmental and genetic factors. The genetic contribution to AL remains modest when compared to the environmental component, which explains approximately 70% of the phenotypic variance.

Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes.

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single pro-inflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could well be explained by the widespread gene expression dysregulation known as "genomic storm" in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this "storm". Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some pro-inflammatory molecules, complement components and endogenous "danger" signals. The improved survival in endotoxemia was associated with serum levels of pro-inflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.