Tissue-specific inflammation and insulin sensitivity in subjects with obesity.

Obesity is associated with low-grade inflammation and insulin resistance (IR). The contribution of adipose tissue (AT) and hepatic inflammation to IR remains unclear. We conducted a study across three cohorts to investigate this relationship. The first cohort consists of six women with normal weight and twenty with obesity. In women with obesity, we found an upregulation of inflammatory markers in subcutaneous and visceral adipose tissue, isolated AT macrophages, and the liver, but no linear correlation with tissue-specific insulin sensitivity. In the second cohort, we studied 24 women with obesity in the upper vs lower insulin sensitivity quartile. We demonstrated that several omental and mesenteric AT inflammatory genes and T cell-related pathways are upregulated in IR, independent of BMI. The third cohort consists of 23 women and 18 men with obesity, studied before and one year after bariatric surgery. Weight loss following surgery was associated with downregulation of multiple immune pathways in subcutaneous AT and skeletal muscle, alongside notable metabolic improvements. Our results show that obesity is characterised by systemic and tissue-specific inflammation. Subjects with obesity and IR show a more pronounced inflammation phenotype, independent of BMI. Bariatric surgery-induced weight loss is associated with reduced inflammation and improved metabolic health.

Liver X receptor alpha ensures blood-brain barrier function by suppressing SNAI2.

In Alzheimer's disease (AD) more than 50% of the patients are affected by capillary cerebral amyloid-angiopathy (capCAA), which is characterized by localized hypoxia, neuro-inflammation and loss of blood-brain barrier (BBB) function. Moreover, AD patients with or without capCAA display increased vessel number, indicating a reactivation of the angiogenic program. The molecular mechanism(s) responsible for BBB dysfunction and angiogenesis in capCAA is still unclear, preventing a full understanding of disease pathophysiology. The Liver X receptor (LXR) family, consisting of LXRα and LXRß, was reported to inhibit angiogenesis and particularly LXRα was shown to secure BBB stability, suggesting a major role in vascular function. In this study, we unravel the regulatory mechanism exerted by LXRα to preserve BBB integrity in human brain endothelial cells (BECs) and investigate its role during pathological conditions. We report that LXRα ensures BECs identity via constitutive inhibition of the transcription factor SNAI2. Accordingly, deletion of brain endothelial LXRα is associated with impaired DLL4-NOTCH signalling, a critical signalling pathway involved in vessel sprouting. A similar response was observed when BECs were exposed to hypoxia, with concomitant LXRα decrease and SNAI2 increase. In support of our cell-based observations, we report a general increase in vascular SNAI2 in the occipital cortex of AD patients with and without capCAA. Importantly, SNAI2 strongly associated with vascular amyloid-beta deposition and angiopoietin-like 4, a marker for hypoxia. In hypoxic capCAA vessels, the expression of LXRα may decrease leading to an increased expression of SNAI2, and consequently BECs de-differentiation and sprouting. Our findings indicate that LXRα is essential for BECs identity, thereby securing BBB stability and preventing aberrant angiogenesis. These results uncover a novel molecular pathway essential for BBB identity and vascular homeostasis providing new insights on the vascular pathology affecting AD patients.

A JAK1 Selective Kinase Inhibitor and Tofacitinib Affect Macrophage Activation and Function.

BACKGROUND: Janus kinases (JAKs) mediate cytokine signaling involved in inflammatory bowel disease. The pan-JAK inhibitor tofacitinib has shown efficacy in the treatment of ulcerative colitis. However, concerns regarding adverse events due to their wide spectrum inhibition fueled efforts to develop selective JAK inhibitors. Given the crucial role of myeloid cells in intestinal immune homeostasis, we evaluated the effect of pan-JAK and selective JAK inhibitors on pro- and anti-inflammatory macrophage polarization and function (M1/M2) and in experimental colitis. METHODS: Murine bone marrow-derived macrophages or human monocytes were treated using JAK1 and JAK3 selective inhibitors (JAK1i;JAK3i) and tofacitinib and were evaluated by transcriptional, functional, and metabolic analyses. In vivo, oral administration of JAK1i and tofacitinib (10 or 30 mg/kg) was tested in both acute and acute rescue dextran sodium sulfate (DSS) colitis. RESULTS: Both tofacitinib and JAK1i but not JAK3i effectively inhibited STAT1 phosphorylation and interferon gamma-induced transcripts in M1 polarized macrophages. Strikingly, transcriptional profiling suggested a switch from M1 to M2 type macrophages, which was supported by increased protein expression of M2-associated markers. In addition, both inhibitors enhanced oxidative phosphorylation rates. In vivo, JAK1i and tofacitinib did not protect mice from acute DSS-induced colitis but ameliorated recovery from weight loss and disease activity during acute rescue DSS-induced colitis at the highest dose. CONCLUSION: JAK1i and tofacitinib but not JAK3i induce phenotypical and functional characteristics of anti-inflammatory macrophages, suggesting JAK1 as the main effector pathway for tofacitinib in these cells. In vivo, JAK1i and tofacitinib modestly affect acute rescue DSS-induced colitis.

De novo gene mutations in normal human memory B cells.

In the past years, the genomes of thousands of tumors have been elucidated. To date however, our knowledge on somatic gene alterations in normal cells is very limited. In this study, we demonstrate that tetanus-specific human memory B lymphocytes carry a substantial number of somatic mutations in the coding regions of the genome. Interestingly, we observed a statistically significant correlation between the number of exome mutations and those present in the immunoglobulin heavy variable regions. Our findings indicate that the majority of these genomic mutations arise in an antigen-dependent fashion, most likely during clonal expansion in germinal centers. The knowledge that normal B cells accumulate genomic alterations outside the immunoglobulin loci during development is relevant for our understanding of the process of lymphomagenesis.

Effect of preoperative biliary drainage on cholestasis-associated inflammatory and fibrotic gene signatures in perihilar cholangiocarcinoma.

Preoperative biliary drainage (PBD) is used routinely in the evaluation of patients with potentially resectable perihilar cholangiocarcinoma to relieve cholestasis and improve the liver's resilience to surgery. Little preclinical or translatational data are, however, currently available to guide the use of PBD in this patient group. The effect of PBD on hepatic gene expression profiles was therefore studied by microarray analysis. Drainage affects inflammatory and fibrotic gene signatures.

Age-related gene expression profiles of immature human oocytes.

STUDY QUESTION: What is the difference between the gene expression profiles of single human germinal vesicle (GV) oocytes from women of different ages? SUMMARY ANSWER: There were no statistically significant differences in gene expression profiles of human GV oocytes from women of different ages (range: 25-43). WHAT IS KNOWN ALREADY: It is well established that reproductive capacity declines as women age, which is attributed to oocyte quality since this decline is counterbalanced in older women receiving young donor oocytes. Altered gene expression of human oocytes at different stages of development in relation to female age is one of the suggested mechanisms that could explain the decrease in oocyte quality. STUDY DESIGN, SIZE, DURATION: Between 2012 and 2014, 40 human GV oocytes of 40 women were obtained during follicular aspiration as part of routine ICSI treatment. Gene expression profiles of 38 GV oocytes were determined in four different age groups: 25-30, 31-35, 36-38 and 39-43 years of age. PARTICIPANTS/MATERIALS, SETTING, METHODS: GV oocytes were donated for research and frozen between 3.5 and 7.5 h after follicular aspiration. Subsequently, GV oocytes were thawed and prepared for gene expression profile analysis using Agilent microarrays containing ~42 000 Human Gene Expression probe-sets. Gene expression profiles were visualized by hierarchical clustering and the top 500 most differing genes were determined by multidimensional scaling (MDS). Transcripts were analysed in a class comparison between the four age groups and for indicators of biological age: antral follicle count (AFC) and the total dosage of FSH needed for ovarian stimulation. Individual transcripts were analysed using linear regression. A false discovery rate <0.05 was considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE: Visualization of gene expression profiles of GV oocytes with hierarchal clustering and MDS demonstrated no clear grouping of samples based on female age, AFC or FSH dosage. The gene expression profile of GV oocytes classified in four age groups revealed no significantly differentially expressed genes between the four different age groups. There were also no significantly differentially expressed genes in the linear regression analysis for individual transcripts against age. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Immature (GV) oocytes obtained from ovarian stimulation cycles were used. Findings may therefore differ for oocytes at other developmental stages and for in-vivo matured oocytes under physiological conditions. Due to our relatively large, but still limited study sample (40 GV oocytes), we cannot exclude that there might be smaller age-related gene-expression differences, i.e. due to a lack of power. WIDER IMPLICATIONS OF THE FINDINGS: We did not find an effect of female age on gene expression profiles of individual human GV oocytes. Other studies have suggested that gene-expression profiles are affected in mature oocytes, which might imply that female age affects oocyte maturation. Alternatively, other mechanisms in human oocytes might cause the age-related fertility decline. STUDY FUNDING/COMPETING INTEREST(S): This study received no external funding and there are no competing interests.

Variation in coagulation and fibrinolysis genes evaluated for their contribution to cerebrovascular complications in adults with bacterial meningitis in the Netherlands.

OBJECTIVE: To study whether genetic variation in coagulation and fibrinolysis genes contributes to cerebrovascular complications in bacterial meningitis. METHODS: We performed a nationwide prospective genetic association study in adult community-acquired bacterial meningitis patients. The exons and flanking regions of 16 candidate genes involved in coagulation and fibrinolysis pathways were sequenced. We analyzed whether genetic variation in these genes resulted in a higher risk of cerebrovascular complications, unfavorable outcome and differences in thrombocyte count on admission. RESULTS: From 2006 to 2011, a total of 1101 bacterial meningitis patients were identified of whom 622 supplied DNA for genotyping and passed genetic quality control steps. In 139 patients (22%) the episode of bacterial meningitis was complicated by cerebral infarction, and 188 (30%) had an unfavorable outcome. We identified the functional variant rs494860 in the protein Z (PROZ) gene as our strongest association with occurrence of cerebral infarction (odds ratio (OR) 0.49 (95% confidence interval 0.33-0.73), p = 5.2 × 10-4). After Bonferroni correction for multiple testing no genetic variant was significantly associated (p-value threshold 2.7 × 10-4). CONCLUSION: Our study suggests a functional genetic variation in the PROZ gene, rs494860, may be of importance in bacterial meningitis pathogenesis and cerebral infarction risk. Replication of this finding in other cohort studies populations is needed.

Macrophages confer survival signals via CCR1-dependent translational MCL-1 induction in chronic lymphocytic leukemia.

Protective interactions with bystander cells in micro-environmental niches, such as lymph nodes (LNs), contribute to survival and therapy resistance of chronic lymphocytic leukemia (CLL) cells. This is caused by a shift in expression of B-cell lymphoma 2 (BCL-2) family members. Pro-survival proteins B-cell lymphoma-extra large (BCL-XL), BCL-2-related protein A1 (BFL-1) and myeloid leukemia cell differentiation protein 1 (MCL-1) are upregulated by LN-residing T cells through CD40L interaction, presumably via nuclear factor (NF)-κB signaling. Macrophages (Mφs) also reside in the LN, and are assumed to provide important supportive functions for CLL cells. However, if and how Mφs are able to induce survival is incompletely known. We first established that Mφs induced survival because of an exclusive upregulation of MCL-1. Next, we investigated the mechanism underlying MCL-1 induction by Mφs in comparison with CD40L. Genome-wide expression profiling of in vitro Mφ- and CD40L-stimulated CLL cells indicated activation of the phosphoinositide 3-kinase (PI3K)-V-Akt murine thymoma viral oncogene homolog (AKT)-mammalian target of rapamycin (mTOR) pathway, which was confirmed in ex vivo CLL LN material. Inhibition of PI3K-AKT-mTOR signaling abrogated MCL-1 upregulation and survival by Mφs, as well as CD40 stimulation. MCL-1 can be regulated at multiple levels, and we established that AKT leads to increased MCL-1 translation, but does not affect MCL-1 transcription or protein stabilization. Furthermore, among Mφ-secreted factors that could activate AKT, we found that induction of MCL-1 and survival critically depended on C-C motif chemokine receptor-1 (CCR1). In conclusion, this study indicates that two distinct micro-environmental factors, CD40L and Mφs, signal via CCR1 to induce AKT activation resulting in translational stabilization of MCL-1, and hence can contribute to CLL cell survival.

Unexpected regulation of miRNA abundance during adaptation of early-somite mouse embryos to diabetic pregnancy.

Maternal diabetes is one of major causes of congenital malformations in offspring, but the underlying mechanism is still unclear. MiRNAs play an important role in transcriptional and post-transcriptional regulation of gene expression. However, no miRNA expression profiling of hyperglycemic offspring are thus far available. Female mice were made diabetic with streptozotocin, treated with slow-release insulin tablets, and mated. MiRNA expression profiling with Next Generation Sequencing on the SOLiD5 platform was performed on 8 control and 5 hyperglycemic embryonic day (ED)8.5 and 9 control and 6 hyperglycemic ED9.5 embryos. Differential expression was analyzed with the Wald test. On ED8.5, the abundance of expressed miRNAs was similar in control and hyperglycemic ED8.5 embryos. The spectrum of expressed miRNAs had not changed in ED9.5 embryos, but the abundance of most miRNAs increased ∼5-fold in control embryos. However, hyperglycemic D9.5 embryos were unable to mount this increase in prevalence. Only 3 miRNAs were differentially expressed in control and hyperglycemic ED9.5 embryos, but their putative target genes were underrepresented in the Jackson database of genes causing cardiovascular or neural malformations.

Direct hydride transfer in the reaction mechanism of quinoprotein alcohol dehydrogenases: a quantum mechanical investigation.

Oxidation of alcohols by direct hydride transfer to the pyrroloquinoline quinone (PQQ) cofactor of quinoprotein alcohol dehydrogenases has been studied using ab initio quantum mechanical methods. Energies and geometries were calculated at the 6-31G(d,p) level of theory. Comparison of the results obtained for PQQ and several derivatives with available structural and spectroscopic data served to judge the feasibility of the calculations. The role of calcium in the enzymatic reaction mechanism has been investigated. Transition state searches have been conducted at the semiempirical and STO-3G(d) level of theory. It is concluded that hydride transfer from the Calpha-position of the substrate alcohol (or aldehyde) directly to the C(5) carbon of PQQ is energetically feasible. Copyright 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1732-1749, 2001

Homology model of the quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni.

A molecular model of QH-ADH, the quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni, has been built by homology modelling. Sequence similarity of N-terminal residues 1-570 with the alpha-subunit of quinoprotein methanol dehydrogenases (MDHs) from Methylophilus methylotrophus W3A1 and Methylobacterium extorquens provided a basis for the design of the PQQ-binding domain of QH-ADH. Minimal sequence similarity with cytochrome c551 from Ectothiorhodospira halophila and cytochrome c5 from Azotobacter vinelandii has been used to model the C-terminal haem c-binding domain, residues 571-677, absent in MDHs. Distance constraints inferred from 19F-NMR relaxation studies of trifluoromethylphenylhydrazine-derivatized PQQ bound to QH-ADH apoenzyme as well as theoretical relations for optimal electron transfer have been employed to position the haem- and PQQ-binding domains relative to each other. The homology model obtained shows overall topological similarity with the crystal structure of cd1-nitrite reductase from Thiosphera pantotropha. The proposed model accounts for the following: (i) the site that is sensitive to in vivo proteolytic attack; (ii) the substrate specificity in comparison with MDHs; (iii) changes of the spectral properties of the haem c upon reconstitution of apo-enzyme with PQQ; (iv) electronic interaction between haem and PQQ; and (v) enantioselectivity in the conversion of a chiral sec alcohol.