Regional effect on the molecular clock rate of protein evolution in Eutherian and Metatherian genomes.

BACKGROUND: Different types of proteins diverge at vastly different rates. Moreover, the same type of protein has been observed to evolve with different rates in different phylogenetic lineages. In the present study we measured the rates of protein evolution in Eutheria (placental mammals) and Metatheria (marsupials) on a genome-wide basis and we propose that the gene position in the genome landscape has an important influence on the rate of protein divergence. RESULTS: We analyzed a protein-encoding gene set (n = 15,727) common to 16 mammals (12 Eutheria and 4 Metatheria). Using sliding windows that averaged regional effects of protein divergence we constructed landscapes in which strong and lineage-specific regional effects were seen on the molecular clock rate of protein divergence. Within each lineage, the relatively high rates were preferentially found in subtelomeric chromosomal regions. Such regions were observed to contain important and well-studied loci for fetal growth, uterine function and the generation of diversity in the adaptive repertoire of immunoglobulins. CONCLUSIONS: A genome landscape approach visualizes lineage-specific regional differences between Eutherian and Metatherian rates of protein evolution. This phenomenon of chromosomal position is a new element that explains at least part of the lineage-specific effects and differences between proteins on the molecular clock rates.

Loss of Furin in ß-Cells Induces an mTORC1-ATF4 Anabolic Pathway That Leads to ß-Cell Dysfunction.

FURIN is a proprotein convertase (PC) responsible for proteolytic activation of a wide array of precursor proteins within the secretory pathway. It maps to the PRC1 locus, a type 2 diabetes susceptibility locus, but its specific role in pancreatic ß-cells is largely unknown. The aim of this study was to determine the role of FURIN in glucose homeostasis. We show that FURIN is highly expressed in human islets, whereas PCs that potentially could provide redundancy are expressed at considerably lower levels. ß-cell-specific Furin knockout (ßFurKO) mice are glucose intolerant as a result of smaller islets with lower insulin content and abnormal dense-core secretory granule morphology. mRNA expression analysis and differential proteomics on ßFurKO islets revealed activation of activating transcription factor 4 (ATF4), which was mediated by mammalian target of rapamycin C1 (mTORC1). ßFurKO cells show impaired cleavage or shedding of vacuolar-type ATPase (V-ATPase) subunits Ac45 and prorenin receptor, respectively, and impaired lysosomal acidification. Blocking V-ATPase pharmacologically in ß-cells increased mTORC1 activity, suggesting involvement of the V-ATPase proton pump in the phenotype. Taken together, these results suggest a model of mTORC1-ATF4 hyperactivation and impaired lysosomal acidification in ß-cells lacking Furin, causing ß-cell dysfunction.

Transcriptional Changes in Kidney Allografts with Histology of Antibody-Mediated Rejection without Anti-HLA Donor-Specific Antibodies.

BACKGROUND: Circulating donor-specific anti-HLA antibodies (HLA-DSAs) are often absent in serum of kidney allograft recipients whose biopsy specimens demonstrate histology of antibody-mediated rejection (ABMR). It is unclear whether cases involving ABMR histology without detectable HLA-DSAs represent a distinct clinical and molecular phenotype. METHODS: In this multicenter cohort study, we integrated allograft microarray analysis with extensive clinical and histologic phenotyping from 224 kidney transplant recipients between 2011 and 2017. We used the term ABMR histology for biopsy specimens that fulfill the first two Banff 2017 criteria for ABMR, irrespective of HLA-DSA status. RESULTS: Of 224 biopsy specimens, 56 had ABMR histology; 26 of these (46.4%) lacked detectable serum HLA-DSAs. Biopsy specimens with ABMR histology showed overexpression of transcripts mostly related to IFNγ-induced pathways and activation of natural killer cells and endothelial cells. HLA-DSA-positive and HLA-DSA-negative biopsy specimens with ABMR histology displayed similar upregulation of pathways and enrichment of infiltrating leukocytes. Transcriptional heterogeneity observed in biopsy specimens with ABMR histology was not associated with HLA-DSA status but was caused by concomitant T cell-mediated rejection. Compared with cases lacking ABMR histology, those with ABMR histology and HLA-DSA had higher allograft failure risk (hazard ratio [HR], 7.24; 95% confidence interval [95% CI], 3.04 to 17.20) than cases without HLA-DSA (HR, 2.33; 95% CI, 0.85 to 6.33), despite the absence of transcriptional differences. CONCLUSIONS: ABMR histology corresponds to a robust intragraft transcriptional signature, irrespective of HLA-DSA status. Outcome after ABMR histology is not solely determined by the histomolecular presentation but is predicted by the underlying etiologic factor. It is important to consider this heterogeneity in further research and in treatment decisions for patients with ABMR histology.

Development and validation of a peripheral blood mRNA assay for the assessment of antibody-mediated kidney allograft rejection: A multicentre, prospective study.

BACKGROUND: Antibody-mediated rejection, a leading cause of renal allograft graft failure, is diagnosed by histological assessment of invasive allograft biopsies. Accurate non-invasive biomarkers are not available. METHODS: In the multicentre, prospective BIOMARGIN study, blood samples were prospectively collected at time of renal allograft biopsies between June 2011 and August 2016 and analyzed in three phases. The discovery and derivation phases of the study (N = 117 and N = 183 respectively) followed a case-control design and included whole genome transcriptomics and targeted mRNA expression analysis to construct and lock a multigene model. The primary end point was the diagnostic accuracy of the locked multigene assay for antibody-mediated rejection in a third validation cohort of serially collected blood samples (N = 387). This trial is registered with ClinicalTrials.gov, number NCT02832661. FINDINGS: We identified and locked an 8-gene assay (CXCL10, FCGR1A, FCGR1B, GBP1, GBP4, IL15, KLRC1, TIMP1) in blood samples from the discovery and derivation phases for discrimination between cases with (N = 49) and without (N = 134) antibody-mediated rejection. In the validation cohort, this 8-gene assay discriminated between cases with (N = 41) and without antibody-mediated rejection (N = 346) with good diagnostic accuracy (ROC AUC 79·9%; 95% CI 72·6 to 87·2, p < 0·0001). The diagnostic accuracy of the 8-gene assay was retained both at time of stable graft function and of graft dysfunction, within the first year and also later after transplantation. The 8-gene assay is correlated with microvascular inflammation and transplant glomerulopathy, but not with the histological lesions of T-cell mediated rejection. INTERPRETATION: We identified and validated a novel 8-gene expression assay that can be used for non-invasive diagnosis of antibody-mediated rejection. FUNDING: The Seventh Framework Programme (FP7) of the European Commission.

COX6A2 variants cause a muscle-specific cytochrome c oxidase deficiency.

OBJECTIVE: Cytochrome c oxidase (COX) deficiency is a major mitochondrial respiratory chain defect that has vast genetic and phenotypic heterogeneity. This study aims to identify novel causative genes of COX deficiency with only striated muscle-specific symptoms. METHODS: Whole exome sequencing was performed in 2 unrelated individuals who were diagnosed with congenital myopathy and presented COX deficiency in muscle pathology. We assessed the COX6A2 variants using measurements of enzymatic activities and assembly of mitochondrial respiratory chain complexes in the samples from the patients and knockout mice. RESULTS: Both patients presented muscle weakness and hypotonia in 4 limbs along with facial muscle weakness. One patient had cardiomyopathy. Neither patient exhibited involvement from other organs. Whole exome sequencing identified biallelic missense variants in COX6A2, which is expressed only in the skeletal muscle and heart. The variants detected were homozygous c.117C > A (p.Ser39Arg) and compound heterozygous c.117C > A (p.Ser39Arg) and c.127T > C (p.Cys43Arg). We found specific reductions in complex IV activities in the skeletal muscle of both individuals. Assembly of complex IV and its supercomplex formation were impaired in the muscle. INTERPRETATION: This study indicates that biallelic variants in COX6A2 cause a striated muscle-specific form of COX deficiency. ANN NEUROL 2019;86:193-202.

Gene and Mirna Regulatory Networks During Different Stages of Crohn's Disease.

BACKGROUND AND AIMS: Early treatment of Crohn's disease [CD] is required in order to optimize patient outcomes. To this end, we need to gain a better understanding of the molecular changes at the onset of CD. METHODS: As a model for the earliest mucosal CD lesions, we study post-operative recurrent CD [Rutgeerts score ≥ i2b]. We are the first to analyse gene and microRNA [miRNA] expression profiles in ileal biopsies from these patients, and compare them with those of newly diagnosed [≤18 months] and late-stage [>10 years after diagnosis] CD patients. RESULTS: Except for one gene [WNT5A], there are no differential genes in CD patients without post-operative recurrence [i0], showing that previous disease did not influence gene expression in the neoterminal ileum, and that this model can be used to study early mucosal CD lesions. Gene expression and co-expression network dysregulation is more pronounced in newly diagnosed and late-stage CD than in post-operative recurrent CD, with most important modules associated with [a]granulocyte adhesion/diapedesis, and cholesterol biosynthesis. In contrast, we found a role for snoRNAs/miRNAs in recurrent CD, highlighting the potential importance of regulatory RNAs in early disease stages. Immunohistochemistry confirmed the expression of key dysregulated genes in damaged/regenerating epithelium and immune cells in recurrent CD. CONCLUSIONS: Aside from regulatory RNAs, there are no clear gene signatures separating post-operative recurrent, newly diagnosed, and late-stage CD. The relative contribution of dysregulated genes and networks differs, and suggests that surgery may reset the disease at the mucosal site, and therefore post-operative recurrent CD might be a good model a good model to study to study early mucosal CD lesions.

Natural killer cell infiltration is discriminative for antibody-mediated rejection and predicts outcome after kidney transplantation.

Despite partial elucidation of the pathophysiology of antibody-mediated rejection (ABMR) after kidney transplantation, it remains largely unclear which of the involved immune cell types determine disease activity and outcome. We used microarray transcriptomic data from a case-control study (n=95) to identify genes that are differentially expressed in ABMR. Given the co-occurrence of ABMR and T-cell-mediated rejection (TCMR), we built a bioinformatics pipeline to distinguish ABMR-specific mRNA markers. Differential expression of 503 unique genes was identified in ABMR, with significant enrichment of natural killer (NK) cell pathways. CIBERSORT (Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts) deconvolution analysis was performed to elucidate the corresponding cell subtypes and showed increased NK cell infiltration in ABMR in comparison to TCMR and normal biopsies. Other leukocyte types (including monocytes/macrophages, CD4 and CD8 T cells, and dendritic cells) were increased in rejection, but could not discriminate ABMR from TCMR. Deconvolution-based estimation of NK cell infiltration was validated using computerized morphometry, and specifically associated with glomerulitis and peritubular capillaritis. In an external data set of kidney transplant biopsies, activated NK cell infiltration best predicted graft failure amongst all immune cell subtypes and even outperformed a histologic diagnosis of acute rejection. These data suggest that NK cells play a central role in the pathophysiology of ABMR and graft failure after kidney transplantation.

Effect of vedolizumab (anti-α4ß7-integrin) therapy on histological healing and mucosal gene expression in patients with UC.

OBJECTIVE: Lymphocyte recruitment to the inflamed gut is increased in UC. Inhibition of this cell trafficking by vedolizumab (VDZ) was successful in inducing and maintaining remission and in induction of endoscopic mucosal healing. There are no data on histological healing with VDZ. We studied histological changes following VDZ therapy and compared gene expression in patients with UC before and after therapy. DESIGN: Forty-one patients with UC from GEMINI I and LTS were studied before and at three time points (weeks 6/12/52) following VDZ therapy. Colonic biopsies were scored using the Geboes index and correlated with Mayo endoscopic subscore. Gene expression was analysed using Affymetrix gene arrays. RESULTS: Fifty-five per cent of patients achieving endoscopic healing (= Mayo endoscopic subscore 0-1) with VDZ at the studied time points also had histological healing (= Geboes grade 0-1). In most healers, some residual histological changes (eg, disturbed architecture and increased mononuclear cell infiltrate) were still observed, although this was less at week 52. VDZ restored expression of many inflammatory genes in patients with endoscopic healing only at week 52 and not before. In VDZ healers, the expression of many genes remained dysregulated at weeks 6/12/52 compared with controls. CONCLUSIONS: VDZ induces histological healing in >50% of patients with endoscopic healing, with maximal effect at week 52. VDZ also restored, although incompletely, the colonic expression of many immune-related genes in patients with UC achieving endoscopic healing at week 52. However, persistent histological and gene dysregulations did remain even in healers, suggesting that maintenance therapy will be necessary to control the intestinal inflammation. TRIAL REGISTRATION NUMBERS: NCT00783718 and NCT00790933; post-results.

How stable is repression of disallowed genes in pancreatic islets in response to metabolic stress?

The specific phenotype of mature differentiated beta cells not only depends on the specific presence of genes that allow beta cell function but also on the selective absence of housekeeping genes ("disallowed genes") that would interfere with this function. Recent studies have shown that both histone modifications and DNA methylation via the de novo methyltransferase DNMT3A are involved in repression of disallowed genes in neonatal beta cells when these cells acquire their mature phenotype. It is unknown, however, if the environmental influence of advanced age, pregnancy and the metabolic stress of high fat diet or diabetes could alter the repression of disallowed genes in beta cells. In the present study, we show that islet disallowed genes-which are also deeply repressed in FACS-purified beta cells-remain deeply repressed in animals of advanced age and in pregnant females. Moreover, the stability of this repression was correlated with strong and stable histone repression marks that persisted in islets isolated from 2 year old mice and with overall high expression of Dnmt3a in islets. Furthermore, repression of disallowed genes was unaffected by the metabolic stress of high fat diet. However, repression of about half of the disallowed genes was weakened in 16 week-old diabetic db/db mice. In conclusion, we show that the disallowed status of islet genes is stable under physiological challenging conditions (advanced age, pregnancy, high fat diet) but partially lost in islets from diabetic animals.

Early differences in islets from prediabetic NOD mice: combined microarray and proteomic analysis.

AIMS/HYPOTHESIS: Type 1 diabetes is an endocrine disease where a long preclinical phase, characterised by immune cell infiltration in the islets of Langerhans, precedes elevated blood glucose levels and disease onset. Although several studies have investigated the role of the immune system in this process of insulitis, the importance of the beta cells themselves in the initiation of type 1 diabetes is less well understood. The aim of this study was to investigate intrinsic differences present in the islets from diabetes-prone NOD mice before the onset of insulitis. METHODS: The islet transcriptome and proteome of 2-3-week-old mice was investigated by microarray and 2-dimensional difference gel electrophoresis (2D-DIGE), respectively. Subsequent analyses using sophisticated pathway analysis and ranking of differentially expressed genes and proteins based on their relevance in type 1 diabetes were performed. RESULTS: In the preinsulitic period, alterations in general pathways related to metabolism and cell communication were already present. Additionally, our analyses pointed to an important role for post-translational modifications (PTMs), especially citrullination by PAD2 and protein misfolding due to low expression levels of protein disulphide isomerases (PDIA3, 4 and 6), as causative mechanisms that induce beta cell stress and potential auto-antigen generation. CONCLUSIONS/INTERPRETATION: We conclude that the pancreatic islets, irrespective of immune differences, may contribute to the initiation of the autoimmune process. DATA AVAILABILITY: All microarray data are available in the ArrayExpress database ( www.ebi.ac.uk/arrayexpress ) under accession number E-MTAB-5264.

Genetic Deletion of Tissue Inhibitor of Metalloproteinase-1/TIMP-1 Alters Inflammation and Attenuates Fibrosis in Dextran Sodium Sulphate-induced Murine Models of Colitis.

BACKGROUND AND AIMS: Increased levels of tissue inhibitor of metalloproteinase-1 [TIMP-1] have been detected in both inflammatory and fibrotic lesions in Crohn's disease. In a murine model of chronic inflammation, fibrosis was associated with an increase in TIMP-1 and inhibition of matrix metalloproteinase [MMP]-mediated degradation. We investigated the effect of TIMP-1 deficiency in acute and chronic murine models of colitis. METHODS: Colitis was induced via oral administration of dextran sodium sulphate [DSS] to B6.129S4-Timp1tm1Pds/J knock-out [KO] and C57BL/6J wild-type [WT] mice. Levels of inflammation and fibrosis were assessed and gelatin zymographies and gene expression microarrays were performed. RESULTS: Compared with WT mice, TIMP-1 KO mice had higher inflammatory parameters after acute DSS administration and developed less fibrosis after chronic DSS administration. MMP-2 levels were increased in WT versus TIMP-1 KO mice with acute colitis, whereas a trend for higher proMMP-9 levels was observed in WT versus TIMP-1 KO mice with chronic colitis. In control conditions, several immune-related genes [e.g Ido1, Cldn8] were differentially expressed between young TIMP-1 KO and WT mice, but to a lesser extent between older TIMP-1 KO and WT mice. In response to DSS, the gene expression pattern was significantly different between young TIMP-1 KO and WT mice, whereas it was similar in older TIMP-1 KO and WT mice. CONCLUSIONS: TIMP-1 deficiency leads to differential expression of immune-related genes and to attenuated development of fibrosis. Unravelling the role of TIMP-1 in intestinal remodelling is necessary to develop more effective and more targeted therapeutic strategies for intestinal fibrosis.

Effect of a transcriptional inactive or absent vitamin D receptor on beta-cell function and glucose homeostasis in mice.

Vitamin D deficiency is associated with beta-cell dysfunction and a higher risk of diabetes, but mice and humans with an absence of the vitamin D receptor (VDR) display normal glucose tolerance. Here, we investigated the direct effects of absence of VDR or absence of ligand activation of VDR on beta-cell function. For this purpose, we generated mice, with a mutation in the AF2 domain of Vdr (VDRΔAF2), preventing ligand-driven transcriptional activation of vitamin D responsive genes. VDRΔAF2 mice were compared to Vdr full knockout (VDR-/-) and wild type (WT) mice. In order to avoid hypocalcemia, which has a direct effect on beta-cell function, mice were fed a high calcium, high lactose diet yielding comparable serum calcium in all mice. While VDR-/- mice developed extensive alopecia by the age of 24 weeks, the fur of VDRΔAF2 remained normal. All VDRΔAF2 mice weighed significantly less than WT, while male but not female VDR-/- mice had a lower body weight than WT mice. Dual-energy X-ray absorptiometry showed that both VDRΔAF2 (17.2% (females) and 16.6% (males)) and VDR-/- (15.7% and 14.8%) mice have a lower percentage of body fat (vs 19.3% and 22.2% in WT). Serum 25(OH)D3 concentrations were lower for both VDRΔAF2 (-4.55 fold, P<0.001) and VDR-/- (-3.7 fold, P<0.001) as compared to 12 week old WT mice, while serum 1,25(OH)2D3 was increased for both strains 94.5 fold (P<0.01) and 92.8 fold (P<0.001) for VDRΔAF2 and VDR-/- vs WT, respectively). In vivo glucose tolerance tests performed at 12 and 24 weeks of age, as well as ex vivo glucose stimulated insulin secretion on freshly isolated islets, revealed no major differences between the three strains. Microarray analysis on freshly isolated islets showed only 1 differentially expressed gene, phosphodiesterase 10a (Pde10a), which was 2.16 and 1.75 fold up-regulated in VDRΔAF2 and VDR-/- islets as compared to WT islets, respectively (P≤0.001). We conclude that in the presence of normocalcemia, absence of VDR or its ligand-activated transcription of genes has no direct regulatory effect on murine glucose homeostasis or gene expression in islets of Langerhans.

Strong Upregulation of AIM2 and IFI16 Inflammasomes in the Mucosa of Patients with Active Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by a chronic inflammation of the gut, partly driven by defects in the innate immune system. Considering the central role of inflammasome signaling in innate immunity, we studied inflammasome components in IBD mucosa. METHODS: Expression of genes encoding inflammasome sensor subunits was investigated in colonic mucosal biopsies from 2 cohorts of patients with IBD and controls. RESULTS: A significant upregulation (>2-fold change in expression, false discovery rate <0.05) of the PYHIN inflammasomes AIM2 and IFI16 in active IBD versus controls was found. Also IFI16 was significantly increased in inactive IBD versus controls. Moreover, responders to anti-tumor necrosis factor therapy showed decreased expression of these inflammasomes although IFI16 remained significantly increased in responders showing endoscopic healing versus controls. AIM2 was mainly expressed in epithelial cells, whereas IFI16 was expressed in both lymphocytes and epithelial cells. Functional activation of predominant AIM2/IFI16-mediated inflammasomes in active IBD colon was shown by the presence of the downstream effectors CASP1 and HMGB-1 in inflamed mucosa. CONCLUSIONS: Our results highlight the importance of PYHIN inflammasome signaling in IBD and also link anti-tumor necrosis factor responsiveness to inflammasome signaling. Together, this points to the potential value of the inflammasome pathway as a new therapeutic target for IBD treatment.

Prolactin receptors and placental lactogen drive male mouse pancreatic islets to pregnancy-related mRNA changes.

Pregnancy requires a higher functional beta cell mass and this is associated with profound changes in the gene expression profile of pancreatic islets. Taking Tph1 as a sensitive marker for pregnancy-related islet mRNA expression in female mice, we previously identified prolactin receptors and placental lactogen as key signalling molecules. Since beta cells from male mice also express prolactin receptors, the question arose whether male and female islets have the same phenotypic resilience at the mRNA level during pregnancy. We addressed this question in vitro, by stimulating cultured islets with placental lactogen and in vivo, by transplanting male or female islets into female acceptor mice. Additionally, the islet mRNA expression pattern of pregnant prolactin receptor deficient mice was compared with that of their pregnant wild-type littermates. When cultured with placental lactogen, or when transplanted in female recipients that became pregnant (day 12.5), male islets induced the 'islet pregnancy gene signature', which we defined as the 12 highest induced genes in non-transplanted female islets at day 12.5 of pregnancy. In addition, serotonin immunoreactivity and beta cell proliferation was also induced in these male transplanted islets at day 12.5 of pregnancy. In order to further investigate the importance of prolactin receptors in these mRNA changes we used a prolactin receptor deficient mouse model. For the 12 genes of the signature, which are highly induced in control pregnant mice, no significant induction of mRNA transcripts was found at day 9.5 of pregnancy. Together, our results support the key role of placental lactogen as a circulating factor that can trigger the pregnancy mRNA profile in both male and female beta cells.

Vitamin D3 Induces Tolerance in Human Dendritic Cells by Activation of Intracellular Metabolic Pathways.

Metabolic switches in various immune cell subsets enforce phenotype and function. In the present study, we demonstrate that the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), induces human monocyte-derived tolerogenic dendritic cells (DC) by metabolic reprogramming. Microarray analysis demonstrated that 1,25(OH)2D3 upregulated several genes directly related to glucose metabolism, tricarboxylic acid cycle (TCA), and oxidative phosphorylation (OXPHOS). Although OXPHOS was promoted by 1,25(OH)2D3, hypoxia did not change the tolerogenic function of 1,25(OH)2D3-treated DCs. Instead, glucose availability and glycolysis, controlled by the PI3K/Akt/mTOR pathway, dictate the induction and maintenance of the 1,25(OH)2D3-conditioned tolerogenic DC phenotype and function. This metabolic reprogramming is unique for 1,25(OH)2D3, because the tolerogenic DC phenotype induced by other immune modulators did not depend on similar metabolic changes. We put forward that these metabolic insights in tolerogenic DC biology can be used to advance DC-based immunotherapies, influencing DC longevity and their resistance to environmental metabolic stress.

Integrated miRNA and mRNA expression profiling in inflamed colon of patients with ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA. METHODOLOGY: Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays. RESULTS: When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression. CONCLUSION: Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.

Impaired islet function in commonly used transgenic mouse lines due to human growth hormone minigene expression.

The human growth hormone (hGH) minigene is frequently used in the derivation of transgenic mouse lines to enhance transgene expression. Although this minigene is present in the transgenes as a secondcistron, and thus not thought to be expressed, we found that three commonly used lines, Pdx1-Cre(Late), RIP-Cre, and MIP-GFP, each expressed significant amounts of hGH in pancreatic islets. Locally secreted hGH binds to prolactin receptors on ß cells, activates STAT5 signaling, and induces pregnancy-like changes in gene expression, thereby augmenting pancreatic ß cell mass and insulin content. In addition, islets of Pdx1-Cre(Late) mice have lower GLUT2 expression and reduced glucose-induced insulin release and are protected against the ß cell toxin streptozotocin. These findings may be important when interpreting results obtained when these and other hGH minigene-containing transgenic mice are used.

A complex Xp11.22 deletion in a patient with syndromic autism: exploration of FAM120C as a positional candidate gene for autism.

We present a male patient with sporadic Aarskog syndrome, cleft palate, mild intellectual disability, and autism spectrum disorder (ASD). A submicroscopic discontiguous deletion was detected on chromosome Xp11.2 encompassing FGD1, FAM120C, and PHF8. That the deletion encompassed FGD1 (exons 2-8) explains the Aarskog features while the deletion of PHF8 most likely explains the cleft palate and mild intellectual disability. We identify FAM120C as a novel X-linked candidate gene for autism for two reasons: first, a larger deletion encompassing FAM120C segregates with autism in a previously reported family and second, there is recent evidence that FAM120C interacts with CYFIP1, part of the FMRP (Fragile X Mental Retardation Protein) network. In the current study, resequencing of FAM120C in 87 Belgian male patients with autism spectrum disorder identified no novel mutations. Expression of Fam120c in mouse tissues showed enriched expression in pituitary, cerebellum, cortex, and pancreatic islets of Langerhans. Additionally, we found a cortical expression pattern of Fam120c similar to that of Fmr1. In conclusion, FAM120C is a novel candidate gene for autism spectrum disorder based on genetic evidence and the brain expression pattern. Thereby we highlight a role for FMRP network genes in ASD.

Infliximab restores the dysfunctional matrix remodeling protein and growth factor gene expression in patients with inflammatory bowel disease.

BACKGROUND: Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), a disintegrin and metalloprotease with thrombospondin motifs [ADAM(TS)s] and growth factors are involved in inflammation and tissue damage and repair, all occurring in inflammatory bowel disease (IBD). We studied the impact of anti-inflammatory therapy with infliximab on mucosal expression of these tissue remodeling genes in patients with IBD. METHODS: Mucosal gene expression of 23 MMPs, 4 TIMPs, 50 ADAM(TS)s, and 158 growth factors was investigated in 61 patients with IBD before and after the first infliximab therapy and in 12 controls, with microarrays and quantitative RT-PCR. Protein localization, mucosal gelatinase levels, and net gelatinolytic activity were investigated by immunohistochemistry, zymography analysis, and gelatin degradation assay, respectively. RESULTS: In patients with active IBD before infliximab versus controls, gene expression of many MMPs, TIMPs, ADAM(TS)s, and growth factors was upregulated, whereas colonic expression of MMP28 and TGFA and ileal expression of ADAMDEC1 and AGT were downregulated. After controlling inflammation with infliximab, most gene dysregulations observed at baseline were restored in responders. Increased ratio of MMP1/TIMP1 expression at baseline in active IBD was restored in responders with colonic mucosal healing. With immunohistochemistry, protein localization differences of MMP1, MMP3, REG1A, and TIMP1 were shown between active IBD and control mucosa. With zymography analysis and gelatin degradation assay, higher gelatinase levels and net gelatinolytic activity were measured before infliximab and levels normalized after infliximab. CONCLUSIONS: Our data suggest that suppression of inflammation results in the arrest of epithelial damage and subsequent mucosal healing. Therefore, the therapeutic potential of agents targeting MMPs or growth factors as primary therapy seems rather complex.

Gene expression profiling of early hepatic stellate cell activation reveals a role for Igfbp3 in cell migration.

BACKGROUND: Scarring of the liver is the result of prolonged exposure to exogenous or endogenous stimuli. At the onset of fibrosis, quiescent hepatic stellate cells (HSCs) activate and transdifferentiate into matrix producing, myofibroblast-like cells. AIM AND METHODS: To identify key players during early HSC activation, gene expression profiling was performed on primary mouse HSCs cultured for 4, 16 and 64 hours. Since valproic acid (VPA) can partly inhibit HSC activation, we included VPA-treated cells in the profiling experiments to facilitate this search. RESULTS: Gene expression profiling confirmed early changes for known genes related to HSC activation such as alpha smooth muscle actin (Acta2), lysyl oxidase (Lox) and collagen, type I, alpha 1 (Col1a1). In addition we noticed that, although genes which are related to fibrosis change between 4 and 16 hours in culture, most gene expression changes occur between 16 and 64 hours. Insulin-like growth factor binding protein 3 (Igfbp3) was identified as a gene strongly affected by VPA treatment. During normal HSC activation Igfbp3 is up regulated and this can thus be prevented by VPA treatment in vitro and in vivo. siRNA-mediated silencing of Igfbp3 in primary mouse HSCs induced matrix metalloproteinase (Mmp) 9 mRNA expression and strongly reduced cell migration. The reduced cell migration after Igfbp3 knock-down could be overcome by tissue inhibitor of metalloproteinase (TIMP) 1 treatment. CONCLUSION: Igfbp3 is a marker for culture-activated HSCs and plays a role in HSC migration. VPA treatment prevents Igfbp3 transcription during activation of HSCs in vitro and in vivo.