Publications by authors named "Ole Kristoffer Olstad"

39 Publications

Uptake of circulating extracellular vesicles from rectal cancer patients and differential responses by human monocyte cultures.

FEBS Open Bio 2021 Mar 8;11(3):724-740. Epub 2021 Feb 8.

Department of Oncology, Akershus University Hospital, Lørenskog, Norway.

Extracellular vesicles (EVs) released by tumor cells can directly or indirectly modulate the phenotype and function of the immune cells of the microenvironment locally or at distant sites. The uptake of circulating EVs and the responses by human monocytes in vitro may provide new insights into the underlying biology of the invasive and metastatic processes in cancer. Although a mixed population of vesicles is obtained with most isolation techniques, we predominantly isolated exosomes (small EVs) and microvesicles (medium EVs) from the SW480 colorectal cancer cell line (established from a primary adenocarcinoma of the colon) by sequential centrifugation and ultrafiltration, and plasma EVs were prepared from 22 patients with rectal adenoma polyps or invasive adenocarcinoma by size-exclusion chromatography. The EVs were thoroughly characterized. The uptake of SW480 EVs was analyzed, and small SW480 EVs were observed to be more potent than medium SW480 EVs in inducing monocyte secretion of cytokines. The plasma EVs were also internalized by monocytes; however, their cytokine-releasing potency was lower than that of the cell line-derived vesicles. The transcriptional changes in the monocytes highlighted differences between adenoma and adenocarcinoma patient EVs in their ability to regulate biological functions, whereas the most intriguing changes were found in monocytes receiving EVs from patients with metastatic compared with localized cancer.
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http://dx.doi.org/10.1002/2211-5463.13098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931235PMC
March 2021

Integrated differential DNA methylation and gene expression of formalin-fixed paraffin-embedded uveal melanoma specimens identifies genes associated with early metastasis and poor prognosis.

Exp Eye Res 2021 02 30;203:108426. Epub 2020 Dec 30.

Center for Eye Research, Department of Ophthalmology, Oslo University Hospital, Oslo, Norway. Electronic address:

Purpose: Uveal melanoma (UM) is an aggressive malignancy, in which nearly 50% of the patients die from metastatic disease. Aberrant DNA methylation is recognized as an important epigenomic event in carcinogenesis. Formalin-fixed paraffin-embedded (FFPE) samples represent a valuable source of tumor tissue, and recent technology has enabled the use of these samples in genome-wide DNA methylation analyses. Our aim was to investigate differential DNA methylation in relation to histopathological classification and survival data. In addition we sought to identify aberrant DNA methylation of genes that could be associated with metastatic disease and poor survival.

Methods: FFPE samples from UM patients (n = 23) who underwent enucleation of the eye in the period 1976-1989 were included. DNA methylation was assessed using the Illumina Infinium HumanMethylation450 array and coupled to histopathological data, Cancer Registry of Norway- (registered UM metastasis) and Norwegian Cause of Death Registry- (time and cause of death) data. Differential DNA methylation patterns contrasting histological classification, survival data and clustering properties were investigated. Survival groups were defined as "Early metastasis" (metastases and death within 2-5 years after enucleation, n = 8), "Late metastasis" (metastases and death within 9-21 years after enucleation, n = 7) and "No metastasis" (no detected metastases ≥18 years after enucleation, n = 8). A subset of samples were selected based on preliminary multi-dimensional scaling (MDS) plots, histopathological classification, chromosome 3 status, survival status and clustering properties; "Subset Early metastasis" (n = 4) vs "Subset No metastasis" (n = 4). Bioinformatics analyses were conducted in the R statistical software. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) in various comparisons were assessed. Gene expression of relevant subgroups was determined by microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR).

Results: DNA methylation analyses identified 2 clusters that separated the samples according to chromosome 3 status. Cluster 1 consisted of samples (n = 5) with chromosome 3 disomy (D3), while Cluster 2 was comprised of samples (n = 15) with chromosome 3 monosomy (M3). 1212 DMRs and 9386 DMPs were identified in M3 vs D3. No clear clusters were formed based on our predefined survival groups ("Early", "Late", "No") nor histopathological classification (Epithelioid, Mixed, Spindle). We identified significant changes in DNA methylation (beta FC ≥ 0.2, adjusted p < 0.05) between two sample subsets (n = 8). "Subset Early metastasis" (n = 4) vs "Subset No metastasis" (n = 4) identified 348 DMPs and 36 DMRs, and their differential gene expression by microarray showed that 14 DMPs and 2 DMRs corresponded to changes in gene expression (FC ≥ 1.5, p < 0.05). RNF13, ZNF217 and HYAL1 were hypermethylated and downregulated in "Subset Early metastasis" vs "Subset No metastasis" and could be potential tumor suppressors. TMEM200C, RGS10, ADAM12 and PAM were hypomethylated and upregulated in "Subset Early metastasis vs "Subset No metastasis" and could be potential oncogenes and thus markers of early metastasis and poor prognosis in UM.

Conclusions: DNA methylation profiling showed differential clustering of samples according to chromosome 3 status: Cluster 1 (D3) and Cluster 2 (M3). Integrated differential DNA methylation and gene expression of two subsets of samples identified genes associated with early metastasis and poor prognosis. RNF13, ZNF217 and HYAL1 are hypermethylated and candidate tumor suppressors, while TMEM200C, RGS10, ADAM12 and PAM are hypomethylated and candidate oncogenes linked to early metastasis. UM FFPE samples represent a valuable source for methylome studies and enable long-time follow-up.
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http://dx.doi.org/10.1016/j.exer.2020.108426DOI Listing
February 2021

Cytokine and Gene Expression Profiling in Patients with HFE-Associated Hereditary Hemochromatosis according to Genetic Profile.

Acta Haematol 2020 Dec 16:1-12. Epub 2020 Dec 16.

Center for Laboratory Medicine, Østfold Hospital Trust, Grålum, Norway,

Background: Hemochromatosis gene (HFE)-associated hereditary hemochromatosis (HH) is characterized by downregulation of hepcidin synthesis, leading to increased intestinal iron absorption.

Objectives: The objectives were to characterize and elucidate a possible association between gene expression profile, hepcidin levels, disease severity, and markers of inflammation in HFE-associated HH patients.

Methods: Thirty-nine HFE-associated HH patients were recruited and assigned to 2 groups according to genetic profile: C282Y homozygotes in 1 group and patients with H63D, as homozygote or in combination with C282Y, in the other group. Eleven healthy first-time blood donors were recruited as controls. Gene expression was characterized from peripheral blood cells, and inflammatory cytokines and hepcidin-25 isoform were quantified in serum. Biochemical disease characteristics were recorded.

Results: Elevated levels of interleukin 8 were observed in a significant higher proportion of patients than controls. In addition, compared to controls, gene expression of ζ-globin was significantly increased among C282Y homozygote patients, while gene expression of matrix metalloproteinase 8, and other neutrophil-secreted proteins, was significantly upregulated in patients with H63D.

Conclusion: Different disease signatures may characterize HH patients according to their HFE genetic profile. Studies on larger populations, including analyses at protein level, are necessary to confirm these findings.
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http://dx.doi.org/10.1159/000511551DOI Listing
December 2020

Sericin-Induced Melanogenesis in Cultured Retinal Pigment Epithelial Cells Is Associated with Elevated Levels of Hydrogen Peroxide and Inflammatory Proteins.

Molecules 2020 Sep 24;25(19). Epub 2020 Sep 24.

Department of Medical Biochemistry, Oslo University Hospital, Kirkeveien 166, P.O. Box 4956, Nydalen, 0424 Oslo, Norway.

We previously demonstrated that the silk protein sericin promotes pigmentation of retinal pigment epithelium (RPE) by activating the NF-κB pathway. Among numerous agents, NF-κB can be activated by hydrogen peroxide. In the present study, we explored possible associations between reactive oxygen species and sericin-induced melanogenesis in RPE. The proteome of human fetal RPE cultured for seven days with or without 1% sericin was analyzed using ingenuity pathway analysis (IPA). The proteomic data was verified by immunofluorescence and immunoblotting. Light microscopy and scanning electron microscopy were used to assess morphology. Dihydroethidium (DHE) and dihydrorhodamine (DHR) assays were used to measure superoxide and hydrogen peroxide species. Expression levels of proteins related to inflammation, differentiation, cell survival and cell adhesion were higher in cells cultured in Dulbecco's Modified Eagle Medium (DMEM) with 1% sericin, whereas cells cultured in DMEM alone showed higher expression levels of proteins associated with Bruch's membrane and cytoskeleton. Despite upregulation of inflammatory proteins, sericin co-cultured RPE yielded significantly higher cell viability compared to cells cultured without sericin. Addition of sericin to culture media significantly increased hydrogen peroxide-levels without significantly affecting superoxide-levels. We suggest that sericin-induced melanogenesis in cultured RPE is associated with elevated levels of superoxide dismutase, hydrogen peroxide and inflammatory proteins.
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http://dx.doi.org/10.3390/molecules25194395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582875PMC
September 2020

Lacrimal Gland Myoepithelial Cells Are Altered in a Mouse Model of Dry Eye Disease.

Am J Pathol 2020 10 15;190(10):2067-2079. Epub 2020 Jul 15.

Schepens Eye Research Institute/Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts; Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts. Electronic address:

The purpose of this study was to determine the pathogenic changes that occur in myoepithelial cells (MECs) from lacrimal glands of a mouse model of Sjögren syndrome. MECs were cultured from lacrimal glands of C57BL/6J [wild type (WT)] and thrombospondin 1 null (TSP1, alias Thbs1) mice and from mice expressing α-smooth muscle actin-green fluorescent protein that labels MECs. MECs were stimulated with cholinergic and α-adrenergic agonists, vasoactive intestinal peptide (VIP), and the purinergic agonists ATP and UTP. Then intracellular [Ca] was measured using fura-2, and contraction was observed using live cell imaging. Expression of purinergic receptors was determined by Western blot analysis, and mRNA expression was analyzed by microarray. The increase in intracellular [Ca] with VIP and UTP was significantly smaller in MECs from TSP1 compared with WT mice. Cholinergic agonists, ATP, and UTP stimulated contraction in MECs, although contraction of MECs from TSP1 mice was reduced compared with WT mice. The amount of purinergic receptors P2Y1, P2Y11, and P2Y13 was significantly decreased in MECs from TSP1 compared with WT mice, whereas several extracellular matrix and inflammation genes were up-regulated in MECs from TSP1 mice. We conclude that lacrimal gland MEC function is altered by inflammation because the functions regulated by cholinergic agonists, VIP, and purinergic receptors are decreased in TSP1 compared with WT mice.
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http://dx.doi.org/10.1016/j.ajpath.2020.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7520664PMC
October 2020

Postmenopausal osteoporosis is a musculoskeletal disease with a common genetic trait which responds to strength training: a translational intervention study.

Ther Adv Musculoskelet Dis 2020 28;12:1759720X20929443. Epub 2020 May 28.

Lovisenberg Diakonale Sykehus, Unger-Vetlesen Institute, Lovisenberggata 17, Oslo 0456, Norway.

Background: Clinical evidence suggests that body muscle mass is positively associated with bone mass, of significance for the elderly population at risk of osteoporosis (OP). Furthermore, muscle and bone interact mechanically and functionally, local interactions as well as remotely secreted components. Thus, it was of interest to compare muscle transcriptomes in postmenopausal OP and healthy women, and study effects of strength training on the muscle transcriptome, muscle stress proteins and bone mineral density (BMD).

Methods: Skeletal muscle histological and genetic properties were compared in postmenopausal healthy ( = 18) and OP ( = 17) women before and after heavy-load strength training for 13-15 weeks. The cohorts were of similar age and body mass index without interfering diseases, medication or difference in lifestyle factors. Muscle biopsies obtained before and after intervention were studied histologically, and stress proteins and transcriptomes analyzed.

Results: The OP women showed distinct muscle transcription profiles when compared with healthy women and had higher levels of the stress proteins HSP70 and α-β-crystalline. A set of 12 muscle transcripts, including ACSS3, FZD4, GNAI1 and IGF1, were differentially expressed before and after intervention (false discovery rate ⩽0.10,  ⩽0.001), and their corresponding bone transcripts were associated with BMD. Experimental data underline and describe the functionality of these genes in bone biology. OP women had 8% ( <0.01) higher proportion of type I fibres, but muscle fibre cross-sectional area did not differ. Muscle strength increased in both groups ( <0.01).

Conclusions: Postmenopausal healthy and OP women have distinct muscle transcriptomes [messenger ribonucleic acids (mRNAs) and microRNAs] that are modulated by strength training, translating into key protein alterations and muscle fibre changes. The function of common skeletal muscle and bone genes in postmenopausal OP is suggestive of a shared disease trait.
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http://dx.doi.org/10.1177/1759720X20929443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7268165PMC
May 2020

Vitamin D metabolites influence expression of genes concerning cellular viability and function in insulin producing β-cells (INS1E).

Gene 2020 Jul 3;746:144649. Epub 2020 Apr 3.

Hormone Laboratory, Dep. of Medical Biochemistry, Oslo University Hospital, Norway.

Background: Studies have shown that vitamin D can enhance glucose-stimulated insulin secretion (GSIS) and change the expression of genes in pancreatic β-cells. Still the mechanisms linking vitamin D and GSIS are unknown.

Material And Methods: We used an established β-cell line, INS1E. INS1E cells were pre-treated with 10 nM 1,25(OH)vitamin D or 10 nM 25(OH)vitamin D for 72 h and stimulated with 22 mM glucose for 60 min. RNA was extracted for gene expression analysis.

Results: Expression of genes affecting viability, apoptosis and GSIS changed after pre-treatment with both 1,25(OH)vitamin D and 25(OH)vitamin D in INS1E cells. Stimulation with glucose after pre-treatment of INS1E cells with 1,25(OH)vitamin D resulted in 181 differentially expressed genes, whereas 526 genes were differentially expressed after pre-treatment with 25(OH)vitamin D.

Conclusion: Vitamin D metabolites may affect pancreatic β-cells and GSIS through changed gene expression for genes involved in β-cell function and viability.
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http://dx.doi.org/10.1016/j.gene.2020.144649DOI Listing
July 2020

Extensive Changes in Transcriptomic "Fingerprints" and Immunological Cells in the Large Organs of Patients Dying of Acute Septic Shock and Multiple Organ Failure Caused by .

Front Cell Infect Microbiol 2020 19;10:42. Epub 2020 Feb 19.

Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.

Patients developing meningococcal septic shock reveal levels of (10-10/mL) and endotoxin (10-10 EU/mL) in the circulation and organs, leading to acute cardiovascular, pulmonary and renal failure, coagulopathy and a high case fatality rate within 24 h. To investigate transcriptional profiles in heart, lungs, kidneys, liver, and spleen and immunostain key inflammatory cells and proteins in post mortem formalin-fixed, paraffin-embedded (FFPE) tissue samples from meningococcal septic shock patients. Total RNA was isolated from FFPE and fresh frozen (FF) tissue samples from five patients and two controls (acute non-infectious death). Differential expression of genes was detected using Affymetrix microarray analysis. Lung and heart tissue samples were immunostained for T-and B cells, macrophages, neutrophils and the inflammatory markers PAI-1 and MCP-1. Inflammatory mediators were quantified in lysates from FF tissues. The transcriptional profiles showed a complex pattern of protein-coding and non-coding RNAs with significant regulation of pathways associated with organismal death, cell death and survival, leukocyte migration, cellular movement, proliferation of cells, cell-to-cell signaling, immune cell trafficking, and inflammatory responses in an organ-specific clustering manner. The canonical pathways including acute phase response-, EIF2-, TREM1-, IL-6-, HMBG1-, PPAR signaling, and LXR/RXR activation were associated with acute heart, pulmonary, and renal failure. Fewer genes were regulated in the liver and particularly in the spleen. The main upstream regulators were TNF, IL-1β, IL-6, RICTOR, miR-6739-3p, and CD3. Increased numbers of inflammatory cells (CD68+, MPO+, CD3+, and CD20+) were found in lungs and heart. PAI-1 inhibiting fibrinolysis and MCP-1 attracting leukocyte were found significantly present in the septic tissue samples compared to the controls. FFPE tissue samples can be suitable for gene expression studies as well as immunostaining of specific cells or molecules. The most pronounced gene expression patterns were found in the organs with highest levels of DNA. Thousands of protein-coding and non-coding RNA transcripts were altered in lungs, heart and kidneys. We identified specific biomarker panels both protein-coding and non-coding RNA transcripts, which differed from organ to organ. Involvement of many genes and pathways add up and the combined effect induce organ failure.
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http://dx.doi.org/10.3389/fcimb.2020.00042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045056PMC
February 2020

Healthy Nordic Diet Modulates the Expression of Genes Related to Mitochondrial Function and Immune Response in Peripheral Blood Mononuclear Cells from Subjects with Metabolic Syndrome-A SYSDIET Sub-Study.

Mol Nutr Food Res 2019 Apr 9:e1801405. Epub 2019 Apr 9.

Department of Nutrition, Institute for Basic Medical Sciences, University of Oslo, 0316, Oslo, Norway.

Scope: To explore the effect of a healthy Nordic diet on the global transcriptome profile in peripheral blood mononuclear cells (PBMCs) of subjects with metabolic syndrome.

Methods And Results: Subjects with metabolic syndrome undergo a 18/24 week randomized intervention study comparing an isocaloric healthy Nordic diet with an average habitual Nordic diet served as control (SYSDIET study). Altogether, 68 participants are included. PBMCs are obtained before and after intervention and total RNA is subjected to global transcriptome analysis. 1302 probe sets are differentially expressed between the diet groups (p-value < 0.05). Twenty-five of these are significantly regulated (FDR q-value < 0.25) and are mainly involved in mitochondrial function, cell growth, and cell adhesion. The list of 1302 regulated probe sets is subjected to functional analyses. Pathways and processes involved in the mitochondrial electron transport chain, immune response, and cell cycle are downregulated in the healthy Nordic diet group. In addition, gene transcripts with common motifs for 42 transcription factors, including NFR1, NFR2, and NF-κB, are downregulated in the healthy Nordic diet group.

Conclusion: These results suggest that benefits of a healthy diet may be mediated by improved mitochondrial function and reduced inflammation.
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http://dx.doi.org/10.1002/mnfr.201801405DOI Listing
April 2019

Differential expression of vitamin D associated genes in the aorta of coronary artery disease patients with and without rheumatoid arthritis.

PLoS One 2018 23;13(8):e0202346. Epub 2018 Aug 23.

Department of Research, Innlandet Hospital Trust, Brumunddal, Norway.

Background: Vitamin D has an important role in the immune system, and has been linked to rheumatoid arthritis (RA) and coronary artery disease (CAD). The exact mechanisms by which vitamin D is involved in these processes are still unclear. Therefore, we wanted to search for differences in expression of genes involved in the vitamin D receptor (VDR) activation pathway and genes that are known to alter upon vitamin D stimulation, in the aortic adventitia of CAD patients with and without RA.

Methods: Affymetrix microarray was used to determine gene expression profile in surgical specimens from the adventitia of the ascending aorta of CAD patients with RA (n = 8) and without RA (n = 8) from the Feiring Heart Biopsy Study.

Results: We identified three vitamin D associated genes that were differentially expressed between RA and non-RA patients: Growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A) (FC = 1.47; p = 0.006), Nuclear Receptor Co-repressor 1 (NCOR1) (FC = 1,21; p = 0.005) and paraoxonases 2 (PON2) (FC = -1.37; p = 0.01). High expression of GADD45A in RA tissues was confirmed by real-time qRT-PCR. GADD45A expression correlated with plasma levels of 1,25(OH)2D3 (rs = 0.69; p = 0.003).

Conclusions: Microarray analyses revealed higher expression of GADD45A and NCOR1; and lower expression of PON2 in the aortic adventitia of RA than non-RA patients. Further studies are needed to elucidate if and how GADD45A, NCOR1 and PON2 are involved in the development of accelerated atherosclerosis in RA. In theory, some of these factors might have proatherogenic effects whereas others might reflect an underlying vascular pathology promoting atherogenesis (such as vascular stress).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202346PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107153PMC
February 2019

Transcription and microRNA Profiling of Cultured Human Tympanic Membrane Epidermal Keratinocytes.

J Assoc Res Otolaryngol 2018 06 5;19(3):243-260. Epub 2018 Apr 5.

Ear, Nose and Throat Department, Division of Surgery, Akershus University Hospital, Lørenskog, Norway.

The human tympanic membrane (TM) has a thin outer epidermal layer which plays an important role in TM homeostasis and ear health. The specialised cells of the TM epidermis have a different physiology compared to normal skin epidermal keratinocytes, displaying a dynamic and constitutive migration that maintains a clear TM surface and assists in regeneration. Here, we characterise and compare molecular phenotypes in keratinocyte cultures from TM and normal skin. TM keratinocytes were isolated by enzymatic digestion and cultured in vitro. We compared global mRNA and microRNA expression of the cultured cells with that of human epidermal keratinocyte cultures. Genes with either relatively higher or lower expression were analysed further using the biostatistical tools g:Profiler and Ingenuity Pathway Analysis. Approximately 500 genes were found differentially expressed. Gene ontology enrichment and Ingenuity analyses identified cellular migration and closely related biological processes to be the most significant functions of the genes highly expressed in the TM keratinocytes. The genes of low expression showed a marked difference in homeobox (HOX) genes of clusters A and C, giving the TM keratinocytes a strikingly low HOX gene expression profile. An in vitro scratch wound assay showed a more individualised cell movement in cells from the tympanic membrane than normal epidermal keratinocytes. We identified 10 microRNAs with differential expression, several of which can also be linked to regulation of cell migration and expression of HOX genes. Our data provides clues to understanding the specific physiological properties of TM keratinocytes, including candidate genes for constitutive migration, and may thus help focus further research.
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http://dx.doi.org/10.1007/s10162-018-0660-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5962475PMC
June 2018

Proliferative Cells Isolated from the Adult Human Peripheral Retina only Transiently Upregulate Key Retinal Markers upon Induced Differentiation.

Curr Eye Res 2018 03 21;43(3):340-349. Epub 2017 Nov 21.

a Center for Eye Research, Department of Ophthalmology , Oslo University Hospital and University of Oslo , Oslo , Norway.

Purpose/Aim: The adult human retina has limited regenerative potential, and severe injury will result in permanent damage. Lower vertebrates handle retinal injury by activating neural stem cells (NSCs) in the ciliary marginal zone (CMZ). Müller glia-like cells expressing markers of NSCs are also present in the peripheral retina (PR) of the adult human eye, leading to the hypothesis that a CMZ-like zone might exists also in humans. In order to shed further light on this hypothesis we investigated the in vitro differentiation potential of proliferative cells isolated from the adult human PR towards a retinal phenotype.

Materials And Methods: Proliferative cells were isolated from the peripheral retina of human eyes (n = 6) within 24 to 48 hours post mortem and further expanded for 2 or 3 passages before being differentiated for 1-3 weeks. Gene expression was analyzed by microarray and qRT-PCR analysis, while protein expression was identified by immunocytochemistry.

Results: A high density of cells co-staining with markers for progenitor cells and Müller glia was found in situ in the PR. Cells isolated from this region and cultured adherently showed fibrillary processes and were positive for the immature marker Nestin and the glial marker GFAP, while a few co-expressed PAX6. After 7 days of differentiation, there was a transient upregulation of early and mature photoreceptor markers, including NRL, CRX, RHO and RCVRN, as well as the Müller cell and retinal pigmented epithelium (RPE) marker CRALBP, and the early RPE marker MITF. However, the expression of all these markers dropped from Day 14 and onwards.

Conclusions: Upon exposure of proliferating cells from the adult human PR to differentiating conditions in culture, there is a widespread change in morphology and gene expression, including the upregulation of key retinal markers. However, this upregulation is only transient and decreases after 14 days of differentiation.
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http://dx.doi.org/10.1080/02713683.2017.1403630DOI Listing
March 2018

Transcriptomic data from two primary cell models stimulating human monocytes suggest inhibition of oxidative phosphorylation and mitochondrial function by N. meningitidis which is partially up-regulated by IL-10.

BMC Immunol 2017 10 27;18(1):46. Epub 2017 Oct 27.

Blood Cell Research Group, Section for Research, Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.

Background: Biological interpretation of DNA microarray data may differ depending on underlying assumptions and statistical tests of bioinformatics tools used. We used Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) to analyze previously generated DNA microarray data from human monocytes stimulated with N. meningitidis and IL-10 ("the model system"), and with meningococcal sepsis plasma before and after immunodepletion of IL-10 ("the patient plasma system"). The objectives were to compare if the two bioinformatics methods resulted in similar biological interpretation of the datasets, and to identify whether GSEA provided additional insight compared with IPA about the monocyte host response to meningococcal activation.

Results: In both experimental models, GSEA and IPA identified genes associated with pro-inflammatory innate immune activation, including TNF-signaling, Toll-like receptor signaling, JAK-STAT-signaling, and type I and type II interferon signaling. GSEA identified genes regulated by the presence of IL-10 with similar gene sets in both the model system and the patient plasma system. In the model system, GSEA and IPA in sum identified 170 genes associated with oxidative phosphorylation/mitochondrial function to be down-regulated in monocytes stimulated with meningococci. In the patient plasma system, GSEA and IPA in sum identified 122 genes associated with oxidative phosphorylation/mitochondrial dysfunction to be down-regulated by meningococcal sepsis plasma depleted for IL-10. Using IPA, we identified IL-10 to up-regulate 18 genes associated with oxidative phosphorylation/mitochondrial function that were down-regulated by N. meningitidis.

Conclusions: Biological processes associated with the gene expression changes in the model system of meningococcal sepsis were comparable with the results found in the patient plasma system. By combining GSEA with IPA, we discovered an inhibitory effect of N. meningitidis on genes associated with mitochondrial function and oxidative phosphorylation, and that IL-10 partially reverses this strong inhibitory effect, thereby identifying, to our knowledge, yet another group of genes where IL-10 regulates the effect of LPS. We suggest that relying on a single bioinformatics tool together with an arbitrarily chosen filtering criteria for data analysis may result in overlooking relevant biological processes and signaling pathways associated with genes differentially expressed between compared experimental conditions.
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http://dx.doi.org/10.1186/s12865-017-0229-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659018PMC
October 2017

Role of Human Corneal Stroma-Derived Mesenchymal-Like Stem Cells in Corneal Immunity and Wound Healing.

Sci Rep 2016 05 19;6:26227. Epub 2016 May 19.

Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary.

Corneal tissue regeneration is of crucial importance for maintaining normal vision. We aimed to isolate and cultivate human corneal stroma-derived mesenchymal stem-like cells (CSMSCs) from the central part of cadaver corneas and study their phenotype, multipotency, role in immunity and wound healing. The isolated cells grew as monolayers in vitro, expressed mesenchymal- and stemness-related surface markers (CD73, CD90, CD105, CD140b), and were negative for hematopoietic markers as determined by flow cytometry. CSMSCs were able to differentiate in vitro into fat, bone and cartilage. Their gene expression profile was closer to bone marrow-derived MSCs (BMMSCs) than to limbal epithelial stem cells (LESC) as determined by high-throughput screening. The immunosuppressive properties of CSMSCs were confirmed by a mixed lymphocyte reaction (MLR), while they could inhibit proliferation of activated immune cells. Treatment of CSMSCs by pro-inflammatory cytokines and toll-like receptor ligands significantly increased the secreted interleukin-6 (IL-6), interleukin-8 (IL-8) and C-X-C motif chemokine 10 (CXCL-10) levels, as well as the cell surface adhesion molecules. CSMSCs were capable of closing a wound in vitro under different stimuli. These cells thus contribute to corneal tissue homeostasis and play an immunomodulatory and regenerative role with possible implications in future cell therapies for treating sight-threatening corneal diseases.
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http://dx.doi.org/10.1038/srep26227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872602PMC
May 2016

Transcriptome Analysis of Cultured Limbal Epithelial Cells on an Intact Amniotic Membrane following Hypothermic Storage in Optisol-GS.

J Funct Biomater 2016 Feb 18;7(1). Epub 2016 Feb 18.

Department of Ophthalmology, Drammen Hospital, Vestre Viken Hospital Trust, Drammen 3004, Norway.

The aim of the present study was to investigate the molecular mechanisms underlying activation of cell death pathways using genome-wide transcriptional analysis in human limbal epithelial cell (HLEC) cultures following conventional hypothermic storage in Optisol-GS. Three-week HLEC cultures were stored in Optisol-GS for 2, 4, and 7 days at 4 °C. Partek Genomics Suite software v.6.15.0422, (Partec Inc., St. Louis, MO, USA) was used to identify genes that showed significantly different (P < 0.05) levels of expression following hypothermic storage compared to non-stored cell sheets. There were few changes in gene expression after 2 days of storage, but several genes were differently regulated following 4 and 7 days of storage. The histone-coding genes HIST1H3A and HIST4H4 were among the most upregulated genes following 4 and 7 days of hypothermic storage. Bioinformatic analysis suggested that these two genes are involved in a functional network highly associated with cell death, necrosis, and transcription of RNA. HDAC1, encoding histone deacetylase 1, was the most downregulated gene after 7 days of storage. Together with other downregulated genes, it is suggested that HDAC1 is involved in a regulating network significantly associated with cellular function and maintenance, differentiation of cells, and DNA repair. Our data suggest that the upregulated expression of histone-coding genes together with downregulated genes affecting cell differentiation and DNA repair may be responsible for increased cell death following hypothermic storage of cultured HLEC. In summary, our results demonstrated that a higher number of genes changed with increasing storage time. Moreover, in general, larger differences in absolute gene expression values were observed with increasing storage time. Further understanding of these molecular mechanisms is important for optimization of storage technology for limbal epithelial sheets.
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http://dx.doi.org/10.3390/jfb7010004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810063PMC
February 2016

Gene expression profiling of Gram-negative bacteria-induced inflammation in human whole blood: The role of complement and CD14-mediated innate immune response.

Genom Data 2015 Sep 30;5:176-83. Epub 2015 May 30.

Research Laboratory, Nordland Hospital, Bodø, Norway ; Faculty of Health Sciences, K.G. Jebsen TREC, University of Tromsø, Tromsø, Norway ; Center of Molecular Inflammation Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway ; Department of Immunology, Oslo University Hospital, K.G. Jebsen IRC, University of Oslo, Oslo, Norway.

Non-sterile pathogen-induced sepsis and sterile inflammation like in trauma or ischemia-reperfusion injury may both coincide with the life threatening systemic inflammatory response syndrome and multi-organ failure. Consequently, there is an urgent need for specific biomarkers in order to distinguish sepsis from sterile conditions. The overall aim of this study was to uncover putative sepsis biomarkers and biomarker pathways, as well as to test the efficacy of combined inhibition of innate immunity key players complement and Toll-like receptor co-receptor CD14 as a possible therapeutic regimen for sepsis. We performed whole blood gene expression analyses using microarray in order to profile Gram-negative bacteria-induced inflammatory responses in an ex vivo human whole blood model. The experiments were performed in the presence or absence of inhibitors of complement proteins (C3 and CD88 (C5a receptor 1)) and CD14, alone or in combination. In addition, we used blood from a C5-deficient donor. Anti-coagulated whole blood was challenged with heat-inactivated Escherichia coli for 2 h, total RNA was isolated and microarray analyses were performed on the Affymetrix GeneChip Gene 1.0 ST Array platform. The initial experiments were performed in duplicates using blood from two healthy donors. C5-deficiency is very rare, and only one donor could be recruited. In order to increase statistical power, a technical replicate of the C5-deficient samples was run. Subsequently, log2-transformed intensities were processed by robust multichip analysis and filtered using a threshold of four. In total, 73 microarray chips were run and analyzed. The normalized and filtered raw data have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible with GEO Series accession number GSE55537. Linear models for microarray data were applied to estimate fold changes between data sets and the respective multiple testing adjusted p-values (FDR q-values). The interpretation of the data has been published by Lau et al. in an open access article entitled "CD14 and Complement Crosstalk and Largely Mediate the Transcriptional Response to Escherichia coli in Human Whole Blood as revealed by DNA Microarray" (Lau et al., 2015).
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http://dx.doi.org/10.1016/j.gdata.2015.05.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4583639PMC
September 2015

Serglycin protects against high fat diet-induced increase in serum LDL in mice.

Glycoconj J 2015 Dec 21;32(9):703-14. Epub 2015 Sep 21.

Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Pb. 1046, Blindern, 0317, Oslo, Norway.

Proteoglycans have been implicated in regulation of lipoprotein metabolism. However, the impact of serglycin, the major proteoglycan expressed by many hematopoietic- and endothelial cells, on lipoprotein metabolism has not been explored. Here we addressed this issue by comparing several parameters of lipid metabolism in wild type (WT) and serglycin-/- mice, both at baseline and after feeding mice the Paigen diet. We show that, after feeding this diet for 20 weeks, serglycin deficient mice exhibited elevated concentrations of serum LDL in comparison with WT mice, thus suggesting that serglycin protects against an elevation of serum LDL levels after intake of a high-fat diet. Body weight increased in both groups, but only significantly in the serglycin-/- group. To explore the mechanism underlying this phenotype, genome-wide expression analysis was performed on liver tissues from WT and serglycin-/- mice. This analysis showed that serglycin-deficiency is associated with differential expression of numerous genes involved in the regulation of lipid metabolism, suggesting that the impact of serglycin on LDL levels may be related to effects at the gene expression level. In particular, several members of the CYP gene family were differently regulated in serglycin-/- compared with WT mice. Moreover, upstream regulator analysis suggested that several pro-inflammatory pathways, including the NFκB pathway, could contribute to the impact of serglycin on LDL. Hence, the elevation of serum LDL seen in serglycin-/- mice may be linked to dysregulated inflammatory responses. Taken together, our findings introduce serglycin as a novel player in processes that regulate lipid metabolism.
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http://dx.doi.org/10.1007/s10719-015-9621-7DOI Listing
December 2015

CD14 and complement crosstalk and largely mediate the transcriptional response to Escherichia coli in human whole blood as revealed by DNA microarray.

PLoS One 2015 23;10(2):e0117261. Epub 2015 Feb 23.

Research Laboratory and Department of Laboratory Medicine, Nordland Hospital, Bodø, Norway; Faculty of Health Sciences, University of Tromsø, Tromsø, Norway; Center of Molecular Inflammation Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway; Institute of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo, Oslo, Norway.

Systemic inflammation like in sepsis is still lacking specific diagnostic markers and effective therapeutics. The first line of defense against intruding pathogens and endogenous damage signals is pattern recognition by e.g., complement and Toll-like receptors (TLR). Combined inhibition of a key complement component (C3 and C5) and TLR-co-receptor CD14 has been shown to attenuate certain systemic inflammatory responses. Using DNA microarray and gene annotation analyses, we aimed to decipher the effect of combined inhibition of C3 and CD14 on the transcriptional response to bacterial challenge in human whole blood. Importantly, combined inhibition reversed the transcriptional changes of 70% of the 2335 genes which significantly responded to heat-inactivated Escherichia coli by on average 80%. Single inhibition was less efficient (p<0.001) but revealed a suppressive effect of C3 on 21% of the responding genes which was partially counteracted by CD14. Furthermore, CD14 dependency of the Escherichia coli-induced response was increased in C5-deficient compared to C5-sufficient blood. The observed crucial distinct and synergistic roles for complement and CD14 on the transcriptional level correspond to their broad impact on the inflammatory response in human blood, and their combined inhibition may become inevitable in the early treatment of acute systemic inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117261PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338229PMC
January 2016

Immunomodulatory effects of the Agaricus blazei Murrill-based mushroom extract AndoSan in patients with multiple myeloma undergoing high dose chemotherapy and autologous stem cell transplantation: a randomized, double blinded clinical study.

Biomed Res Int 2015 18;2015:718539. Epub 2015 Jan 18.

Institute of Clinical Medicine, University of Oslo, 0372 Oslo, Norway ; Department of Immunology and Transfusion Medicine, Oslo University Hospital, 0424 Oslo, Norway.

Forty patients with multiple myeloma scheduled to undergo high dose chemotherapy with autologous stem cell support were randomized in a double blinded fashion to receive adjuvant treatment with the mushroom extract AndoSan, containing 82% of Agaricus blazei Murrill (19 patients) or placebo (21 patients). Intake of the study product started on the day of stem cell mobilizing chemotherapy and continued until the end of aplasia after high dose chemotherapy, a period of about seven weeks. Thirty-three patients were evaluable for all study endpoints, while all 40 included patients were evaluable for survival endpoints. In the leukapheresis product harvested after stem cell mobilisation, increased percentages of Treg cells and plasmacytoid dendritic cells were found in patients receiving AndoSan. Also, in this group, a significant increase of serum levels of IL-1ra, IL-5, and IL-7 at the end of treatment was found. Whole genome microarray showed increased expression of immunoglobulin genes, Killer Immunoglobulin Receptor (KIR) genes, and HLA genes in the Agaricus group. Furthermore, AndoSan displayed a concentration dependent antiproliferative effect on mouse myeloma cells in vitro. There were no statistically significant differences in treatment response, overall survival, and time to new treatment. The study was registered with Clinicaltrials.gov NCT00970021.
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http://dx.doi.org/10.1155/2015/718539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4312620PMC
October 2015

IL-10 immunodepletion from meningococcal sepsis plasma induces extensive changes in gene expression and cytokine release in stimulated human monocytes.

Innate Immun 2015 May 18;21(4):429-49. Epub 2014 Sep 18.

Blood Cell Research Group, Section for Research, Department of Medical Biochemistry, Oslo University Hospital, University of Oslo, Oslo, Norway.

The severity of systemic meningococcal disease (SMD) correlates to plasma concentrations of LPS and IL-10, with the highest levels detected in non-survivors. Here, plasma from patients with SMD containing high and low concentrations of LPS were incubated with human monocytes before and after immunodepletion of IL-10 to study the effect of IL-10 on gene expression and cytokine release. Patient plasma containing IL-10 induced the expression of 1657 genes in human monocytes when compared with gene expression induced by low LPS plasma. After immunodepletion of IL-10, this number increased to 2260. By directly comparing the gene expression profiles induced before and after immunodepletion of IL-10, the presence of IL-10 differentially regulated 373 genes. Functional classes associated with these genes were cellular function and maintenance, cellular development, cellular growth and proliferation, cell-cell signaling and interaction and cellular movement. Immunodepletion of IL-10 resulted in down-regulation of genes of the leukocyte immunoglobulin-like receptor family, and up-regulation of genes of type I IFN signaling, TLR signaling, the inflammasomes, coagulation and fibrinolysis. Finally, immunodepletion of IL-10 increased the protein levels of IL-1β, IL-8, TNF-α, MIP-1α and MIP-1β. Data suggest that IL-10 in meningococcal sepsis plasma regulates a variety of genes and signaling pathways, likely leading to an overall inhibitory effect on the inflammatory response induced in meningococcal sepsis.
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http://dx.doi.org/10.1177/1753425914547743DOI Listing
May 2015

Methylation of bone SOST, its mRNA, and serum sclerostin levels correlate strongly with fracture risk in postmenopausal women.

J Bone Miner Res 2015 Feb;30(2):249-56

Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway; Lovisenberg Diakonale Hospital, Oslo, Norway; Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.

Inhibition of sclerostin, a glycoprotein secreted by osteocytes, offers a new therapeutic paradigm for treatment of osteoporosis (OP) through its critical role as Wnt/catenin signaling regulator. This study describes the epigenetic regulation of SOST expression in bone biopsies of postmenopausal women. We correlated serum sclerostin to bone mineral density (BMD), fractures, and bone remodeling parameters, and related these findings to epigenetic and genetic disease mechanisms. Serum sclerostin and bone remodeling biomarkers were measured in two postmenopausal groups: healthy (BMD T-score > -1) and established OP (BMD T-score < -2.5, with at least one low-energy fracture). Bone specimens were used to analyze SOST mRNAs, single nucleotide polymorphisms (SNPs), and DNA methylation changes. The SOST gene promoter region showed increased CpG methylation in OP patients (n = 4) compared to age and body mass index (BMI) balanced controls (n = 4) (80.5% versus 63.2%, p = 0.0001) with replication in independent cohorts (n = 27 and n = 36, respectively). Serum sclerostin and bone SOST mRNA expression correlated positively with age-adjusted and BMI-adjusted total hip BMD (r = 0.47 and r = 0.43, respectively; both p < 0.0005), and inversely to serum bone turnover markers. Five SNPs, one of which replicates in an independent population-based genomewide association study (GWAS), showed association with serum sclerostin or SOST mRNA levels under an additive model (p = 0.0016 to 0.0079). Genetic and epigenetic changes in SOST influence its bone mRNA expression and serum sclerostin levels in postmenopausal women. The observations suggest that increased SOST promoter methylation seen in OP is a compensatory counteracting mechanism, which lowers serum sclerostin concentrations and reduces inhibition of Wnt signaling in an attempt to promote bone formation.
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http://dx.doi.org/10.1002/jbmr.2342DOI Listing
February 2015

RNA-sequencing analysis of HepG2 cells treated with atorvastatin.

PLoS One 2014 25;9(8):e105836. Epub 2014 Aug 25.

Fürst Medical Laboratory, Oslo, Norway.

The cholesterol-lowering drug atorvastatin is among the most prescribed drug in the world. Alternative splicing in a number of genes has been reported to be associated with variable statin response. RNA-seq has proven to be a powerful technique for genome-wide splice variant analysis. In the present study, we sought to investigate atorvastatin responsive splice variants in HepG2 cells using RNA-seq analysis to identify novel candidate genes implicated in cholesterol homeostasis and in the statin response. HepG2 cells were treated with 10 µM atorvastatin for 24 hours. RNA-seq and exon array analyses were performed. The validation of selected genes was performed using Taqman gene expression assays. RNA-seq analysis identified 121 genes and 98 specific splice variants, of which four were minor splice variants to be differentially expressed, 11 were genes with potential changes in their splicing patterns (SYCP3, ZNF195, ZNF674, MYD88, WHSC1, KIF16B, ZNF92, AGER, FCHO1, SLC6A12 and AKAP9), and one was a gene (RAP1GAP) with differential promoter usage. The IL21R transcript was detected to be differentially expressed via RNA-seq and RT-qPCR, but not in the exon array. In conclusion, several novel candidate genes that are affected by atorvastatin treatment were identified in this study. Further studies are needed to determine the biological significance of the atorvastatin responsive splice variants that have been uniquely identified using RNA-seq.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105836PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143339PMC
November 2015

Global miRNA expression analysis of serous and clear cell ovarian carcinomas identifies differentially expressed miRNAs including miR-200c-3p as a prognostic marker.

BMC Cancer 2014 Feb 11;14:80. Epub 2014 Feb 11.

Department of Gynecological Oncology, Oslo University Hospital (OUH), The Norwegian Radium Hospital, Postbox 4953 Nydalen 0424, Oslo, Norway.

Background: Improved insight into the molecular characteristics of the different ovarian cancer subgroups is needed for developing a more individualized and optimized treatment regimen. The aim of this study was to a) identify differentially expressed miRNAs in high-grade serous ovarian carcinoma (HGSC), clear cell ovarian carcinoma (CCC) and ovarian surface epithelium (OSE), b) evaluate selected miRNAs for association with clinical parameters including survival and c) map miRNA-mRNA interactions.

Methods: Differences in miRNA expression between HGSC, CCC and OSE were analyzed by global miRNA expression profiling (Affymetrix GeneChip miRNA 2.0 Arrays, n = 12, 9 and 9, respectively), validated by RT-qPCR (n = 35, 19 and 9, respectively), and evaluated for associations with clinical parameters. For HGSC, differentially expressed miRNAs were linked to differentially expressed mRNAs identified previously.

Results: Differentially expressed miRNAs (n = 78) between HGSC, CCC and OSE were identified (FDR < 0.01%), of which 18 were validated (p < 0.01) using RT-qPCR in an extended cohort. Compared with OSE, miR-205-5p was the most overexpressed miRNA in HGSC. miR-200 family members and miR-182-5p were the most overexpressed in HGSC and CCC compared with OSE, whereas miR-383 was the most underexpressed. miR-205-5p and miR-200 members target epithelial-mesenchymal transition (EMT) regulators, apparently being important in tumor progression. miR-509-3-5p, miR-509-5p, miR-509-3p and miR-510 were among the strongest differentiators between HGSC and CCC, all being significantly overexpressed in CCC compared with HGSC. High miR-200c-3p expression was associated with poor progression-free (p = 0.031) and overall (p = 0.026) survival in HGSC patients. Interacting miRNA and mRNA targets, including those of a TP53-related pathway presented previously, were identified in HGSC.

Conclusions: Several miRNAs differentially expressed between HGSC, CCC and OSE have been identified, suggesting a carcinogenetic role for these miRNAs. miR-200 family members, targeting EMT drivers, were mostly overexpressed in both subgroups, among which miR-200c-3p was associated with survival in HGSC patients. A set of miRNAs differentiates CCC from HGSC, of which miR-509-3-5p and miR-509-5p are the strongest classifiers. Several interactions between miRNAs and mRNAs in HGSC were mapped.
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http://dx.doi.org/10.1186/1471-2407-14-80DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928323PMC
February 2014

Comparison of upstream regulators in human ex vivo cultured cornea limbal epithelial stem cells and differentiated corneal epithelial cells.

BMC Genomics 2013 Dec 17;14:900. Epub 2013 Dec 17.

Stem Cells and Eye Research Laboratory, University of Debrecen, Debrecen, Hungary.

Background: The surface of the human eye is covered by corneal epithelial cells (CECs) which regenerate from a small population of limbal epithelial stem cells (LESCs). Cell therapy with LESCs is a non-penetrating treatment for preventing blindness due to LESC deficiency or dysfunction. Our aim was to identify new putative molecular markers and upstream regulators in the LESCs and associated molecular pathways.

Results: Genome-wide microarray transcriptional profiling was used to compare LESCs to differentiated human CECs. Ingenuity-based pathway analysis was applied to identify upstream regulators and pathways specific to LESCs. ELISA and flow cytometry were used to measure secreted and surface expressed proteins, respectively. More than 2 fold increase and decrease in expression could be found in 1830 genes between the two cell types. A number of molecules functioning in cellular movement (381), proliferation (567), development (552), death and survival (520), and cell-to-cell signaling (290) were detected having top biological functions in LESCs and several of these were confirmed by flow cytometric surface protein analysis. Custom-selected gene groups related to stemness, differentiation, cell adhesion, cytokines and growth factors as well as angiogenesis could be analyzed. The results show that LESCs play a key role not only in epithelial differentiation and tissue repair, but also in controlling angiogenesis and extracellular matrix integrity. Some pro-inflammatory cytokines were found to be important in stemness-, differentiation- and angiogenesis-related biological functions: IL-6 and IL-8 participated in most of these biological pathways as validated by their secretion from LESC cultures.

Conclusions: The gene and molecular pathways may provide a more specific understanding of the signaling molecules associated with LESCs, therefore, help better identify and use these cells in the treatment of ocular surface diseases.
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http://dx.doi.org/10.1186/1471-2164-14-900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3880589PMC
December 2013

Attenuated RORC expression in the presence of EMT progression in somatotroph adenomas following treatment with somatostatin analogs is associated with poor clinical recovery.

PLoS One 2013 25;8(6):e66927. Epub 2013 Jun 25.

Section of Specialized Endocrinology, Department of Endocrinology, Oslo University Hospital, Oslo, Norway.

Somatostatin analogs (SA) have been established as the first line medical treatment for acromegaly, but following long-term treatment, SA normalizes GH and IGF-I levels in only 40-60% of patients. The epithelial marker E-cadherin plays a crucial role in the epithelial mesenchymal transition (EMT) and is associated with a poor response to SA treatment. We hypothesized that the characterization of transcripts regulated by SA in somatotroph adenomas with high and low E-cadherin expression may identify signaling pathways and mediators that can explain the poor response to SA treatment. We performed a microarray analysis of sixteen adenomas with different levels of E-cadherin and SA treatment to identify regulated transcripts. Candidate transcripts were further explored in vivo in sixty-five adenomas, and interactions between SA treatment and EMT progression on mRNA expression profiles and associations with clinical recovery were assessed. Finally, the effects of SA treatment on adenoma cells in vitro from acromegalic patients were determined. Microarray analysis of selected adenomas with differential E-cadherin expression, as a marker of EMT progression, identified 172 genes that displayed differential expression that was dependent on SA treatment. The validation of selected candidates in the entire cohort identified 9 transcripts that showed an interaction between E-cadherin expression and SA treatment. Further analysis of the impact of these genes suggests that attenuated RORC expression in somatotroph adenomas is associated with increased tumor size and a blunted clinical response. Our study indicates that attenuated RORC may be involved in the poor clinical response to SA treatment in patients with acromegaly.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066927PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3692554PMC
February 2014

Epithelial splicing regulator protein 1 and alternative splicing in somatotroph adenomas.

Endocrinology 2013 Sep 3;154(9):3331-43. Epub 2013 Jul 3.

Section of Specialized Endocrinology and Research Institute for Internal Medicine, Oslo University Hospital, Rikshospitalet, University of Oslo, PO Box 4950 Nydalen, 0424 Oslo, Norway.

Somatotroph adenomas secrete supraphysiological amounts of GH, causing acromegaly. We have previously hypothesized that epithelial mesenchymal transition (EMT) may play a central role in the progression of these adenomas and that epithelial splicing regulator 1 (ESRP1) may function prominently as a master regulator of the EMT process in pituitary adenomas causing acromegaly. To further elucidate the role of ESRP1 in somatotroph adenomas and in EMT progression, we used RNA sequencing (RNAseq) to sequence somatotroph adenomas characterized by high and low ESRP1 levels. Transcripts identified by RNAseq were analyzed in 65 somatotroph adenomas and in GH-producing pituitary rat cells with a specific knockdown of Esrp1. The clinical importance of the transcripts was further investigated by correlating mRNA expression levels with clinical indices of disease activity and treatment response. Many of the transcripts and isoforms identified by RNAseq and verified by quantitative PCR were involved in vesicle transport and calcium signaling and were associated with clinical outcomes. Silencing Esrp1 in GH3 cells resulted in changes of gene expression overlapping the data observed in human somatotroph adenomas and revealed a decreased granulation pattern and attenuated GH release. We observed an alternative splicing pattern for F-box and leucine-rich repeat protein 20, depending on the ESPR1 levels and on changes in circulating IGF-I levels after somatostatin analog treatment. Our study indicates that ESRP1 in somatotroph adenomas regulates transcripts that may be essential in the EMT progression and in the response to somatostatin analog treatment.
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http://dx.doi.org/10.1210/en.2013-1051DOI Listing
September 2013

TFPI alpha and beta regulate mRNAs and microRNAs involved in cancer biology and in the immune system in breast cancer cells.

PLoS One 2012 5;7(10):e47184. Epub 2012 Oct 5.

Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway.

Emerging evidence indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s) mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIα or TFPIβ cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA) expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIα or TFPIβ, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion) and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. The expression profiles also correlated significantly with clinical phenotype and outcome. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor, small GTPases, and the NF-κB and JAK/STAT cascades when TFPI was overexpressed, and increased activity of the transcription factors NF-κB and Elk-1 and phospho-Akt levels was observed. Integrated mRNA-miRNA analyses showed that 19% and 32% of the differentially expressed genes in cells overexpressing TFPIα or TFPIβ, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and miRNAs involved in processes facilitating cancer cell growth and immunologic response, possibly by signal transduction involving the EGFR pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047184PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465304PMC
April 2013

Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.

PLoS One 2012 9;7(10):e47187. Epub 2012 Oct 9.

Department of Ophthalmology, Medical and Health Science Center, University of Debrecen, Hungary.

A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2 × 2 × 0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047187PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3467238PMC
May 2013

ZNF385B and VEGFA are strongly differentially expressed in serous ovarian carcinomas and correlate with survival.

PLoS One 2012 28;7(9):e46317. Epub 2012 Sep 28.

Department of Gynaecological Oncology, Oslo University Hospital, Oslo, Norway.

Background: The oncogenesis of ovarian cancer is poorly understood. The aim of this study was to identify mRNAs differentially expressed between moderately and poorly differentiated (MD/PD) serous ovarian carcinomas (SC), serous ovarian borderline tumours (SBOT) and superficial scrapings from normal ovaries (SNO), and to correlate these mRNAs with clinical parameters including survival.

Methods: Differences in mRNA expression between MD/PD SC, SBOT and SNO were analyzed by global gene expression profiling (n = 23), validated by RT-qPCR (n = 41) and correlated with clinical parameters.

Results: Thirty mRNAs differentially expressed between MD/PD SC, SBOT and SNO were selected from the global gene expression analyses, and 21 were verified (p<0.01) by RT-qPCR. Of these, 13 mRNAs were differentially expressed in MD/PD SC compared with SNO (p<0.01) and were correlated with clinical parameters. ZNF385B was downregulated (FC = -130.5, p = 1.2×10(-7)) and correlated with overall survival (p = 0.03). VEGFA was upregulated (FC = 6.1, p = 6.0×10(-6)) and correlated with progression-free survival (p = 0.037). Increased levels of TPX2 and FOXM1 mRNAs (FC = 28.5, p = 2.7×10(-10) and FC = 46.2, p = 5.6×10(-4), respectively) correlated with normalization of CA125 (p = 0.03 and p = 0.044, respectively). Furthermore, we present a molecular pathway for MD/PD SC, including VEGFA, FOXM1, TPX2, BIRC5 and TOP2A, all significantly upregulated and directly interacting with TP53.

Conclusions: We have identified 21 mRNAs differentially expressed (p<0.01) between MD/PD SC, SBOT and SNO. Thirteen were differentially expressed in MD/PD SC, including ZNF385B and VEGFA correlating with survival, and FOXM1 and TPX2 with normalization of CA125. We also present a molecular pathway for MD/PD SC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046317PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460818PMC
February 2013

Global effect of interleukin-10 on the transcriptional profile induced by Neisseria meningitidis in human monocytes.

Infect Immun 2012 Nov 10;80(11):4046-54. Epub 2012 Sep 10.

Blood Cell Research Unit, Section for Research, Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.

In meningococcal septic shock, the dominant inducer of inflammation is lipopolysaccharide (LPS) in the outer membrane of Neisseria meningitidis, while interleukin-10 (IL-10) is the principal anti-inflammatory cytokine. We have used microarrays and Ingenuity Pathway Analysis to study the global effects of IL-10 on gene expression induced by N. meningitidis, after exposure of human monocytes (n = 5) for 3 h to N. meningitidis (10(6) cells/ml), recombinant human IL-10 (rhIL-10) (25 ng/ml), and N. meningitidis combined with rhIL-10. N. meningitidis and IL-10 differentially expressed 3,579 and 648 genes, respectively. IL-10 downregulated 125 genes which were upregulated by N. meningitidis, including NLRP3, the key molecule of the NLRP3 inflammasome. IL-10 also upregulated 270 genes which were downregulated by N. meningitidis, including members of the leukocyte immunuglobulin-like receptor (LIR) family. Fifty-three genes revealed a synergistically increased expression when N. meningitidis and IL-10 were combined. AIM2 (the principal molecule of the AIM2 inflammasome) was among these genes (fold change [FC], 18.3 versus 7.4 and 9.4 after stimulation by N. meningitidis and IL-10, respectively). We detected reduced concentrations (92% to 40%) of six cytokines (IL-1b, IL-6, IL-8, tumor necrosis factor alpha [TNF-α], macrophage inflammatory protein alpha [MIP-α], MIP-β) in the presence of IL-10, compared with concentrations with stimulation by N. meningitidis alone. Our data analysis of the effects of IL-10 on gene expression induced by N. meningitidis suggests that high plasma levels of IL-10 in meningococcal septic shock plasma may have a profound effect on a variety of functions and cellular processes in human monocytes, including cell-to-cell signaling, cellular movement, cellular development, antigen presentation, and cell death.
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http://dx.doi.org/10.1128/IAI.00386-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486056PMC
November 2012