Biomedical subjects

Hans Geir Eiken

Publications and source records attributed to Hans Geir Eiken.

8 recordsLinked to original sources

PubMed2006

Circadian expression of clock genes in purified hematopoietic stem cells is developmentally regulated in mouse bone marrow.

OBJECTIVE: Clock genes are known to mediate circadian rhythms in the central nervous system and peripheral organs. Although they are expressed in mouse hematopoietic progenitor and stem cells, it is unknown if they are related to circadian rhythms in these cells. We therefore investigated the 24-hour patterns in the activity of several clock genes in the bone marrow (BM) side population (SP) primitive stem cells, and compared these 24-hour patterns to clock gene variations in the whole BM and liver. METHODS: Cells were obtained from 84 B6D2F(1) mice in three replicate experiments on the second day after release into constant darkness from a standardizing light-dark schedule. mRNA expression of clock genes was measured with quantitative reverse transcriptase polymerase chain reaction. RESULTS: mPer2 displayed circadian rhythms in SP cells, whole BM, and liver cells. mPer1 and mRev-erb alpha showed a circadian rhythm in whole BM and liver, but not SP cells. mBmal1 was not expressed rhythmically in SP cells, nor in the whole BM, contrary to rhythms observed in the liver. CONCLUSIONS: With the exception of mPer2, most clock genes studied in primitive hematopoietic SP stem cells were not oscillating in a fully organized circadian manner, which is similar to immature cells in rapidly proliferating organs, such as the testis and thymus. These findings indicate that circadian clock gene expression variations in BM are developmentally regulated.

Animals↗

PubMed2005

Clock gene expression in purified mouse hematopoietic stem cells.

OBJECTIVE: Circadian genes have recently been characterized in many tissues, but not in hematopoietic stem cells. These cells are rare in the bone marrow (BM), which makes it difficult to collect enough cells for detailed molecular analysis in a short period of time without reduced RNA quality. The aim was to improve methodology and reliability of clock gene expression analysis in purified mouse hematopoietic stem cells. METHODS: Stem cells were highly enriched by high-speed flow cytometric cell sorting of the side population (SP) cells from Hoechst 33342 (Hoechst)-stained mouse BM. Total RNA was isolated from sorted SP and whole BM cells and exposed to DNase treatment. The relative mRNA levels of major clock genes mPer1, mPer2, mBmal1, mCry1, mClock, and mRev-erb alpha were measured with real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) and normalized to m36B4, used as a reference gene. The clonogenity of sorted SP cells and whole BM; cells taken before and after sorting, were tested in colony-formation assay. RESULTS: Clock gene activity in sorted SP cells showed pronounced relative differences compared with whole BM for mPer1 and mCry1. The high-speed sorting procedure did not influence clock gene expression or cell clonogenity, even when this was performed with a delay period up to 24 hours. CONCLUSIONS: We demonstrated expression of six clock genes in mouse hematopoietic stem cells. A combination of high-speed flow cytometric sorting and Q-RT-PCR was shown to be useful and reliable for analysis of clock gene activity in small stem cell fractions.

ARNTL Transcription Factors↗

PubMed2005

Complete mutation screening and haplotype characterization of the BRCA1 gene in 61 familial breast cancer patients from Norway.

Mutations in the Breast-Cancer-1 (BRCA1) gene are the major cause of familial breast/ovarian cancer. Among familial breast cancer only, 15-20% have been suggested to have a deleterious mutation in BRCA1. A highly sensitive method (REF-SSCP) was applied to screen the open reading frame and the 5'UTRs of BRCA1 for mutations. The patient cohort comprised 61 unrelated moderate to high risk breast cancer patients from Western-Norway. Only one known deleterious BRCA1 mutation (c.816-817delGT) was found in two of the 61 patients (3.3%). Four haplotypes were established based on nine known single nucleotide polymorphisms. Two patients had a novel deletion (c.-33_-29delAAAAA) in the 5'UTR, and a novel amino acid substitution (L523W) was found in one patient. Size variations analysis in the 5'UTR was repeated in a cohort of 159 unrelated familial breast/ovarian cancer patients and 94 healthy blood donors. Two patients were identified with 5'UTR (c.-30 to -60) variations (CAAAA)5 and (CAAAA)7, instead of the (CAAAA)6-repeat. All of the identified 5'UTR size variations were localized between the start codon and the most stable secondary structures previously proposed for the exon 1b transcript. No such alterations were found among the healthy blood donors but association studies of the 5'UTR variations within the respective families were not conclusive.

5' Untranslated Regions↗

PubMed2004

Cytokine networks are pre-activated in T cells from HIV-infected patients on HAART and are under the control of cAMP.

OBJECTIVE: Cytokines seem to play a critical role in HIV infection. The cAMP/protein kinase A (PKA) type I pathway is shown to be hyper-activated and contributes to T-cell immune dysfunction in HIV infection. Here, we analysed firstly the levels of cytokine gene expression in unstimulated CD3+T cells from HIV-infected patients on HAART, and secondly the regulation of cytokine and cytokine-related genes by cAMP agonist and antagonist in anti-CD3 activated T cells in order to understand their effects on cytokine networks. METHODS: Cytokine Macro Array and real-time RT-PCR techniques were used to study cytokine gene expression in T cells of HIV-positive patients. RESULTS: Of the cytokine-related genes analysed 45% were expressed at twofold or higher levels in unstimulated T cells from HIV-infected patients as compared with healthy controls, and one-third of these genes were hypo-responsive upon activation as compared with controls. Furthermore, cAMP modulated levels of expression of a number of cytokine-related genes differently in patient and control T cells. CXCR4, CCR5 and amphiregulin were up-regulated by cAMP agonist, whereas other cytokine-related genes including macrophage inflammatory protein 1 beta, tumour necrosis factor-alpha and lymphotoxin-beta were markedly down-regulated by cAMP agonist in T cells from both HIV-infected patients and controls. Moreover, members of the chemokine/chemokine receptor family were over-represented among genes regulated by cAMP agonist/antagonist in patient T cells. CONCLUSIONS: Our data indicate that T cells from HIV-infected patients are in a pre-activated state and that a set of cytokine genes is hypo-responsive to activation and under tonic regulation by cAMP in these T cells.

Adult↗

PubMed2003

Evaluation of five different cDNA labeling methods for microarrays using spike controls.

BACKGROUND: Several different cDNA labeling methods have been developed for microarray based gene expression analysis. We have examined the accuracy and reproducibility of such five commercially available methods in detection of predetermined ratio values from target spike mRNAs (A. thaliana) in a background of total RNA. The five different labeling methods were: direct labeling (CyScribe), indirect labeling (FairPlay--aminoallyl), two protocols with dendrimer technology (3DNA Array 50 and 3DNA submicro), and hapten-antibody enzymatic labeling (Micromax TSA). Ten spike controls were mixed to give expected Cy5/Cy3 ratios in the range 0.125 to 6.0. The amounts of total RNA used in the labeling reactions ranged from 5-50 microg. RESULTS: The 3DNA array 50 and CyScribe labeling methods performed best with respect to relative deviation from the expected values (16% and 17% respectively). These two methods also displayed the best overall accuracy and reproducibility. The FairPlay method had the lowest total experimental variation (22%), but the estimated values were consistently higher than the expected values (36%). TSA had both the largest experimental variation and the largest deviation from the expected values (45% and 48% respectively). CONCLUSION: We demonstrate the usefulness of spike controls in validation and comparison of cDNA labeling methods for microarray experiments.

Arabidopsis↗

PubMed2003

Increased salivary gland tissue expression of Fas, Fas ligand, cytotoxic T lymphocyte-associated antigen 4, and programmed cell death 1 in primary Sjögren's syndrome.

OBJECTIVE: To assess salivary gland tissues obtained from patients with primary Sjögren's syndrome (SS) for the gene expression profile of the candidate genes TNFRSF6 (Fas), TNFSF6 (FasL), SSA1 (Ro52 alpha and the splice variant Ro52 beta), SSB (La), CTLA4, PDCD1 (PD-1), and ORM2, which were selected on the basis of their putative participation in salivary gland inflammation. METHODS: Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the expression of messenger RNA (mRNA). Tissue localization of the expressed proteins was detected by immunohistochemistry. RESULTS: Expression of mRNA was increased for Fas (5.1-fold; P < 0.001), FasL (8.8-fold; P < 0.05), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (11.2-fold; P < 0.01), programmed cell death 1 (PD-1) (15.2-fold; P < 0.05), Ro52 alpha (3.0-fold; P < 0.01), La (2.3-fold; P < 0.05), and orosomucoid 2 (ORM2) (4.4-fold; P < 0.05) in patients compared with controls when GAPDH was used as endogenous standard in duplex runs. In single runs using 2 other endogenous standards (18S and beta-actin), statistically significant differences between patients and controls were confirmed for expression of Fas, FasL, CTLA-4, and PD-1, but this difference was not observed for Ro52 alpha, La, and ORM2. Expression of Ro52 beta mRNA was similar in patients and controls. CONCLUSION: The present study demonstrates a substantial increase in expression of the negative regulator molecules PD-1 and CTLA-4 and the apoptotic signal molecules Fas and FasL in patients with primary SS compared with controls, which corresponded to the immunomorphologic pattern. The results strongly indicate that these molecules have central roles in the inflammatory process in the salivary glands of patients with primary SS.

Abatacept↗

PubMed2002

Stromal cell-derived factor-1alpha in unstable angina: potential antiinflammatory and matrix-stabilizing effects.

BACKGROUND: Chemokines play a pathogenic role in atherogenesis and plaque destabilization by activating and directing leukocytes into the atherosclerotic plaque. However, stromal cell-derived factor (SDF)-1 was recently found to have antiinflammatory effects, and we hypothesized that this chemokine could play a beneficial role in coronary artery disease. METHODS AND RESULTS: Plasma levels of SDF-1alpha were significantly decreased in patients with stable (n=30) and unstable angina (n=30) compared with healthy control subjects (n=20), particularly in those with unstable disease. By flow cytometry and RNase protection assay, we found decreased surface expression but increased gene expression of the SDF-1alpha receptor CXCR-4 in peripheral blood mononuclear cells (PBMC) from patients with stable angina and patients with unstable angina. In vitro, SDF-1alpha (500 ng/mL) reduced both unstimulated and endotoxin/mitogen-stimulated mRNA and protein levels of monocyte chemoattractant protein-1, interleukin-8, matrix metalloproteinase-9, and tissue factor while increasing tissue inhibitor of metalloproteinases-1 in PBMC from patients with unstable angina. The SDF-1alpha-mediated suppression of monocyte chemoattractant protein-1 and interleukin-8 appears to involve cAMP/protein kinase A type I-dependent pathways. Finally, although SDF-1alpha suppressed the spontaneous release of these inflammatory mediators in unstable angina, enhancing effects were seen in unstimulated PBMC from healthy control subjects, possibly reflecting that PBMC in unstable angina are preactivated in vivo. CONCLUSIONS: In contrast to several other chemokines, our findings suggest that SDF-1alpha, at least in high concentrations, may mediate antiinflammatory and matrix-stabilizing effects in unstable angina. These effects may promote plaque stabilization, and therapeutic intervention that enhances SDF-1alpha activity could potentially be beneficial in acute coronary syndromes.

Angina, Unstable↗

PubMed2002

Increased gene expression of tumor necrosis factor superfamily ligands in peripheral blood mononuclear cells during chronic heart failure.

OBJECTIVE: Inflammation may play a pathogenic role in chronic heart failure (CHF). The objective of the study was to characterise the imbalance in the cytokine network in CHF. METHODS: cDNA expression arrays were used to analyse the gene expression of cytokines and related mediators in peripheral blood mononuclear cells (PBMC) from CHF patients (n=8) and healthy controls (n=8). Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the gene expression of individual genes in additional 12 patients and eight controls. RESULTS: From 375 genes, 34 were upregulated and two downregulated in CHF patients in the cDNA expression array experiments. Regulated genes included chemokines/-receptors, members of the transforming growth factor beta superfamily, orphan receptors and in particular several members of the tumor necrosis factor (TNF) superfamily. Thus, 4-1BB ligand (L), APRIL, CD27L, CD40L, FasL, LIGHT, TRAIL-receptor 4 were upregulated, while TRAIL-receptor 3 was downregulated. Real-time quantitative RT-PCR confirmed significantly upregulated gene expression of APRIL, LIGHT, FasL and CD27L in CHF patients and showed in addition significantly enhanced gene expression of TNFalpha and TRAIL. CONCLUSION: The present study demonstrates differential gene expression in PBMC of several members of the cytokine network in CHF. In particular, the enhanced expression of several ligands in the TNF superfamily may reflect a potential pathogenic role of these cytokines in CHF.

4-1BB Ligand↗