Ole Olstad

Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha. Purified hPTH from yeast-culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses. The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone. In both the O-glycosylated forms that were separated by isocratic reverse-phase HPLC, two mannose-linked residues were localized to Thr79. In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66. Lysine is N-terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for O-glycosylation in yeast. The two glycosylated forms ...

Directional tag PCR subtractive hybridization was applied to construct a cDNA library generated from three different human osteosarcoma (OS) target cell lines (OHS, SaOS-2 and KPDXM) from which normal osteoblast (NO) sequences were subtracted. After two consecutive subtractive steps more than 98% of the common mRNAs species were depleted, leading to effective enrichment of the remaining target sequences. After differential screening of 960 clones, 81 candidates were further studied by Northern blot analysis and 73 represented separate mRNA species. Fifty-three of these showed enriched mRNA levels, of which 36 represented known and 17 not previously published cDNAs or EST sequences. The mRNAs showed a 1.4- to 504-fold enrichment compared to the mRNA levels in NO cells. The known mRNAs are: Ribosomal protein S11, KSP-37, Tethering factor SEC34, FXYD6, Alpha enolase, G-s-alpha, GPR85, DAF, RPL35A, GIF, TAPA-1, ANAPC11, DCI, hsp27, MRPS7 homolog, eIF p110 subunit, DPH2L, HMG-14, FB1 pro...

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).

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...

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 ...

During the past years, we and others discovered a series of human ATP-binding cassette (ABC) transporters, now referred to as ABC A-subfamily transporters. Recently, a novel testis-specific ABC A transporter, Abca17, has been cloned in rodent. In this study, we report the identification and characterization of the human ortholog of rodent Abca17. The novel human ABC A-transporter gene on chromosome 16p13.3 is ubiquitously expressed with highest expression in glandular tissues and the heart. The new ABC transporter gene exhibits striking nucleotide sequence homology with the recently cloned mouse (58%) and rat Abca17 (51%), respectively, and is located in the syntenic region of mouse Abca17 indicating that it represents the human ortholog of rodent Abca17. However, unlike in the mouse, the full-length ABCA17 transcript (4.3 kb) contains numerous mutations that preclude its translation into a bona fide ABC transporter protein strongly suggesting that the human ABCA17 gene is a transcr...

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.

Postmenopausal hormone therapy is associated with many diseases and conditions, e.g., cardiovascular diseases and asthma, but the underlying molecular mechanisms are incompletely understood. The aim of the current study was to investigate the effect of four different postmenopausal hormone therapy regimens on gene transcription. Twenty-four healthy postmenopausal women (six women in four groups) were randomly allocated to conventional-dose 17β-estradiol/norethisterone acetate (NETA), low-dose 17β-estradiol/NETA, tibolone, or raloxifene hydrochloride. RNA was isolated from whole blood before and after 6weeks of treatment. The changes in mRNA were assessed with a microarray chip. The genes FKBP5, IL13RA1, TPST1, and TLR2 were up-regulated and among the most significantly changed genes in the groups treated with conventional-dose 17β-estradiol/NETA and tibolone. Up-regulation of TPST1 was associated with reduction of tissue factor pathway inhibitor in plasma. Nine biological pathways were associated with conventional-dose 17β-estradiol/NETA, most significantly the pathways for asthma, toll-like receptor signaling, cell adhesion molecules, and MAPK signaling. Transcriptional changes with false discovery rate below 0.10 were found in 10 genes in the conventional-dose 17β-estradiol/NETA group, 7 genes in the tibolone group, and zero genes in the women on low-dose 17β-estradiol/NETA. No genes or pathways were associated with raloxifene treatment. The difference between low-dose and conventional-dose17β-estradiol/NETA indicates an effect of dose on transcriptional response. Several genes and pathways related to cell adhesion molecules and immunity related cell surface receptors were influenced by conventional-dose 17β-estradiol/NETA.

To investigate the early apoptosis that may be detected by Annexin V binding to phosphatidylserine and propidium iodide (PI) exclusion in human monocytes. When studying monocytes in culture, less than 40 % of these cells survive after 7 days. In the first 4 h, 24 % of monocytes in culture develop into Annexin V(+)/PI(-) cells. Human monocytes were investigated at 0 h and sorted into Annexin V(+) and Annexin V(-) by FACS after 4 h. Gene expression was examined by microarray analyses. At 4 h, Annexin V(+) monocytes versus Annexin V(-) cells showed 1220 differentially expressed genes. Ingenuity Pathway Analysis disclosed 153 genes related to cell death. Among these were caspase activators, caspase 6, Apaf 2 and FAS, as well as the autophagy gene ATG5. In addition, examination of the most up-regulated or down-regulated genes among the 1220 revealed genes involved in other biological processes, as well as genes not yet annotated. These included the non-annotated genes LOC28480 (fold change: 82) and 225767-at (fold change: 68) and the transcription factor SOX 4 (fold change: 24). conclusions: We suggest that apoptosis in cultured monocytes, as evidenced by Annexin V(+), operates through genes well known in apoptosis, but that the process also involves additional genes not commonly associated with apoptosis.

The number of circulating B-cells in peripheral blood plateaus between 2 and 24 months of age, and thereafter declines gradually. How this reflects the kinetics of the precursor B-cell pool in the bone marrow is of clinical interest, but has not been studied thoroughly in humans. The authors analyzed bone marrow (n = 37) from healthy children and adults (flow cytometry) searching for age-related changes in the total precursor B-cell compartment. In an age-matched cohort (n = 25) they examined age-related global gene expression changes (Affymetrix) in unsorted bone marrow with special reference to the recombination activating gene 1, RAG1. Subsequently, they searched the entire gene set for transcripts correlating to the RAG1 profile to discover other known and possibly new precursor B-cell related transcripts. Both methods disclosed a marked, transient increase of total precursor B-cells at 6-20 months, followed by a rapid decrease confined to the first 2 years. The decline thereafter was considerably slower, but continued until adulthood. The relative composition of total precursor B-cells, however, did not change significantly with age. The authors identified 54 genes that were highly correlated to the RAG1 profile (r >or= .9, p < 1 x 10(-8)). Of these 54 genes, 15 were characteristically B-lineage associated like CD19, CD79, VPREB, EBF1, and PAX5; the remaining 39 previously not described as distinctively B-lineage related. The marked, transient increase in precursor B-cells and RAG1 transcriptional activity is not reflected by a similar peak in B-cells in peripheral blood, whereas the sustained plateau concurs in time.

The objective of these studies was to investigate genes of importance in the pathogenesis of Aspergillus infections. To do so, we employed microarray methodology to explore gene expression in human monocytes infected with Aspergillus conidia as compared with unstimulated monocytes and those stimulated with lipopolysaccharide (LPS) signaling through TOLL-like receptor 4 (TLR4). We found 997 (P<or=0.05) differentially expressed (DE) genes in monocytes stimulated with conidia from Aspergillus fumigatus and 4976 (P<or=0.05) DE genes in monocytes stimulated with LPS. A total of 454 genes were only regulated by A. fumigatus stimulation. Selected genes were verified with qRT-PCR. Our study revealed significant changes in the expression of a number of genes of potential importance in the interaction between Aspergillus conidia and monocytes, including immune response genes and genes involved in apoptosis and initiation of Th1 response. Several of these changes in gene expression were unique to Aspergillus conidia and were not seen with LPS stimulation. These results do not support a role for TLR4 in the response to conidia. Further studies of genes regulated only by Aspergillus conidia may be of interest in the exploration of the pathogenesis of Aspergillus infections in man.

Background. Oral intake (60 ml daily) over 12 days in eight healthy volunteers of an immunostimulatory extract based on the medicinal mushroom Agaricus blazei Murill (AbM (AndoSan(™))), reduced the monocyte and granulocyte release of mainly proinflammatory cytokines in vivo, suggesting an anti-inflammatory effect. In this foremost in vivo study, the aim was to examine the effect of such AndoSan(™) consumption on the expression of adhesion molecules CD11b, CD11c and CD62L and production of reactive oxygen species (ROS) in leukocytes. Methodology/Principal findings. As shown by flow cytometry, there was a significant increase of CD62L expression on monocytes and granulocytes from before (day 0) compared with 12 days after daily AndoSan(™) consumption. However, only minor alterations and no clear trend in the expression of CD11b and CD11c were detected. Intracellular ROS (mainly superoxide ion) were significantly reduced in these cells from days 0 to 12. Conclusions/Significance. These...

Explore the role of viral factors and immune response in patients with severe pandemic pdmH1N1 illness without significant co-morbidity. Seven patients with pdmH1N1 influenza, bilateral chest X-rays infiltrates, requiring mechanical ventilator support were consecutively recruited. Seven age- and gender-matched healthy individuals served as controls. Four patients were viremic, two with the mutant D222G/N pdmH1N1.Microarray analyses of peripheral blood leukocytes suggested a marked granulocytes activation, but no up-regulation of inflammatory cytokine mRNA. Patients with severe pdmH1NI had a marked systemic complement activation, and in contrast to the lack of cytokine mRNA up-regulation in blood leukocytes, plasma levels of a broad range of inflammatory mediators, including IP-10, and mediators involved in pulmonary remodelling were markedly elevated. Patients with mutant virus had particularly high IP-10 levels, and the most pronounced complement activation. In severe pdmH1N1, viremia was common and the D222G/N mutant was found in half of the viremic patients. Host immune response was characterized by strong activation of the innate immune system, including complement and granulocytes activation, increased serum levels of inflammation and pulmonary remodelling markers, possibly contributing to the observed tissue damage. However, few patients were included and further studies are needed to characterize the immune response in severe pdmH1N1 infection.

Precursor B cell production from bone marrow in mice and humans declines with age. Because the mechanisms behind are still unknown, we studied five precursor B cell subsets (ProB, PreBI, PreBII large, PreBII small, immature B) and their differentiation-stage characteristic gene expression profiles in healthy individual toddlers and middle-aged adults. Notably, the composition of the precursor B cell compartment did not change with age. The expression levels of several transcripts encoding V(D)J recombination factors were decreased in adults as compared with children: RAG1 expression was significantly reduced in ProB cells, and DNA-PKcs, Ku80, and XRCC4 were decreased in PreBI cells. In contrast, TdT was 3-fold upregulated in immature B cells of adults. Still, N-nucleotides, P-nucleotides, and deletions were similar for IGH and IGK junctions between children and adults. PreBII large cells in adults, but not in children, showed highly upregulated expression of the differentiation inhibitor, inhibitor of DNA binding 2 (ID2), in absence of changes in expression of the ID2-binding partner E2A. Further, we identified impaired Ig locus contraction in adult precursor B cells as a likely mechanism by which ID2-mediated blocking of E2A function results in reduced bone marrow B cell output in adults. The reduced B cell production was not compensated by increased proliferation in adult immature B cells, despite increased Ki67 expression. These findings demonstrate distinct regulatory mechanisms in B cell differentiation between adults and children with a central role for transcriptional regulation of ID2.

The epithelial marker E-cadherin plays a crucial role in epithelial-mesenchymal transition (EMT). Decreased protein content in somatotroph adenomas has been associated with increased tumor size, invasion, and poor response to somatostatin analog (SA) treatment, but the potential mechanisms of EMT progression in these adenomas are lacking. We hypothesized that characterization of EMT-related transcripts in somatotroph adenomas could identify novel therapeutic targets in individuals with poor response to SA treatment and provide more knowledge of the mechanism of EMT progression. Fifty-three patients with acromegaly participated in the study. We performed microarray analysis of 16 adenomas, eight with high expression and eight with low expression of E-cadherin, in order to identify EMT-related transcripts. Candidate transcripts were further explored in vivo in 53 adenomas and in vitro in a rat pituitary GH-producing cell (GH3) after exploring three models for reducing E-cadherin and inducing a mesenchymal phenotype. In vivo E-cadherin mRNA expression in tumor tissue is associated negatively with tumor size and invasiveness and positively with GH and IGF-I levels in serum and response to SA treatment. Microarray and subsequent PCR analysis identify several EMT-related genes associated with E-cadherin expression. In vitro, few of these EMT-related genes were regulated by silencing E-cadherin or by TGF-β1 treatment in GH3 cells. In contrast, silencing Esrp1 in GH3 cells regulated many of the EMT-related transcripts. These results indicate that ESRP1 could be a master regulator of the EMT process in pituitary adenomas causing acromegaly.

To investigate whether human limbal epithelial cells (HLECs) derived from various regions of the limbus exhibit differences in gene expression and epithelial characteristics. HLECs were derived from explants taken from the superior, nasal, inferior, and temporal limbus and cultured for 21 days. Whole genome transcript profiling was performed with a gene microarray. The microarray results were validated by using RT-PCR. Epithelial morphology was studied with light microscopy and transmission electron microscopy, and phenotype was evaluated by immunohistochemistry. Epithelial outgrowth was present in most cultures of superior origin (88%) in contrast to cultures of temporal origin (38%). The epithelial thickness and number of cell layers were significantly greater in cultures of superior origin than in cultures from inferior and temporal areas. TRIM36, OSR2, and RHOU, which are involved in morphogenesis, were significantly differentially expressed in the superior region, compared with the other regions. Proposed limbal stem cell, progenitor, and differentiation markers were not differentially expressed. The uniform gene expression of ocular surface markers correlated with homogeneous immunostaining of corresponding protein markers in HLEC cultures from all regions, demonstrating an undifferentiated phenotype (p63(+), DeltaNp63alpha(+), ABCG2(+), K19(+), vimentin(+), integrin beta1(+), nestin(-), K3(-), K5(+), and E-cadherin(+)). No major transcriptional or phenotypic differences were observed in cultured HLECs derived from different regions of the limbus. However, explants of superior origin demonstrated the highest outgrowth success rate and generated epithelia with greater epithelial thickness and number of cell layers, which may prove useful for transplantation purposes.

Lipopolysaccharide (LPS) in the outer membrane of Neisseria meningitidis plays a dominant role as an inflammation-inducing molecule in meningococcal disease. We have used microarray analysis to study the global gene expression after exposure of human monocytes for 3 h to wild-type N. meningitidis (10(6)), LPS-deficient N. meningitidis (10(6) and 10(8)), and purified N. meningitidis LPS (1 ng [33 endotoxin units]/ml) to identify LPS-inducible genes. Wild-type N. meningitidis (10(6)) induced 4,689 differentially expressed genes, compared with 72 differentially expressed genes induced by 10(6) LPS-deficient N. meningitidis organisms. However, 10(8) LPS-deficient N. meningitidis organisms induced 3,905 genes, indicating a dose-response behavior of non-LPS cell wall molecules. A comparison of the gene expression patterns from 10(6) wild-type N. meningitidis and 10(8) LPS-deficient N. meningitidis organisms showed that 2,401 genes in human monocytes were not strictly LPS dependent. A list of "particularly LPS-sensitive" genes (2,288), differentially induced by 10(6) wild-type N. meningitidis but not by 10(8) LPS-deficient N. meningitidis organisms, showed an early expression of beta interferon (IFN-beta), most likely through the Toll-like receptor-MyD88-independent pathway. Subsequently, IFN-beta may activate the type I IFN signaling pathway, and an unknown number of IFN-beta-inducible genes, such as those for CXCL9, CXCL10, CXCL11, IFIT1, IFIT2, IFIT3, and IFIT5, are transcribed. Supporting this, human monocytes secreted significantly higher levels of CXCL10 and CXCL11 when stimulated by 10(6) wild-type N. meningitidis organisms than when stimulated by 10(8) LPS-deficient N. meningitidis organisms. Plasma CXCL10, but not CXCL11, was positively correlated (r = 0.67; P < 0.01) to LPS in patients (n = 24) with systemic meningococcal disease. Thus, new circulating biomarkers in meningococcal disease may be suggested through LPS-induced gene expression changes in human monocytes.

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.

A full-length cDNA encoding human parathyroid hormone (hPTH) containing the prepro region was cloned into Bombyx mori baculovirus under the control of the polyhedrin promoter and polyadenylation sequences. After transfection and generation of the recombinant baculovirus, hPTH production was examined in silkworm larvae and BmN cell cultures. The larvae synthesized and efficiently secreted the correctly processed and authentic hPTH (9.4 kDa) with no sign of internal degradation. In BmN cells, the major secreted form was the correctly sized protein, but small amounts of degraded hPTH could also be detected in the medium by immunoblotting. Unlike the situation in larvae, prepro-hPTH could also be demonstrated intracellularly in BmN cells. The concentration of hPTH in the larval hemolymph was about 70 mg/l, as compared to approx. 55 micrograms/l in the medium per 7.5 x 10(6) cells. Recombinant hPTH (re-hPTH) from the hemolymph was purified by reverse-phase HPLC and subjected to chemical and biological analyses. The authenticity of the purified re-hPTH was confirmed by N-terminal sequencing, amino acid composition and a mass of 9425 Da, close to the theoretical value. The hormone showed high-affinity receptor binding and full biological potency in increasing cellular cAMP.